In Mun Ology

RESEARCH HIGHLIGHTS
Nature Reviews Immunology | AOP, published online 7 May 2013; doi:10.1038/nri3462
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INFECTION
Resistance is futile
Secondary bacterial pneumonia is a common complication of influenza virus infection in humans. But what accounts for the increased morbidity that is associated with viralbacterial co-infection? Now, Medzhitov and colleagues describe a mouse model of co-infection with influenza virus and Legionella pneumophila that can be used to discriminate between the ability of the host to detect and eliminate a pathogen (resistance) and the ability to maintain homeostasis with a given level of pathogen burden (tolerance). All mice that had been infected intranasally with a sublethal dose of influenza virus followed 3days later by a sublethal dose of L.pneumophila died within a week of co-infection, whereas all mice survived a single infection with the virus or the bacteria. Importantly, there was no significant difference in the viral or bacterial burden after single infection or co-infection, and co-infection was not associated with systemic dissemination of either pathogen. Furthermore, an attenuated strain of L. pneumophila that is unable to secrete virulence factors still resulted in 100% mortality in co-infected mice. So, bacterial growth or virulence is not required for the effects of co-infection, which confirms that this is not a failure of immune resistance. Using various mouse strains with genetic deletions of key immune molecules, as well as antibodymediated depletion of neutrophils or natural killer cells, the authors showed that co-infection still results in mortality in the absence of all major immune and inflammatory pathways that might be triggered by the virus or bacteria. They suggest that these results show that an excessive inflammatory response leading to immunopathology is not the cause of death after co-infection. Even in the absence of all controllable immunostimulatory signals when immunodeficient (Toll-like receptor2 (Tlr2)/Tlr4/) mice were infected with influenza virus and a severely attenuated L. pneumophila strain that lacked flagellin and was unable to replicate or secrete effectors most mice died following co-infection but not after a single infection with either the virus or the bacteria. So, if resistance is not involved in co-infection-associated mortality in terms of the failure of the immune response to control pathogen growth and spread, increased pathogen virulence or the effects of immuno pathology the alternative scenario is that the host is unable to tolerate the tissue damage that results from co-infection. The lungs of co-infected mice had significantly increased necrosis of airway epithelial cells compared with singly infected mice, and genes involved in tissue protection and repair were downregulated in co-infected mice compared with singly infected mice. Administration of amphiregulin an epidermal growth factor family member that has a role in maintaining lung homeostasis during influenza virus infection to Tlr2/Tlr4/ mice that had been infected with influenza virus and the severely attenuated L. pneumophila strain significantly decreased lung damage and mortality but had no effect on viral or bacterial burden. Together, the results show that tolerance can determine the outcome of an infection independently of resistance and is, thus, a bona fide host defence strategy that could be targeted for therapeutic purposes.
Kirsty Minton
ORIGINAL RESEARCH PAPER Jamieson, A. M. et al. Role of tissue protection in lethal respiratory viralbacterial coinfection. Science 25 Apr 2013 (doi:10.1126/science.1233632) FURTHER READING Schneider, D. S. & Ayres, J. S. Two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases. Nature Rev. Immunol. 8, 889895 (2008)
tolerance can determine the outcome of an infection independently of resistance and is, thus, a bona fide host defence strategy
NATURE REVIEWS | IMMUNOLOGY 2013 Macmillan Publishers Limited. All rights reserved
VOLUME 13 | JUNE 2013
RESEARCH HIGHLIGHTS
INFECTION
The interferon paradox

The type I interferon (IFN) response, which results in the expression of diverse IFN-stimulated genes (ISGs), is known to contribute to the control of viral infections. However, two research groups now report in Science that type I IFN signalling is also involved in the establishment of persistent viral infections and their associated immune suppression. In both studies, mice were infected with one of two strains of lymphocytic choriomeningitis virus (LCMV): the Armstrong LCMV strain, which induces robust virus-specific CD8+ T cell responses, was used to model acute viral infection; and the C113 LCMV strain, which persists system ically for more than 70 days postinfection, was used to mimic chronic viral infection. Wilson etal. observed long-term expression of type I IFN and ISGs in mice with chronic LCMV infection but not in those with acute LCMV infection. Moreover, Teijaro etal. found that C113 LCMV (but not Armstrong LCMV) targeted plasmacytoid dendritic cells (DCs) to induce the expression of type I IFN very early after infection. So, the type I IFN response seems to be elevated during chronic infection, and both groups associated this with the increased expression of immunosuppressive genes, such as interleukin-10 (Il10) and programmed cell death 1 ligand 1 (Pdl1), in C113 LCMV-infected mice. Treatment of mice with an IFN/ receptor 1 (IFNAR1)-specific blocking antibody starting one day before infection with persistent C113 LCMV reduced the levels of IL-10 in the blood and the numbers of the immunosuppressive PDL1+ DCs in the spleen, increased the splenic numbers of other immune cells (including natural killer cells, Bcells and IFNproducing CD4+ Tcells) and restored lymphoid tissue architecture (which is otherwise disturbed during chronic viral infection). Notably, IFNAR1 blockade compromised viral control in Armstrong LCMV-infected mice, which was consistent with the idea that type I IFN signalling has potent antiviral activity during acute viral infections. However, in C113 LCMV-infected mice, IFNAR1 blockade facilitated viral clearance approximately 30 days after infection, which indicates that the typeI IFN response might have a role in the establishment of chronic viral infections. Importantly, CD4+ T cells and IFN expression were essential for viral control in C113 LCMV-infected mice that had been treated with IFNAR1specific antibodies. Next, the two groups tested the therapeutic potential of IFNAR1 blockade in chronically infected mice. Administration of the IFNAR1specific antibody either 10 days (Teijaro et al.) or 25 days (Wilson etal.) after viral infection promoted clearance of the persistent infection. This suggests that the therapeutic potential of inhibitors of type I IFN signalling should now be investigated in patients with chronic viral infections, including HIV or hepatitisC virus infection. Taken together, these findings indicate a dual role for type I IFN signalling in viral infections it has a potent antiviral activity during the early stages of acute viral infection but it also contributes to the establishment and maintenance of chronic viral infection.
Maria Papatriantafyllou
ORIGINAL RESEARCH PAPERS Wilson, E. B. et al. Blockade of chronic type I interferon signalling to control persistent LCMV infection. Science 340, 202207 (2013) | Teijaro, J. R. et al. Persistent LCMV infection is controlled by blockade of type I interferon signalling. Science 340, 207211 (2013)
the therapeutic potential of inhibitors of type I IFN signalling should now be investigated in patients with chronic viral infections
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RESEARCH HIGHLIGHTS
NPG
LY M P H O C Y T E R E S P O N S E S
Stand by for action!

During an adaptive immune response, T follicular helper (TFH) cells enter germinal centres and support the affinity maturation, isotype class-switching and memory responses of antigen-specific B cells. Previous studies have focused on how cognate interactions shape germinal centre responses; Qi and colleagues now report that costimulatory signals delivered by bystander B cells are essential for TFH cell recruitment to the germinal centre. Deficiency of inducible T cell costimulator (ICOS) leads to defective germinal centre responses. This was thought to be owing to the inability of ICOS-deficient T cells to upregulate CXC-chemokine receptor5 (CXCR5), which is required for migration into B cell follicles and, as such, for recruitment to germinal centres. The authors transduced ICOS-deficient ovalbumin (OVA)specific CD4+ T cells (OT-II cells) with CXCR5 and found that they were still unable to migrate into the follicles after immunization with haptenated OVA. Instead, these cells accumulated at the T cellB cell (TB) border at the follicular edge. ICOS-deficient OT-II cells that were transduced with the transcription factor B cell lymphoma 6 (BCL-6; a master regulator of TFH cell development) also failed to enter the follicles.
Therefore, ICOS signalling promotes TFH cell recruitment by a mechanism that is distinct from CXCR5 or BCL-6 upregulation. Surprisingly, recruitment of wildtype OT-II cells was not impaired if OVA-presenting dendritic cells or OVA-presenting B cells were defective in ICOS ligand (ICOSL) expression. This suggested that ICOS signalling promotes T cell entry into the follicles in an antigen-independent manner. In support of this theory, Tcells that had been activated invitro could migrate deep into follicles, even if they were not specific for the immunizing antigen; however, their entry was strictly dependent on ICOS expression. Therefore, the authors examined whether ICOS signalling provided by bystander B cells promotes T cell entry into the follicles. In mice with a B cell-specific deficiency in ICOSL, activated OT-II cells were unable to enter the follicles. By contrast, these T cells were recruited into follicles in control mice in which Bcells expressed ICOSL, even if the Bcells were deficient in MHC classII expression. Closer analyses showed that ICOSL-expressing bystander B cells do not alter chemokine gradient sensing by T cells; instead, they promote persistent random motility in Tcells in a
bystander B cells are essential for TFH cell recruitment to the germinal centre
phosphoinositide3-kinase (PI3K)dependent manner. Two-photon intravital imaging showed that activated T cells at the TB border extended frequent pseudopods and maintained a polarized state, which is crucial for directional cell migration. By contrast, in animals with ICOS-deficient Tcells or ICOSL-deficient B cells, Tcells were depolarized and showed decreased motility at the follicular border. Finally, the authors showed that the lack of expression of ICOSL by bystander B cells led to defective germinal centre responses following immunization, even when antigenspecific T cells and B cells were present and competent for ICOS and ICOSL expression, respectively. The authors conclude that bystander follicular B cells at the TB border provide an ICOS-dependent signal to activated CD4+ T cells, thereby promoting their eventual recruitment to germinal centres. Notably, this study shows that stimulatory signals that are delivered to T cells by B cells in an antigenindependent manner also support germinal centre responses.
Yvonne Bordon
ORIGINAL RESEARCH PAPER Xu, H. et al. Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility. Nature 25 Apr 2013 (doi: 10.1038/nature12058)
RESEARCH HIGHLIGHTS
MUCOSAL IMMUNOLOGY
TLRs get rhythm

As well as detecting pathogenic organisms, Toll-like receptors (TLRs) coordinate homeostatic responses to commensal bacteria. Chambon and colleagues now report that TLR expression by intestinal epithelial cells (IECs) is under circadian control, leading to oscillatory immune signalling in response to the microbiota. Notably, in the absence of the microbiota the circadian clock is disrupted in IECs and mice develop metabolic disturbances. To examine whether commensal bacteria are involved in circadian responses, the authors compared temporal gene expression profiles in IECs from control and microbiota-depleted mice. IECs from microbiota-depleted mice showed disrupted circadian control of several nuclear receptors, including PPAR (peroxisome proliferator-activated receptor-), REV-ERB and ROR (retinoic acid receptor-related orphan receptor-), and of components of the circadian clock machinery. Notably, depletion of the microbiota also led to systemic metabolic effects, with animals showing increased blood levels of glucose, triglycerides and free fatty acids, and decreased production of insulin. Further analyses showed that these metabolic disturbances resulted from increased corticosterone synthesis by ileal IECs, which was caused by increased PPAR expression in these cells. Indeed, microbiotadepleted mice with an IEC-specific deletion of PPAR showed normal regulation of circadian clock components and did not show any metabolic disturbances. Microbial products promoted the activation of JUN N-terminal kinase (JNK) and the activator protein 1 (AP-1) transcription factor JUN in IECs, and JUN was shown to repress PPAR. However, although JNK and JUN showed a circadian mode of activation, their levels of gene and protein expression did not change over time. Instead, the authors found that the expression of TLR1 to TLR5 and also TLR9 (but not TLR6 and TLR7) is under circadian control in IECs. This explains how microbiotaderived products can activate JNK and JUN and repress PPAR in a circadian manner. The temporal transcription of TLRs in IECs was shown to be activated and repressed by the alternate binding of ROR or REV-ERB, respectively, to a conserved site in the TLR gene promoters. Using a bioinformatics approach, the authors found that a large number of genes (>2,000) that are expressed by IECs contain the ROR and REV-ERB DNA-binding site. This suggests that many of the genes that are involved in homeostatic IEC responses are controlled in a microbiota-dependent circadian manner. Indeed, the authors confirmed that several immune mediators that are important for maintaining epithelial barrier integrity are regulated in this way in IECs. Taken together, the data show a key role for the microbiota and TLRs in controlling circadian responses in IECs. Such circadian control of IEC responses could be important for coordinating homeostatic IEC functions with behavioural activities in mice.
Yvonne Bordon
COMSTOCK IMAGES
ORIGINAL RESEARCH PAPER Mukherji, A. et al. Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs. Cell 153, 812827 (2013) FURTHER READING Sheiermann, C., Kunisaki, Y. & Frenette, P. S. Circadian control of the immune system. Nature Rev. Immunol. 13, 190198 (2013)
TLR expression by intestinal epithelial cells ... is under circadian control
RESEARCH HIGHLIGHTS
IN BRIEF
MAST CELLS
Distal signalling in mast cell degranulation

Ligation of the high-affinity Fc receptor for IgE (FcRI) promotes mast cell activation. Now, a role for a truncated splice variant of the FcRI -subunit (tFcRI) in mast cell degranulation has been identified. Findings by Cruse et al. suggest that full-length FcRI might inhibit mast cell degranulation. By contrast, tFcRI promoted mast cell degranulation and the release of interleukin-8, acting downstream of calcium mobilization. Notably, mast cell activation promoted the translocation of the tFcRI complex to the Golgi apparatus, where it initiated microtubule formation. Thus, tFcRI could be targeted in the future to inhibit the microtubule-dependent degranulation of mast cells and their release of pro-inflammatory mediators in patients with allergies.
ORIGINAL RESEARCH PAPER Cruse, G. et al. A truncated splice-variant of the FcRI receptor subunit is critical for microtubule formation and degranulation in mast cells. Immunity 2 May 2013 (doi:10.1016/j.immuni.2013.04.007)
TECHNIQUE
A genetically encoded T cell calcium indicator

For the first time, the genetically encoded calcium indicator TN-XXL has been used for in vivo imaging of T cell activation. As TN-XXL is self-replenishing, it should enable longer-term invivo work to be carried out than the synthetic calcium indicator dyes that have been previously used. TN-XXL comprises a fluorophore donor attached to a fluorophore acceptor by a calcium-sensitive linker. Binding of free calcium to the linker results in energy tranfer from the donor to the acceptor. The resulting change in the fluorescence ratio is a direct indicator of a change in intracellular calcium concentration. The previously developed form of TN-XXL was optimized by the authors through codon diversification to enable the retroviral transduction of Tcells. A linker with increased calcium affinity was used to increase sensitivity and to enable the measurement of physiologically relevant changes in intracellular calcium levels that occur in Tcells after antigen stimulation. As a proof of principle, the resulting construct (Twitch-1CD) was used to transduce myelin-specific 2D2 T cells and to assess their activation in response to antigen in vivo.
ORIGINAL RESEARCH PAPER Mues, M. et al. Real-time in vivo analysis of T cell activation in the central nervous system using a genetically encoded calcium indicator. Nature Med. 12 May 2013 (doi:10.1038/nm.3180)
M AC R O P H AG E S
Metabolites mangle microbes

Macrophages express high levels of immunoresponsive gene1 (IRG1) under inflammatory conditions but the function of this gene has been unknown. This study now identifies IRG1 as an enzyme that catalyses the production of itaconic acid by decarboxylating the Krebs cycle intermediate cis-aconitate. When mouse macrophages were activated with lipopolysaccharide (LPS), they upregulated Irg1 transcripts in 2 hours and showed increased levels of itaconic acid by 6 hours. Human monocyte-derived macrophages showed a similar response to LPS. Why do activated macrophages upregulate this metabolite? Itaconic acid can inhibit the glyoxylate shunt this metabolic process is not found in animals but it is essential for bacterial growth when fatty acids or acetate are a limiting carbon source. Notably, when Mycobacteria spp. or Salmonella spp. were cultured under such conditions, itaconic acid inhibited their growth. Finally, silencing of Irg1 in mouse macrophages prior to infection with Salmonella spp. increased intracellular bacterial loads.
ORIGINAL RESEARCH PAPER Michelucci, A. et al. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production. Proc. Natl Acad. Sci. USA 110, 78207825 (2013)
RESEARCH HIGHLIGHTS
TO L E R A N C E
The origins of colonic TReg cells
It has been previously suggested that thymus-derived regulatory T (TReg) cells primarily recognize self antigens, whereas peripherally derived TReg cells respond to foreign antigens. Indeed, conversion of forkhead box P3 (FOXP3)CD4+ T cells to FOXP3+ TReg cells is known to be favoured by the tolerogenic microenvironment of the gut, where there is an abundance of innocuous foreign antigens, including food- and microbiotaderived components. However, Cebula et al. now suggest that most colonic TReg cells, including TReg cells that recognize intestinal commensal bacteria, develop in the thymus. The authors used transgenic mice that express a limited but diverse T cell receptor (TCR) repertoire (TCRmini mice) to investigate the origin of colonic TReg cells on the basis of high-throughput TCR sequencing analyses. T cells from TCRmini mice express a single TCR
colonic TReg cells seem to derive predominantly from FOXP3+CD4+ thymocytes
-chain combined with a variety of TCR -chains, and their development and function was found to be comparable to that of non-transgenic T cells. Sorted individual FOXP3+ T cells and FOXP3CD4+ T cells from the thymus, peripheral lymph nodes and intestines of TCRmini mice were analysed in terms of the complementarity determining region3 (CDR3) sequence of the TCR -chain that they expressed. It was found that the most abundant TCRs did not overlap between the FOXP3+ and FOXP3CD4+ T cell subsets, irrespective of the organ of origin. Moreover, 86% of the TCRs from colonic TReg cells were identical to 50% of the TCRs from FOXP3+CD4+ thymocytes. This suggests that in all organs that were analysed, including the intestines, peripheral conversion of FOXP3CD4+ T cells to FOXP3+ TReg cells did not account for the
majority of TReg cells. A similar result was observed in transgenic mice that had an even broader TCR repertoire. So, colonic TReg cells seem to derive predominantly from FOXP3+CD4+ thymocytes. But do these thymus-derived intestinal TReg cells respond to foreign antigens such as those derived from commensal bacteria? Treatment of TCRmini mice with antibiotics altered the frequency of the most dominant colonic TReg cell TCRs, which probably reflects the antigen-specific expansion or contraction of colonic TReg cell populations. However, the diversity of the colonic TReg cell TCR repertoire was not reduced following the changes in the composition of colonic microbiota. Moreover, most colonic TReg cell TCRs were shared with FOXP3+CD4+ thymocytes even after treatment with antibiotics. This indicates that changes in the composition of the population of intestinal commensal bacteria did not promote substantial recruitment of peripherally derived TReg cells. Finally, hybridomas that were generated from colonic TReg cells showed responsiveness to sterile filtrates of caecal contents and to sonicates of individual commensal bacterial species. As 90% of the TCRs of hybridomas that responded to caecal contents were expressed by FOXP3+CD4+ thymocytes, the authors conclude that thymusderived TReg cells have a leading role in establishing intestinal T cell tolerance against microbiota.
Maria Papatriantafyllou
ORIGINAL RESEARCH PAPER Cebula, A. et al. Thymus-derived regulatory T cells contribute to tolerance to commensal microbiota. Nature 497, 258262 (2013)
Photod isc/Getty I mages
RESEARCH HIGHLIGHTS
AL PIXT
N AT U R A L K I L L E R C E L L S
Adaptive control of NK cells

Forkhead box P3 (FOXP3)+ regulatory T (TReg) cells maintain peripheral self-tolerance by suppressing the responsiveness of other immune cells, including natural killer (NK) cells. Three recent studies have discovered a mechanism by which TReg cells regulate NK cells. In the first study by Gasteiger etal., the authors found that NKcell reactivity to strong activating signals, as well as their tolerance to self ligands, was not affected by the absence of TReg cells. However, NKcell-mediated targeting of MHC classI-deficient cells (which lack NKcell inhibitory receptors; also known as the missing-self response) was greatly enhanced in the absence of TReg cells. Interleukin-2 (IL-2) neutralization reversed this enhanced missing-self response, as did the depletion of CD4+ T cells, which readily produce IL-2 in TReg cell-deficient mice. These data suggest that TReg cells specifically suppress NK cell reactivity to missing-self targets by limiting the availability of CD4+ T cell-derived IL-2. Of note, IL-2, which was shown to increase the adhesiveness of NKcells to missing-self targets, also enhanced NK cell adhesion to, and NKcell-mediated killing of, weak targets that is, cells that are normally inefficiently killed by NK cells. In the second study by this group, the minor CD127+ NK cell splenic population was shown to increase in the absence of TReg cells. This expanded population comprised mainly immature NK cells that gave rise to mature CD11b+ NK cells following their transfer to lymphopenic hosts. CD127+ NK cells were the only NK cells that expressed CD25 (the high-affinity receptor for IL-2). The expression of CD25 by these cells was upregulated in response to the pro-inflammatory cytokine IL-12, levels of which are increased following TReg cell depletion. The expansion of the CD127+ NK cell population in the absence of TReg cells depended on the presence of CD4+ T cells and on IL-2, which suggests that TReg cells control the homeostasis of this immature NKcell population by restricting T cell-derived IL-2 availability. Interestingly, immature CD127+ NKcells also accumulated in tumourbearing and chronically infected mice. In a third study, Sitrin etal. showed that following acute TReg cell depletion in BDC2.5/NOD mice (a model of type 1 diabetes), pancreas-infiltrating NK cells were activated and had enhanced IL-2-induced gene expression. IL-2 neutralization in TReg celldepleted BDC2.5/NOD mice reduced NK cell accumulation and their production of interferon- (IFN), which the group had previously shown to promote disease in this model. Correspondingly, supplementation of TReg cell-sufficient BDC2.5/NOD mice with IL-2 induced pancreatic NK cell proliferation and IFN production. The main source of IL-2 in this model was found to be CD4+ Tcells in the pancreas. Taken together, these studies suggest that TReg cells control the activity of NK cells by limiting their exposure to T cell-derived IL-2. These findings have important implications for the therapeutic manipulation of NK cells and for IL-2-based immunotherapies.
Olive Leavy
ORIGINAL RESEARCH PAPERS Gasteiger, G. et al. IL-2-dependent tuning of NK cell sensitivity for target cells is controlled by regulatory T cells. J. Exp. Med. 6 May 2013 (doi:10.1084/jem.20122462) | Gasteiger, G. et al. IL-2-dependent adaptive control of NK cell homeostasis. J. Exp. Med. 6 May 2013 (doi:10.1084/jem.20122571) | Sitrin, J. et al. Regulatory T cells control NK cells in an insulitic lesion by depriving them of IL-2. J. Exp. Med. 6 May 2013 (doi:10.1084/jem.20122248)
TReg cells control the activity of NK cells by limiting their exposure to T cell-derived IL-2
RESEARCH HIGHLIGHTS
TYPE 2 IMMUNITY
Regenerating muscles the type 2 way

Innate immune cells such as macro phages have previously been associ ated with tissue repair responses, including muscle regeneration after injury. However, a direct molecular link between an innate immune response and the progenitor cells that give rise to or support muscle regen eration remains to be established. Now, reporting in Cell, Heredia, Mukundan et al. delineate a pathway for the role of type 2 innate immune signals in muscle regeneration. The type 2 cytokines interleukin-4 (IL-4) and IL-13 are known to have a role in tissue repair, so the authors investigated the functions of these cytokines in muscle regeneration following cardiotoxin-induced injury of the tibialis anterior muscles of mice. In contrast to wild-type mice, Il4/Il13/ mice failed to repair their injured muscles these muscles lacked regenerative myofibres and instead contained cell debris and inflammatory cells. Using 4get reporter mice, in which IL-4-producing cells can be readily identified through their expression of green fluorescent protein, the source of IL-4 in regenerating muscles was found to be mainly eosinophils, with a small contribution from mast cells. Furthermore, eosinophil-deficient mice were unable to regenerate injured muscles. But which cells in the regenerat ing muscles respond to IL-4 and/or IL-13? IL-4 receptor- (IL-4R), which is a shared component of the IL-4 and IL-13 receptors, was shown to be specifically expressed by fibrocyteadipocyte progenitors (FAPs) and myeloid cells in muscle. Surprisingly, loss of IL-4R expres sion specifically on myeloid cells did not greatly affect muscle regen eration. However, loss of IL-4R expression in mice (or a deficiency in IL-4 and IL-13, or a deficiency in eosinophils) resulted in decreased proliferation of FAPs and defective muscle repair, indicating that FAPs may be the responding cells. FAPs are stromal cells that are thought to support myogenesis. However, these cells can also give rise to ectopic adipocytes that accumulate in degenerating muscles. Analysis invitro showed that IL-4 induced the proliferation of FAPs through the activation of signal transducer and activator of transcription 6 (STAT6), it increased their expression of myogenic transcription factors and it inhibited their differentiation into adipocytes. Further analysis invivo showed that administration of IL-4 prevented fatty degeneration of injured muscles in response to glycerol injection. These data suggest that IL-4 functions as a molecular switch that controls the fate of FAPs in regenerating muscles to support myogenesis and to prevent fatty degeneration of muscle. Finally, the authors showed that FAPs were the primary cell type that cleared necrotic cell debris in injured muscle, which is a process that is necessary for muscle regeneration. Furthermore, the clearance of cell debris was impaired in mice lacking IL-4R expression on FAPs, as well as in mice lacking eosinophils, which indicates that there is a dependence on type 2 innate immune signals. So, following muscle injury, infiltrating eosinophils secrete the type 2 cytokine IL-4, which induces the proliferation of FAPs to promote clearance of necrotic debris.
Olive Leavy
Dover images
ORIGINAL RESEARCH PAPER Heredia, J. E. et al. Type 2 innate signals stimulate fibro/adipogenic progenitors to facilitate muscle regeneration. Cell 153, 376388 (2013)
IL-4 functions as a molecular switch that controls the fate of FAPs in regenerating muscles to support myogenesis and to prevent fatty degeneration of muscle
REVIEWS
Activation and regulation of the inflammasomes
Eicke Latz14, T.Sam Xiao5 and Andrea Stutz1
Abstract | Inflammasomes are key signalling platforms that detect pathogenic microorganisms and sterile stressors, and that activate the highly pro-inflammatory cytokines interleukin1 (IL1) and IL18. In this Review, we discuss the complex regulatory mechanisms that facilitate a balanced but effective inflammasome-mediated immune response, and we highlight the similarities to another molecular signalling platform the apoptosome that monitors cellular health. Extracellular regulatory mechanisms are discussed, as well as the intracellular control of inflammasome assembly, for example, via ion fluxes, free radicals and autophagy.
ASC
An adaptor protein that was originally found to form protein precipitates in apoptotic cells that are termed protein specks.
Institute of Innate Immunity, University Hospital, University of Bonn, Bonn 53127, Germany. 2 Department of Medicine and Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester 01655, Massachusetts, USA. 3 Deutsches Zentrum fr Neurodegenerative Erkrankungen (DZNE), Bonn 53175, Germany. 4 Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim NO-7491, Norway. 5 Structural Immunobiology Unit, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Bethesda 20892 Maryland, USA. Correspondence to E.L. email: eicke.latz@uni-bonn.de doi:10.1038/nri3452
1
A major role of the immune system is to maintain homeostatic tissue function. For example, sterile tissue damage that occurs after trauma needs to be detected and repaired. Pathogens that can invade and cause harm to tissues should be eliminated while our commensal microbiome must be tolerated, as it fulfils functions that are required for host survival. The innate immune system has a number of signalling receptors that recognize foreign molecular structures as well as self molecules that are altered, that have become too abundant or that emerge in areas normally devoid of these molecules1,2. Innate immune signalling receptors monitor the extracellular space as well as many subcellular compartments for signs of infection, damage or other cellular stressors. The inflammasomes are a group of multimeric protein complexes that consist of an inflammasome sensor molecule, the adaptor protein ASC and caspase 1. Inflammasome formation is triggered by a range of substances that emerge during infections, tissue damage or metabolic imbalances. Once the protein complexes have formed, the inflammasomes activate caspase 1, which proteolytically activates the pro-inflammatory cytokines interleukin1 (IL1)3 and IL18. In addition, inflammasome activation causes a rapid, pro-inflammatory form of cell death called pyroptosis4. With the discovery of pattern recognition receptors (PRRs), such as inflammasome sensor molecules, their signalling pathways and their ability to programme cellular immune responses, we are beginning to understand how the immune system protects the host at a molecular level. At the same time, we have learnt that immune dysregulation contributes to prevalent diseases in Western societies such as atherosclerosis, type2 diabetes, cancer
and neurodegenerative diseases. Thus, a fine balance must be maintained between the activation and inhibition of inflammation to allow the immune system to remove any sources of danger without causing harm to thehost. In this Review, we present an overview of the current understanding of inflammasome activation and regulation, and we discuss the recent findings about noncanonical processing of IL1. We also compare the structures and the regulation of inflammasomes with that of the apoptosome.
The inflammasomes and their coreceptors Inflammasome components. At first glance, the inflammasomes are organized in a very simple manner: inflammasome sensor molecules (see below) connect to caspase 1 via ASC, which is an adaptor protein encoded by PYCARD that is common to all inflammasomes. ASC consists of two death-fold domains: one pyrin domain and one caspase activation and recruitment domain (CARD). ASC interacts with the upstream inflammasome sensor molecules via the pyrin domain5. This interaction triggers the assembly of ASC into a large protein speck consisting mainly of multimers of ASC dimers6,7. Using its CARD, ASC brings monomers of pro-caspase 1 into close proximity, which initiates caspase 1 self-cleavage and the formation of the active heterotetrameric caspase1. Active caspase 1 proteolytically activates a number of proteins8, including proIL1 and proIL18 (REFS9,10), and induces their release via a non-classical secretion pathway11. The transcription of proIL1 is induced by the activation of the transcription factor nuclear factorB (NFB), whereas proIL18 is constitutively expressed and its expression is increased after cellular activation. Therefore, these potent pro-inflammatory cytokines are
VOLUME 13 | JUNE 2013 | 397
REVIEWS
controlled by two checkpoints: transcription as well as maturation and release11. Caspase 1mediated activation of members of the IL1 cytokine family leads to the recruitment and the activation of other immune cells, such as neutrophils, at the site of infection and/or tissuedamage. Several inflammasome sensor molecules can trigger the formation of inflammasomes. Most of the inflammasomes that have been described to date contain a NOD-like receptor (NLR) sensor molecule, namely NLRP1 (NOD-, LRR- and pyrin domain-containing 1), NLRP3, NLRP6, NLRP7, NLRP12 or NLRC4 (NOD-, LRR- and CARD-containing 4; also known as IPAF). The NLR proteins, with the exception of NLRP1, have a tripartite domain organization; they contain an aminoterminal death-fold domain (NLRPs contain a pyrin domain, whereas NLRC4 contains a CARD), a central NACHT nucleotide-binding domain and carboxyterminal leucine-rich repeats (LRRs)12. The NACHT domain has ATPase activity and is thought to have a role in the oligomerization of the proteins, whereas the LRRs have regulatory functions and might be involved in ligand interaction. The death-fold domains of the NLR proteins interact with those of ASC and/or caspase 1. In addition to these domains, human NLRP1 contains a function-to-find domain (FIIND) and a Cterminal CARD. In the original description of the inflammasome, human NLRP1 was shown to recruit and to activate an additional inflammatory caspase, namely caspase 5, via itsCARD3. NLRC4 and NLRP1 can both activate caspase 1 through their CARDs without recruiting ASC; however, the recruitment of ASC greatly enhances the formation of the complex and the processing of IL17,1316. Exactly how ASC is recruited to these inflammasomes remains unclear, as NLRC4 and mouse NLRP1B do not have pyrin domains. In a mammalian two-hybrid analysis, the CARD of NLRC4 was found to interact with the CARD of ASC17. We speculate that a CARDCARD interaction between the NLR and ASC recruits a first layer of ASC, which in turn interacts with a second layer of ASC via pyrinpyrin domain interactions. Two other inflammasomes have been described that contain the PYHIN (pyrin and HIN domain-containing protein) family members absent in melanoma 2 (AIM2) and IFN-inducible protein 16 (IFI16) rather than an NLR18. AIM2 consists of a pyrin domain to recruit ASC and a DNA-binding HIN domain, whereas IFI16 has one pyrin domain and two HIN domains for DNA binding. Retinoic acid-inducible geneI (RIGI) protein is also thought to assemble an inflammasome with ASC and caspase 1 (REF.19), possibly via its CARDs. However, for some of the inflammasome sensors (including NLRP6, NLRP12, RIGI and IFI16), the potential to form inflammasomes has not been well established and other functions have been described for these molecules. Indeed, NLRP12 can function as a positive regulator of dendritic cell migration or as a negative regulator of non-canonical NFB signalling 20,21, and NLRP6 can negatively regulate innate immunity 22. RIGI is widely known as a PRR that senses RNA and that signals via mitochondrial antiviral signalling protein (MAVS) to induce an interferon (IFN) response2, and IFI16 has been suggested to be a DNA sensor that signals via the protein STING (stimulator of IFN genes; also known as TMEM173) to generate an IFN response23. Numerous activators of the inflammasomes and several different activation pathways have been described (reviewed in REF.24). The PYHIN proteins and RIGI recognize nucleic acids18,19,25, whereas NLRC4 is activated by microbial proteinaceous ligands26. NLRP1 recognizes muramyl dipeptide, which is a bacterial peptido glycan, and murine NLRP1B can also be activated by the lethal toxin from Bacillus anthracis 13,27. Many triggers, including crystalline material, peptide aggregates and bacterial toxins, can stimulate NLRP3 (REF. 24). NLRP7, which is not expressed in mice, is activated by bacterial lipo peptides28, and the triggers for NLRP6 and NLRP12 remain to be identified2931. In summary, the inflammasome sensor molecules can detect a broad range of molecular signatures to sense microorganisms and tissue stress. They are best known for triggering a robust inflammatory response via the activation of inflammatory caspases. However, it should also be noted that not all NLR molecules form inflammasomes and that other functions of NLRs, which are not as well understood, might be important contributors to an inflammatory response. Cofactors for inflammasome activation. Some inflammasome sensor molecules require coreceptors to recognize their ligands or to be stabilized in their activated states. NLRP1 has been shown to bind directly to its ligand muramyl dipeptide invitro and this was demonstrated to be sufficient to activate the assembly of an inflammasome. However, a requirement for the interaction of NLRP1 with nucleotide-binding oligomerization domain-containing protein 2 (NOD2), which is another receptor for muramyl dipeptide, has been described. This suggests that NOD2 is a vital component of the NLRP1 inflammasome32,33. Recent studies by the groups of Vance34 and Shao35 have determined that the activation of NLRC4 also requires co-receptors34,35. In mice, NAIP (NLR family, apoptosis inhibitory protein) family proteins sense the proteinaceous NLRC4 activators and, in turn, activate the assembly of the NLRC4 inflammasome. NAIP5 and NAIP6 have been shown to detect flagellin, and NAIP2 has been shown to sense the typeIII secretion system component PrgJ34,35. Only one NAIP orthologue has been found in humans (known as NAIP) and this is necessary to sense a typeIII secretion needle protein35; however, it remains to be determined whether and how human NLRC4 senses flagellin without another NAIP homologue. Some human cell lines (for example, U937) do not respond to flagellin35. However, studies using primary human cells that were infected with Legionella pneumophilia have determined that small interfering RNA (siRNA)-mediated knockdown of NLRC4 results in enhanced bacterial growth only when L.pneumophilia expresses flagellin36, which indicates that human NLRC4 might be able to sense flagellin. It is possible that NAIP could have dual specificity in humans and that it could recognize both a
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Pyroptosis
A rapid form of cell death following caspase 1 activation, which shares characteristics with both apoptosis (such as DNA fragmentation) and necrosis (such as cell swelling and rupture).
Apoptosome
A large multimeric protein complex of apoptotic proteaseactivating factor 1 (APAF1) that recognizes cytochromec release from damaged mitochondria and activates caspase 9.
Death-fold domains
Commonly found in proteins that are involved in cell death pathways and in inflammasomes. The four main death-fold domains the pyrin domain, caspase activation and recruitment domain (CARD), death domain and death effector domain associate with each other through homotypic interactions.
NOD-like receptor
(NLR). A protein that contains aminoterminal pyrin, caspaserecruitment domains or other signalling domains, followed by a NACHT domain and carboxyterminal leucine-rich repeats. Some NLR proteins are involved in forming inflammasomes.
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Non-canonical NLRP3 inflammasome
An inflammasome-like complex containing NLRP3 (NOD-, LRRand pyrin domain-containing 3), the adaptor protein ASC, caspase 1 and caspase 11. The term non-canonical inflammasome is used loosely to describe an inflammasome-like complex that does not conform to the three canonical components of a canonical inflammasome: an inflammasome sensor molecule, ASC and caspase 1. Two other non-canonical inflammasomes have also been described so far: one containing dectin 1, MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), ASC and caspase 8 (termed the non-canonical caspase 8 inflammasome), and a caspase 11activating platform that has not yet been fully described.
typeIII secretion needle protein35 and flagellin36. Dual specificity could also be used by other inflammasome sensors that recognize a variety of structurally and chemically diverse activators. Differential requirements for various NLRP3 activa tors. NLRP3 is unique among the NLRs in that its basal expression is not sufficient for inflammasome activation in resting cells37,38. Similarly to IL1 generation, a twocheckpoint activation mechanism is in place for NLRP3 inflammasome activation; it requires a priming step (see below) and a second activation step that can be induced by various triggers. Given the broad range of NLRP3 activators24, direct binding of these activators to NLRP3 seems to be unlikely. Indeed, as for NLRC4, it is possible that ligand specificity can be achieved via recruitment of different cofactors. There is evidence to support this theory, as the formation of the NLRP3 inflammasome in response to non-crystalline activators can be promoted by guanylate-binding protein 5 (GBP5)39. In addition, oxidized mitochondrial DNA, which is released into the cytosol under cellular stress and is a possible NLRP3 inflammasome activator, was recently reported to interact with NLRP3 (REF.40) (BOX1). However, whether NLRP3 directly interacts with oxidized mitochondrial DNA or whether a cofactor is required for this interaction remains unknown.
Box 1 | Sensing of mitochondrial stress

Mitochondria are required to maintain cellular energy levels and so their health is crucial for cell viability. Mitochondrial dysfunction can trigger cell autonomous pathways that lead to apoptosis or to an inflammatory response. One of the molecules that signals that there is mitochondrial dysfunction is cytochromec, which is an inner mitochondrial membrane protein and an essential component of the electron transport chain. After cytochromec has leaked into the cytosol, it binds to apoptotic proteaseactivating factor 1 (APAF1), which is a scaffold protein for the activation of the initiator caspase 9, forming the apoptosome (REF.137). This intrinsic pathway of apoptosis is regulated by B cell lymphoma 2 (BCL2) proteins138. Pro-apoptotic members of the family (such as BCL2associated X protein (BAX) and BCL2 homologous antagonist/killer (BAK)) promote cytochromec release from the mitochondria, whereas the anti-apoptotic family members (such as BCL2 and BCLXL) inhibit cytochromec release138. Interestingly, these anti-apoptotic proteins also regulate the NLRP1 (NOD-, LRR- and pyrin domain-containing 1) inflammasome. BCL2 and BCLXL bind to NLRP1 via their loop domains, thereby suppressing the ability of NLRP1 to bind to ATP and to oligomerize139,140. Mitochondrial stress can also be sensed by the NLRP3 inflammasome. Indeed, excessive production of reactive oxygen species (ROS)102 and release of oxidized mitochondrial DNA40 from stressed mitochondria have been suggested to activate the NLRP3 inflammasome. In addition, impaired mitochondrial homeostasis leads to changes in metabolites that are crucial for the function of the cell, such as the coenzyme NAD+ (REF.141). Low NAD+ levels inactivate the tubulin deacetylase sirtuin 2 (REF.142), which results in the accumulation of acetylated tubulin. Acetylated tubulin regulates the transport of mitochondria and helps the formation of an efficient interaction between the adaptor protein ASC and NLRP3 (REF.143). Furthermore, the RIGI-like receptor (RLR) adaptor molecule mitochondrial antiviral signalling protein (MAVS), which is localized on the outer mitochondrial membrane, is required for optimal NLRP3 activation144. Another intriguing mechanism by which lipopolysaccharide-activated macrophages can increase their output of interleukin1 (IL1) is through a substantial increase in levels of the tricarboxylic acid cycle intermediate succinate145. This metabolite stabilizes the transcription factor hypoxia-inducible factor1, which leads to more sustained IL1 transcription145. Therefore, monitoring of mitochondrial stress by apoptosomes and inflammasomes seems to directly link mitochondrial health and activity to cell death and inflammation.
The activation of caspase 1 following the recognition of live Gram-negative bacteria depends on inflammatory pro-caspase 11 in mice (human orthologues are pro-caspase 4 and pro-caspase 5)41, which results in non-canonical NLRP3 inflammasome activation. Pyroptosis in response to live Gram-negative bacteria but not in response to other inflammasome triggers is entirely dependent on caspase11, whereas caspase 1 is dispensable for this process41. Therefore, in contrast to the standard two-checkpoint model of priming and activation, three signals are required for full NLRP3 inflammasome activation in response to Gram-negative bacteria42,43. First, a bacterial Toll-like receptor (TLR) activator leads to cellular priming and upregulation of NLRP3 and proIL1 expression (the priming checkpoint in the standard model)37,38. The second activation checkpoint can be divided into two parts in response to bacteria. One checkpoint is that bacterial mRNA from live bacteria (also known as vita-PAMPs)44 activates NLRP3; the other checkpoint is that TLR4- and TRIF (TIR domain-containing adaptor protein inducing IFN)-dependent signalling which is triggered by bacterial lipopolysaccharide (LPS) mediate the secretion of typeI IFNs, inducing pro-caspase 11 expression and activation by triggering the IFN/ receptor (IFNAR) (FIG.1). The mechanisms by which caspase 11 is required to activate caspase 1 in response to bacteria are still under investigation and the exact timing of the different signals is not yet clear. In the context of NLRC4 activation, caspase 11 was shown to interact with cofilin. This interaction regulates actin polymerization and triggers the fusion of bacteria-containing phagosomes with lysosomes. These events facilitate the release of flagellin into the cytosol45, where NAIP family proteins can interact with flagellin and induce the activation of the NLRC4 inflammasome. It is possible that a similar caspase 11dependent release of bacterial mRNA from the phagolysosomal compartment could mediate the activation of the NLRP3 inflammasome. There is also a debate about how pro-caspase 11 expression is induced. One study found that the expression completely depends on IFN signalling 42, whereas another study suggested that its expression could be induced independently of TRIF and IFN signalling 46. Even if the expression of pro-caspase 11 could be induced independently of IFN signalling, caspase 11 activation in macrophages in response to Salmonella enterica subsp. enterica serovar Typhimurium infection is dependent on IFNAR1 as well as signal tranducer and activator of transcription 1 (STAT1)46. Therefore, an IFN-inducible factor might be required for the activation of caspase 11 during S. Typhimurium infection46. Another recent study suggests a role for caspase11 in defence against cytosolic bacteria that is independent of the known inflammasomes. These data have led to the development of a model in which a non-canonical caspase 11containing inflammasome can be formed after cytoplasmic bacteria are sensed, which leads to pyroptosis but not to IL1 release47. Further investigation is needed to determine when this complex is
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Type I IFN PRR IFNAR Cytoplasm ? ? TRIF TLR4 NLRP3 ? Fusion Lysosome ? Bacterium Bacterial mRNA Phagosome Pro-IL-1 ? Pro-caspase 11 ? AutoCaspase 11 processing? Caspase 1 Pro-IL-18 IL-1 secretion Pyroptosis Canonical NLRP3 inammasome Non-canonical caspase 8 inammasome Ripoptosomeinduced IL-1 processing TNFR-induced IL-1 processing IL-18 IL-1 Negative feedback SYK Mitochondrion MALT1 ASC Ripoptosome FADD RIP1 XIAP IAP1 IAP2 Degradation Sequestration RIP3 Dectin 1 TNFR family member such as CD95
Caspase 8 ASC
Type I IFN
Actin mobilization
? ?
IFN-inducible factor Nucleus
Non-canonical NLRP3 inammasome in response to Gram-negative bacteria
Figure 1 | Canonical and non-canonical activation of IL1. NLRP3 (NOD-, LRR- and pyrin domain-containing 3) needs additional cofactors for the processing of interleukin1 (IL1) in response to Gram-negative bacteria; this pathway has been Nature Reviews | Immunology termed the non-canonical NLRP3 inflammasome. Toll-like receptor 4 (TLR4) signalling via TIR domain-containing adaptor protein inducing IFN (TRIF) induces the secretion of typeI interferons (IFNs), which lead to the activation of caspase 11 via autocrine signalling through the IFN/ receptor (IFNAR), possibly involving additional (not yet known) IFN-inducible factors. Caspase 11 is necessary for the activation of caspase 1 and has an independent role in IL1 secretion and pyroptosis in response to Gram-negative bacteria. A possible mechanism for caspase 11 action might be its role in actin mobilization via cofilin, which might lead to increased fusion of lysosomes to phagosomes and, potentially, to increased leakage of bacterial mRNA (also termed vita-PAMPs) to the cytoplasm to activate NLRP3 (not shown). A platform consisting of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), caspase 8 and the adaptor protein ASC (termed the non-canonical caspase 8 inflammasome) is formed in response to stimulation of dectin 1. Caspase 8 might also be activated by TLRs using the signalling adaptor TRIF in the presence of cycloheximide. In addition, the formation of the ripoptosome is triggered by the loss of inhibitor of apoptosis proteins (IAPs) with concurrent TLR stimulation. The ripoptosome consisting of FAS-associated death domain protein (FADD) and receptor-interacting protein 1 (RIP1) activates caspase 8 via RIP3. However, caspase 8 can also limit ripoptosome action on NLRP3. Members of the tumour necrosis factor receptor (TNFR) family might also induce proIL1 cleavage, as has been shown for CD95 (also known as FAS). Question marks show pathways that are still speculative. PRR, pattern recognition receptor; XIAP, X-linked inhibitor of apoptosis protein.
formed, under which circumstances caspase11 activation will lead to NLRP3 activation and when it will cause cell death independently of the canonical inflamma somes. A recent review discusses caspase 11 activation in more detail48.
Non-inflammasome processing of IL1 The inflammasomes and IL1 processing invitro and invivo. Studies that have been carried out in vitro using cells that are deficient in inflammasome components have undoubtedly been instrumental in identifying
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several inflammatory triggers as activators of inflammasomes. However, the contribution of inflammasomes to IL1 activation invivo has not, at times, been nearly as convincing. The reduction in inflammation in mice that are deficient in the inflammasome components ASC or caspase 1 can be much less pronounced than that in mice lacking the IL1 receptor (IL1R) or IL1. It is probable that inflammasome-independent IL1 activation mechanisms are involved invivo that could account for the observed differences between the invitro and invivo studies. For example, the invivo
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REVIEWS
responses to particulates (diesel exhaust particles or silica) and to Mycobacterium tuberculosis have substantial caspase 1independent but IL1dependent components4951. Indeed, a number of non-caspase proteases such as proteinase 3 have the ability to activate IL1 in an inflammasome-independent manner 11. In addition, IL1, which also activates IL1R, could contribute to the inflammatory response invivo. This could partly explain why IL1Rdeficient mice have a more severe phenotype than inflammasome component-deficient mice, in which only IL1 production is affected. In contrast to IL1, IL1 is constitutively expressed in many cell types and does not need to be processed for its biological activity; thus, biologically active IL1 can be released during necrosis11. In addition, IL1 can be found on the membrane of some cell types, where it is active11. IL1 is also secreted in response to numerous NLRP3 inflammasome activators; however, its secretion is not strictly dependent on the inflammasome. Indeed, particulate activators of NLRP3, such as crystals, can induce IL1 secretion in an NLRP3independent manner 52. Therefore, the discrepancies between the invivo and invitro data may be partially explained by inflammasome-independent processing of IL1 and by the contribution of IL1, the secretion of which is regulated differently from that of IL1 release. The role of caspase 8 in IL1 processing. One additional factor that can mediate IL1 processing and activation is caspase 8 (FIG.1). Recent studies indicate that caspase8 might be able to cleave proIL1 during immune responses. For example, following sensing of fungal components by the PRR dectin 1 that is expressed on human dendritic cells, signalling via the kinase SYK leads to the formation of a complex that is composed of caspase 8 and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1); this complex binds to ASC, which possibly recruits cleavage substrates 53. The MALT1ASCcaspase 8 complex directly mediates IL1 maturation53. Thus, caspase8 can be activated by a multiprotein complex that contains the canonical inflammasome adaptor ASC this MALT1ASCcaspase 8 inflamma some protein complex has therefore been termed a noncanonical caspase 8 inflammasome. However, exactly how MALT1, caspase 8 and ASC interact is not understood, as these proteins all have different death-fold domains. In addition, NLRP3 has been implicated in dectin 1mediated IL1 release in mice54 and it therefore remains to be established whether both of these mechanisms can function in parallel or whether they represent differences between the two species. Other PRRs can also activate caspase 8. In the presence of the translation inhibitor cycloheximide, TRIF signalling that is downstream of TLR3 or TLR4 leads to proIL1 processing by caspase 8 (REF.55). Furthermore, in response to genotoxic stress, the ripoptosome assembles and activates caspase 8. The ripoptosome contains caspase 8, FAS-associated death domain protein (FADD) and receptor-interacting protein1 (RIP1; also known as RIPK1), and forms spontaneously in response to loss or inhibition of the anti-apoptotic proteins X-linked inhibitor of apoptosis protein (XIAP), inhibitor of apoptosis protein1 (IAP1; also known as BIRC3) and IAP2 (also known as BIRC2) (REF.56). The formation of this complex activates RIP3, which is necessary for the cleavage of proIL1 by both the NLRP3 caspase 1 and the caspase8 pathways56 (FIG.1). However, two other studies contradict these findings. One study showed that the NLRP3 inflamma some is more active in caspase 8deficient dendritic cells, which indicates that caspase 8 might limit ripoptosome signalling that would otherwise trigger NLRP3caspase 1 activation57. Another study found that IAP1, IAP2 and TNF receptor-associated factor 2 (TRAF2) are important for the non-degradative polyubiquitylation of caspase1, which enhances its activity 58. Other caspase 8activating receptors of the tumour necrosis factor receptor (TNFR) family can also induce IL1 activation. Indeed, activation of the TNFR family member CD95 (also known as FAS) can induce IL1 processing that is independent of inflammasomes and of caspase 1 (REF.59). In addition, it was recently shown that CD95 signalling mediates IL1 and IL18 processing through the activation of caspase 8 (REF.60) (FIG.1). Thus, more non-inflammasome pathways for the maturation of proIL1 are emerging, and these pathways could contribute to the effects of IL1 activation invivo.
Cell-extrinsic inflammasome regulation Cell-autonomous regulatory feedback loops, as well as complex networks of direct cellcell signals and indirect signals via messenger substances, regulate cellular activation. Many important immune responses can only be efficiently induced when two or more signals act on the responding cell in a timely manner for example, as has been described for IL1 maturation and NLRP3 activation. One level at which inflammasomes are regulated is at the level of expression of the individual inflammasome components61.
Positive regulation and priming. Many innate immune signalling or cytokine receptors, such as the TNFR, activate transcription of NLRP3 and thereby influence the susceptibility of immune cells to NLRP3 inflammasome triggers37,38 (FIG.2). In addition to the transcriptional regulation of NLRP3, deubiquitylation of NLRP3 has been identified as an early priming event, which only occurs in response to PRR stimulation, possibly involving the production of reactive oxygen species (ROS)62. NLRP3 deubiquitylation is mediated by the K63specific deubiquitinase BRCC3 (REF.63). Therefore, the NLRP3 activation threshold is regulated by both a fast-acting post-translational mechanism and a slower-acting transcriptional activation of NLRP3 gene expression. An early priming event has also been described for the AIM2 inflammasome, although the molecular mechanisms that are involved remain to be fully elucidated64. In addition, AIM2 expression can be induced by IFN and IFN65,66. NLRC4 can only be activated by S. Typhimurium after a specific serine residue has been phosphorylated by protein kinase C (PKC; encoded
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Ripoptosome
A cytosolic multiprotein complex that induces cell death following genotoxic stress or depletion of inhibitor of apoptosis protein (IAP). The core ripoptosome contains receptor-interacting protein 1 (RIP1), FAS-associated death domain protein (FADD) and caspase 8, but it can also recruit other proteins such as RIP3.
REVIEWS
Activatory TNFR Inhibitory T cell IL-1R PRR CD40 CD40L ? PKC NLRC4 Priming via expression levels P ? ROS ? BRCC3 Nitrosylation iNOS NO IFN IFNGR ? ? PRR Type I IFN IFNAR
IFN IFNGR Cytoplasm
Type I IFN IFNAR
Priming via deubiquitylation
NLRP3 inammasome Ub Caspase 1
Nitrosylation
Pro-IL-1 NLRP3 NLRP3 mRNA miR-223 Degradation NLRP3 pro-IL-1 AIM2
IL-1
Nucleus
Figure 2 | Extracellular signals regulate the inflammasomes. Prointerleukin1 (pro-IL1) and NLRP3 (NOD-, LRRand pyrin domain-containing 3) expression are induced by transcriptionally active pattern recognition receptors (PRRs) NatureBRCC3 Reviews | Immunology or by cytokine receptors. Furthermore, NLRP3 deubiquitylation by the K63specific deubiquitinase is crucial for its activation. Direct contact with mature or memory T cells inhibits the inflammasomes, probably via tumour necrosis factor receptor (TNFR) superfamily interactions. TypeI interferons (IFNs) inhibit the transcription of proIL1, but also upregulate the expression of absent in melanoma 2 (AIM2). Both typeI IFNs and IFN inhibit NLRP3 through the induction of nitric oxide (NO) via inducible nitric oxide synthase (iNOS), possibly with the requirement of concomitant priming by PRRs. Ub shows ubiquitylated proteins. Question marks show pathways that are still speculative. CD40L, CD40 ligand; IFNAR, interferon-/ receptor; IFNGR, interferon- receptor; IL1R, IL1 receptor; miR223, microRNA223; NLRC4, NOD-, LRR- and CARD-containing 4; ROS, reactive oxygen species; PKC, protein kinase C.
Amyloid
An endogenous peptide that is generated by proteases in the brain. It is prone to aggregation and plaque formation. Amyloid plaques are a hallmark of Alzheimers disease and can activate the NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome.
Cryopyrin-associated periodic syndrome

(CAPS). Characterized by NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome hyperactivity and the excessive release of interleukin1, which leads to an autoinflammatory disease phenotype with periodic fever episodes, urticaria and often severe arthritis.
by PRKCD), which depends on the recognition of both flagellin and the typeIII secretion system. This indicates that PKC might be activated by PRR signalling67. Therefore, at least some inflammasomes are only fully capable of responding to danger signals in situations in which either pro-inflammatory cytokines are present or innate signalling molecules sense noxious signals. A few molecules, such as amyloid, can induce both NLRP3 priming through TLR activation and NLRP3 inflammasome activation68. However, the response to aggregated amyloid can be dramatically augmented when innate sensors or cytokine receptors become activated by additional stimuli. These priming mechanisms might be of great relevance in determining the magnitude of an inflammatory response to danger signals. Conversely, genetic differences that influence the threshold of inflammasome activation might contribute to the development of chronic inflammatory
or autoinflammatory diseases. It is known that about 60% of patients with cryopyrin-associated periodic syndrome (CAPS) carry activating mutations in the coding sequence of NLRP3 itself or in other inflammasome components69. This indicates that inflammasome hyperactivity may be influenced by additional mechanisms. Indeed, single nucleotide polymorphisms in the promoter region of NLRP3 were found in patients with CAPS69. In these patients, an increase in NLRP3 expression as opposed to the presence of a more active protein (as a result of mutations in the coding sequence) could conceivably result in a lower threshold of NLRP3 inflamma some activation in response to danger signals and this could trigger the development of CAPS. Therefore, several mechanisms ensure that inflammasomes are not accidentally triggered and that an appropriately balanced immune response to danger signals can occur in a regulatedmanner.
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Negative regulation of inflammasome activation. An inflammatory response should subside once it has carried out its function. For example, the release of proinflammatory cytokines such as IL1 and IL18 might not be required after an adaptive immune response has been initiated. Thus, it is not surprising that effector and memory CD4+ Tcells have the capacity to inhibit the activation of the NLRP1 and NLRP3 inflammasomes in a contact-dependent manner, possibly via TNFR superfamily molecules such as CD40 ligand (CD40L)70. In addition, Tcell-derived IFN has been shown to downregulate the activity of NLRP3 via activation of inducible nitric oxide synthase (iNOS) in a mouse model of tuberculosis71; nitric oxide (NO) induces NLRP3 nitrosylation and thereby inhibits NLRP3 activity. However, it seems to be crucial that TLR priming and IFN stimulation occur simultaneously, as IFN loses its inhibitory activity after sequential priming with IFN and a TLR stimulus70,72. In addition, typeI IFNs can reduce IL1 and IL18 release by functioning at two levels. First, typeI IFNs inhibit the production of the pro-forms of these cytokines; second, they repress the cleavage of the cytokine proforms by the NLRP1 and NLRP3 inflammasomes72. For IFN, the mechanism of NLRP3 inhibition might also involve the induction of NO73. Timing is also important for the inhibitory effect of IFN; although LPS-induced IFN is necessary for caspase 11 expression and, thus, NLRP3 activation in response to bacteria42 (see above), IFN blocks NLRP3 inflammasome activation when the cells sense it before they have been primed, for example, by a TLR stimulus72. It is worth noting that IFNs can have seemingly opposing effects on different inflammasomes. IFNs upregulate AIM2 expression but they downregulate IL1 expression and inhibit the NLRP3 inflammasome. These effects could be adapted to generate optimal antiviral responses, in which excessive responses to danger signals could be harmful for thehost. Furthermore, the amount of NLRP3 mRNA is tightly regulated by the microRNA miR223, which leads to decreased NLRP3 protein levels and, thus, influences the threshold of NLRP3 activation74,75. Pro-inflammatory signals do not regulate miR223; rather, miR223 is expressed in a cell type-specific manner in innate immune cells the lowest levels of expression are found in dendritic cells and the highest levels of expression are found in neutrophils. This might explain why dendritic cells have a comparatively low threshold for NLRP3 stimuli. Taken together, these studies show that the inflammasome-mediated response can be adjusted by the activities of cell-extrinsic negative regulators and amplifiers. This rheostat-like fine-tuning facilitates the adjustment of inflammasome activation in response to danger signals and indicates that both systemic signals and the tissue context might influence the threshold of inflammasome activation (FIG.2). Thus, priming and negative regulation might strengthen or limit the local response, respectively. In fact, it is conceivable that the effectiveness of certain anti-cytokine therapies that are used to treat chronic inflammatory diseases is partly mediated by alterations in the local responsiveness of inflammasomes to danger signals. Extrinsic regulators of inflammasome activity can also affect the overall responsiveness of the cell by influencing the cell-intrinsic regulatory mechanisms of inflammasome activation, which are described in the following section.
Cell-intrinsic inflammasome regulation When the function of a cell is compromised or the survival of the cell poses a danger to the whole organism, it should be eliminated. As mentioned above, inflammasomes are localized in the cytosol and can be activated when microbial molecules are generated in this compartment. It is crucial that pathogens or protein aggregates that have been phagocytosed are subsequently neutralized in the phagolysosomal compartment. However, if the degradative function of lysosomes is defective or if the capacity of phagocytosis is overwhelmed, lysosomal damage occurs. This can activate the NLRP3 inflammasome, possibly involving the activity of lysosomal proteases76. In addition, the integrity of mitochondria is monitored by inflammasomes and apoptosomes, and mitochondrial stress can lead to inflammation and cell death (BOX1). Indeed, inflammasomes (which induce pyroptosis through caspase 1 or caspase 11 activation) and apoptosomes (which activate caspase 9 in response to cytochromec release from mitochondria) are two mechanisms by which compromised cells are eliminated. In order to be activated, the complexes need to oligomerize (BOX2); the resulting different receptor complexes share many similarities in their regulation in response to cellular stress (discussed below).
Regulation by ion fluxes, oxidative state and autophagy. Much of a cells energy is used to maintain ion gradients between the cytosol and the extracellular environment. Many enzymes and signalling pathways are regulated by dynamic changes in ion concentrations. In addition to its crucial role in cellular signalling, an intact ion gradient in healthy cells represents a mechanism that protects against celldeath. Indeed, the binding of cytochromec that has been released from mitochondria to apoptotic protease-activating factor 1 (APAF1) and the subsequent assembly of the apoptosome only occurs when subphysiological K+ concentrations are reached in compromised cells77,78. Similarly, activation of the NLRP1B and NLRP3 inflammasomes depends on low K+ concentrations in intracellular compartments79, and a low K+ concentration promotes the assembly of the ASC speck6. In addition, high extracellular levels of K+ can block IL1 release after NLRC4 and AIM2 inflammasome formation80,81, which indicates that low intracellular K+ levels might also be required for the activation of these inflammasomes. However, the concentrations of K+ that are necessary to block NLRC4 and AIM2 inflammasome formation are higher than those that are needed to block NLRP3 inflammasome formation. Interestingly, for NLRP7, a high extracellular K+ concentration only slightly reduces IL1 release28. Why some inflammasomes are more sensitive to high extracellular concentrations of K+ than others is not understood. It was thought that the danger signal ATP and bacterial pore-forming toxins, which activate the NLRP3 inflammasome, could directly mediate K+ efflux
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MicroRNA
Small, endogenous RNA molecules that can recruit the RNA-induced silencing complex to an mRNA, leading to inhibition of translation or to degradation of the mRNA.
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Box 2 | Oligomerization of death-inducing molecular platforms
Receptors of the death-inducing molecular platforms are activated by ligands such as cytochromec for the apoptosome component apoptotic protease-activating factor 1 (APAF1), and double-stranded DNA (dsDNA) for the absent in melanoma 2 (AIM2) inflammasome. By contrast, the NLRP1 (NOD-, LRR- and pyrin domain-containing 1) inflammasome can be activated by self-proteolysis in its function-to-find (FIIND) domain131133, which might be relevant to its proteolytic activation by the anthrax lethal toxin136. The cleaved fragments remain associated with each other but the proteolytic event enables NLRP1 to recruit the adaptor protein ASC and caspase 1. For the other receptors, ligand binding is thought to induce conformational changes, promoting oligomerization of the receptors into signalling platforms that recruit adaptors and activate effector caspases. Oligomerization of APAF1 and NOD-like receptors (NLRs) is mediated by their NACHT domains, which bind and hydrolyse deoxyadenosine triphosphate (dATP) or ATP146,147. However, AIM2 seems to use the multivalent ligand dsDNA as its oligomerization platform130. Both receptor oligomerization and ligand binding might be modulated by the regulatory domains of the receptors, including the WD40 repeats of APAF1, the HIN domain of AIM2 and the leucine-rich repeats (LRRs) of the NLRs. Two examples of the death-inducing platforms are shown in the box figure. The apoptosome consists of seven APAF1 molecules that are activated by cytochromec; this then recruits pro-caspase 9molecules137,148,149 (see figure, part a). Six- or eight-fold symmetry of the apoptosomes has also been observed, which suggests that there are variable oligomerization states137,148,149. Two hypothetical models of the AIM2 inflammasome highlight the positioning of the activating DNA either at the centre (see figure, part b) or the periphery (see figure, part c) of the complex. It is possible that the physiological AIM2 inflammasome may contain characteristics of both models, and the oligomerization states can be variable owing to the sequence-independent nature of the DNA-binding. For clarity, only one caspase dimer is shown for each oligomeric platform. According to a recent model, the pyrin domain (PYD) of ASC can interact with itself in a helical manner to form filamentous structures5, which might mediate ASC pyroptosome formation6.
Apoptosome APAF1 AIM2 inammasome
b
AIM2 CARD of APAF1 90
Cytochrome c 90
Pro-caspase 1 dsDNA ASC
Pro-caspase 9 90
Apoptosome Ligand-binding domain Oligomerization domain Signalling domain Catalytic domain WD40 repeats NACHT CARD Caspase 9
AIM2 inammasome HIN dsDNA PYD Caspase 1
CARD, caspase activation and recruitment domain.
Nature Reviews | Immunology
through the hemichannel pannexin1 (REFS8284); however, pannexin 1deficient mice do not show diminished caspase 1 activation85. Thus, the mechanisms by which several inflammasome triggers can induce K + efflux remain unclear. In addition to the requirement for decreased K+ levels, osmotic pressure regulates NLRP3 inflammasome activation86. Cells that have been subjected to hypotonic solutions undergo cellular swelling concomitant with a decrease in intracellular K+ and Cl concentrations. Interestingly, cell swelling induces a regulatory volume decrease response through transient receptor potential cation channels (TRPM7 and TRPV2) that triggers intracellular Ca2+ mobilization. The mobilized Ca 2+ has many molecular targets, including TGF-activated kinase 1 (TAK1; also known as MAP3K7) (REF. 86). Although TAK1 might have a role in a PRR-dependent non-transcriptional priming pathway 87 that is, in the signalling pathway that leads to the deubiquitylation, for example, of NLRP3 further investigation into its role is required. Another transient receptor potential channel TRPM2 has also been implicated in NLRP3 activation in response to crystalline substances88. This channel senses intracellular ROS and responds by opening itself to facilitate Ca2+ influx into the cell; this is intriguing considering that both ion fluxes and the oxidative state (see below) have important roles in NLRP3 inflammasome activation. Another regulator of Ca2+, namely C/EPB-homologous protein (CHOP; also known as DDIT3), has been implicated in NLRP3 inflammasome activation89. However, the activation of CHOP following the induction of the unfolded protein response via inhibition of translation is insufficient for NLRP3 inflammasome activation90 and, therefore, the exact role of CHOP in NLRP3 inflammasome activation is unclear. Two recent studies have implicated calcium-sensing Gprotein coupled receptors (GPCRs) in the activation of NLRP3: calcium-sensing receptor (CASR) and GPRC6A signal via Gi and Gq, which inhibit adenylyl cyclase and activate phospho lipase C, respectively 91,92. Thus, sensing of extracellular Ca2+ leads to reduced cyclic AMP (cAMP) levels through adenylyl cyclase inhibition and to increased cytoplasmic Ca2+ concentrations via the activation of phospholipase C. It is worth noting that the GPCR platelet activating factor receptor can also stimulate inflammasome formation92, which indicates that other GPCR pathways could control inflammasome activation. The role of cAMP in inflammasome activation remains unclear as one study has reported that cAMP can directly inhibit NLRP3 (REF.91), whereas another study reported that cAMP levels had no direct influence on inflammasome activation92. Taken together, these studies show that NLRP3 activation is regulated by the ion content of the cell (FIG.3); however, the molecular targets and the mechanisms by which ion fluxes regulate NLRP3 remain to be fully elucidated. The redox state of the cell is another important indicator of the viability of cells, and many signalling pathways are influenced by changes in the redox state. In particular, ROS facilitate the assembly of the apoptosome in several ways. For example, oxidative modifications to
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Autophagy
A homeostatic process during which cellular components are recycled through the lysosomal compartment. Its main triggers include nutrient starvation, defective organelles and infection.
caspase 9 enable its recruitment to the apoptosome93. By contrast, nitrosylation of caspase 9 inhibits its function94. Similarly, caspase 1 can be inhibited by nitrosylation95, which suggests that modification by reactive molecules is a general mechanism for the regulation of caspase activity. The exact role of the redox state in NLRP3 inflammasome activation remains controversial. Originally, it was thought that ROS produced by NADPH oxidases following the phagocytosis of crystals activate the NLRP3 inflammasome9698. However, studies that have been carried out either using cells that were deficient in NADPH protein components or using cells from patients with defective NADPH oxidase subunits have challenged this idea by showing that these patients have normal, or even increased, caspase 1 activity 99101. In addition, ROS are produced by stressed mitochondria and these mitochondria-derived ROS have been implicated in the activation of the NLRP3 inflammasome102. However, inhibitors of ROS that have been used in previous studies can inhibit NLRP3 priming rather than the actual activation of the inflammasome103. As ROS are involved in several signalling pathways, defining the exact roles of ROS in NLRP3 priming and activation remains challenging.
Furthermore, autophagy can regulate both apoptosis and inflammasome assembly, and, depending on the conditions, can be either pro- or anti-apoptotic104. For inflammasome activation and IL1 release, autophagy is a negative regulator: mice that are deficient in autophagyrelated protein 16like 1 (ATG16L1) an essential component of the autophagy machinery show higher IL1 levels in response to stimulation. This indicates that autophagy limits IL1 activation or release105. The mechanisms by which autophagy regulates inflammasome activation are still under debate. One hypothesis suggests that autophagy is involved in the removal of ubiquitylated inflammasomes106 or proIL1 molecules107. An alternative or additional mechanism could be that autophagy removes damaged mitochondria, which suggests that autophagy could prevent the release of mitochondrial ROS and DNA into the cytoplasm and thereby limit NLRP3 inflammasome formation102. These studies show that inflammasomes are tightly regulated by intracellular ion concentrations (FIG.3), by the redox state of the cell and by the nutritional situation of the cell. It seems that inflammasome activation is regulated at multiple levels and that several requirements need to be met for full inflammasome activation tooccur.
Regulatory volume decrease response induced by cell swelling K+ Unknown pore Cytoplasm ATP K+ Cl TRPM7 TRPV2 TRPM2 Adenylyl cyclase ROS
K+ Cl Ca2+
CASR or GPRC6A
P2RX7 Pore formed by bacterial toxins
Gi Gq
PLC
InsP3
? ?
NLRP3 ?
cAMP
InsP3R ER Ca2+ Unfolded protein response
Unknown CHOP-independent ER stress response ?
CHOP
ER stress
Figure 3 | Ion fluxes and cell stress regulate the NLRP3 inflammasome. K+ and Cl efflux, as well as Ca2+ mobilization, have a role in the regulation of NLRP3 (NOD-, LRR- and pyrin domain-containing 3). K+ effluxNature is either achieved directly by Reviews | Immunology pore-forming toxins such as nigericin or indirectly for example, via the purinergic receptor for ATP P2RX7. Cl and Ca2+ ion fluxes can be regulated during the regulatory volume decrease response by transient receptor potential receptors (TRPV2 or TRPM7). In addition, reactive oxygen species (ROS) can activate TRPM2 for Ca2+ influx. Ca2+ is also regulated by the unfolded protein response via C/EPB-homologous protein (CHOP). The unfolded protein response is also implicated in other pathways that regulate NLRP3. Gprotein coupled receptors (GPCRs) such as calcium-sensing receptor (CASR) and GPRC6A regulate both Ca2+ levels and cyclic AMP (cAMP) levels via activation of phospholipase C (PLC) or inhibition of adenylyl cyclase, respectively. High cAMP levels might directly inhibit NLRP3 (not shown). Inositol-1,4,5-trisphosphate (InsP3) that is generated by PLC leads to release of Ca2+ from the endoplasmic reticulum (ER). How Ca2+ influences NLRP3 activation is not fully understood but it has many molecular targets. Question marks show pathways that are still speculative. InsP3R, InsP3 receptor.
NATURE REVIEWS | IMMUNOLOGY 2013 Macmillan Publishers Limited. All rights reserved VOLUME 13 | JUNE 2013 | 405
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Table 1 | Regulatory proteins of the inflammasomes
Regulatory protein Inflammasome component targeted
NLRP1 NLRC4 NLRC4 NLRC4
Mechanism of action
Sources of experimental evidence
Refs
Accessory NLR proteins

NOD2 NAIP2* NAIP5 or NAIP6* NAIP Enhances NLRP1 inflammasome formation Knockout mice and biochemical interaction 32,33 34,35 34,35 35,36
Necessary for recognition of the typeIII secretion system RNAi and biochemical interaction by the NLRC4 inflammasome Necessary for recognition of flagellin by the NLRC4 inflammasome Necessary for recognition of flagellin and/or the typeIII secretion needle protein by the NLRC4 inflammasome Inhibits caspase 1 activity, probably by sequestration Inhibits caspase 1 activity, probably by sequestration RNAi and biochemical interaction RNAi, biochemical interaction and overexpression Knockout mice and biochemical interaction Biochemical interaction and overexpression Biochemical interaction and overexpression Biochemical interaction and overexpression Biochemical interaction and overexpression Biochemical interaction and overexpression Biochemical interaction, colocalization and overexpression Inhibitor: knock-in mice, biochemical evidence and overexpression Adaptive immunity: knockout mice Inhibitor: overexpression and biochemical evidence Inflammasome: RNAi and biochemical evidence Inhibitor: Knockout mice Activator: knock-in mice of FMF-mutated pyrin Knockout mice and biochemical evidence RNAi and biochemical evidence Knockout mice and biochemical evidence Knockout mice, biochemical evidence and overexpression Biochemical evidence and overexpression
CARD-containing regulatory proteins

Caspase 12 and caspase 12L CARD18 (also known as ICEBERG) Caspase 1 Caspase 1 110,111 113
CARD16 (also Caspase 1 known as COP1) CARD17 (also known as INCA) POP1 POP2 ASC splice variant NLRP10 Caspase 1
Inhibits caspase 1 activity, probably by sequestration Inhibits caspase 1 activity, probably by sequestration
113 114
PYD-containing regulatory proteins

ASC ASC ASC or caspase 1 ASC Inhibits inflammasome assembly, probably by sequestration Inhibits inflammasome assembly, probably by sequestration Inhibits inflammasome assembly, probably by sequestration Inhibits inflammasome assembly, probably by sequestration Role in adaptive immunity Inhibits inflammasome assembly by sequestration Activates the formation of the NLRP7 inflammasome 115 116,117 118 119122
NLRP7
ASC
28,123
Pyrin
ASC or IL1
Inhibits IL1 release FMF-associated pyrin activates the formation of an inflammasome Enhances NLRP3 inflammasome formation in response to non-crystalline activators Deubiquitylates NLRP3 as a priming step Phosphorylates NLRC4 as a priming step Inhibits NLRP1 inflammasome activation by inhibiting nucleotide binding Inhibits NLRP1 inflammasome activation by inhibiting nucleotide binding
124,125
Other regulators
GBP5 BRCC3 PKC BCL2 BCLXL IAPs IAPs PKR NLRP3 NLRP3 NLRC4 NLRP1 NLRP1 Caspase 1 NLRP3 NLRP1, NLRC4, AIM2 and NLRP3 39 63 67 139,140 139,140 58 56 127, 128
Increases the activity of caspase 1by monoubiquitylation Knockout mice (for IAP1 or IAP2) and biochemical evidence Loss of IAPs leads to activation of the ripoptosome, which is necessary for caspase 1 activation Activated by interacting with the inflammasome sensor molecules Does not have an effect IAP1, IAP2 and XIAP triple-knockout mouse Activator: knockout mice (kinase domain knocked out) and biochemical evidence No role: knockout mice (kinase and RNA-binding domain knocked out)
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Table 1 (cont.) | Regulatory proteins of the inflammasomes
Regulatory protein Inflammasome component targeted
NLRP3 NLRP3
Mechanism of action
Source of experimental evidence
Refs
Other regulators (cont.)

HSP90 SGT1 Necessary for activation, possibly owing to its chaperone Biochemical evidence and inhibitor of function HSP90 Necessary for activation, possibly owing to its chaperone RNAi and biochemical evidence function 129 129
AIM2, absent in melanoma 2; BCL, Bcell lymphoma; CARD, caspase activation and recruitment domain; FMF, familial Mediterranean fever; GBP5, guanylate-binding protein 5; HSP90, heat shock protein 90; IAP, inhibitor of apoptosis protein; IL1, interleukin1; NAIP, NLR family, apoptosis inhibitory protein; NLR, NOD-like receptor; NLRC4, NOD-, LRR- and CARD-containing 4; NLRP, NOD-, LRR- and pyrin domain-containing; NOD2, nucleotide-binding oligomerization domain-containing protein 2; PKC, protein kinase C; PKR, protein kinase R; POP, pyrin domain-only protein; PYD, pyrin domain; RNAi, RNA interference; XIAP, X-linked IAP. *Expressed only in mice. Expressed only in humans. Two contradictory studies have been published about this interactor.
Regulation by interacting host proteins. In addition to general cellular conditions such as ion concentrations and the nutritional state, specific regulatory proteins have evolved that regulate inflammasome formation (TABLE 1) . The formation of the inflammasome can be controlled both at the level of inflammasome sensor molecules and, further downstream, at the level of ASC and caspase 1 interaction. As mentioned above, inflammasomes form large protein aggregates and there are two levels at which death-fold domains are used to recruit effector inflammasome components. First, ASC is recruited to the inflammasome receptors by homotypic pyrin domain interactions. Next, in a second death-fold domain interaction, ASC binds to and activates pro-caspase 1 via its CARD. Studies have elucidated inflammasome regulation at the level of death-fold domain interactions and regulatory proteins. Caspase activity during cell death is regulated by CARD-containing proteins such as CARD8, which has also been shown to be a binding partner of NLRP3 (REF.108). CARD8 has multiple functions in regulating apoptosis, one of which is to directly bind to procaspase 9 and to suppress its activation109. Similarly, mouse caspase 12, which is a paralogue of caspase1, interacts with caspase 1 to reduce its activity 110. A polymorphism in the caspase 12 (CASP12) gene in humans leads to either a truncated protein or to a full-length protein (caspase 12L)111. Similarly to mouse caspase 12, caspase12L reduces cytokine production in response to LPS111, which at high doses can activate the NLRP3 inflammasome and thus caspase 1 without the need for a second signal. Therefore, caspase 12L and mouse caspase 12 probably function as decoy proteins for caspase1, thereby limiting its activation. CARDonly proteins (COPs) can also inhibit caspase1 activation 112. CARD18 (also known as ICEBERG) and CARD16 (also known as pseudo-ICE and COP1) were the first decoy COPs to be described113. Through their CARDCARD interaction, these two inhibitors sequester pro-caspase 1 and inhibit its activation by the inflammasome. CARD17 (also known as INCA), which is another decoy protein, is upregulated by IFN to suppress IL1 generation114. Thus, proteins containing a CARD can sequester caspase1, thereby blocking the formation of a functional inflammasome complex.
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An additional level of fine-tuning and regulation of the inflammasomes can be achieved by interfering with the pyrinpyrin interaction in the inflamma somes. Several pyrin domain-only proteins (POPs) and other pyrin-containing proteins have been characterized. POP1 (also known as PYDC1 and ASC2) and POP2 (also known as PYDC2) inhibit the interactions between the inflammasome sensor molecules and ASC115117. However, COPs and POPs have not been identified in the mouse genome, which indicates that a more complex regulatory system might be present in humans. The fact that COPs and POPs have not been identified in the mouse genome makes the study of these proteins difficult, and many of the COPs and POPs have only been investigated using overexpression studies and not using genetic deletion or RNA interference. Alternative splicing of ASC can provide an additional level of regulation by giving rise to ASC variants that negatively regulate the assembly of the inflamma somes118. Furthermore, NLRP10 (also known as PYNOD) was suggested to negatively regulate inflammasomes by sequestering ASC119,120, but deletion of Nlrp10 in mice did not confirm this hypothesis121,122. NLRP7 has been suggested to be a negative regulator of inflamma somes; however, more recent findings indicate that NLRP7 might assemble an inflammasome in response to microbial acylated lipopeptides28,123. The role of pyrin, which is a protein that is encoded by MEFV (Mediterranean fever gene), in inflammasome regulation also remains unclear. One study found that Mefv deletion in mice lead to increased IL1 release without influencing caspase 1 activity or inflammasome assembly. These findings suggested that pyrin can inhibit IL1 release downstream of the inflammasomes124. However, another study found that short-term IL1 release was not impaired after Mefv gene deletion and that IL1 was only inhibited after several days of stimulation125. In addition, mice carrying Mefv mutations that have been identified in patients with familial Mediterranean fever (FMF) showed ASCdependent but NLRP3independent release of IL1125. Therefore, whether pyrin is an inhibitor at the level of IL1 release or whether it forms a pyrin inflammasome remains to be determined.
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In addition to death domain interactions targeting the formation of the inflammasome complex, regulation can occur at the level of the inflammasome sensor molecules. A recent report linked the inhibition of translation with NLRP3 inflammasome activation126. Protein synthesis could be inhibited by the phosphorylation of the initiation factor eukaryotic translation initiation factor2 subunit- (EIF2; also known as EIF2S1). This phosphorylation event could be mediated by different kinases that sense endoplasmic reticulum (ER) stress, or by the presence of heavy metals or double-stranded RNA (dsRNA). Protein kinase R (PKR; also known as EIF2AK), which is activated by dsRNA and can phosphorylate EIF2, has been suggested to be involved in NLRP3, NLRP1, NLRC4 and AIM2 inflammasome activation, and this was found to be dependent on its kinase activity127. However, another study concluded that PKR does not have any role in inflammasome activation128. The reasons for these seemingly contradictory results could be attributed to differences in mouse strain backgrounds and will need to be further evaluated. In another study, ER stress was shown to activate the NLRP3 inflammasome90 and, although the classical unfolded protein response was shown to activate EIF2 phosphorylation (consistent with the above observations), this pathway was not found to be necessary 90. The authors proposed that another, not yet characterized, ER stress pathway activates the NLRP3 inflammasome90. Finally, heat shock proteins (HSPs) also have important roles in the regulation of cell death, and it has been shown that HSP90 and the cochaperone ubiquitin ligase-associated protein SGT1 are required for NLRP3 activation (REF.129).
Box 3 | Pharmacological interference of inflammasome activation
The fact that the NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome in particular is under the control of numerous regulatory mechanisms provides several opportunities for therapeutic intervention. It is conceivable that the biological effectiveness of certain anti-inflammatory agents is at least partly due to their ability to interfere with NLRP3 activation. Recently, the mechanism of action of certain anti-inflammatory therapies that have been in use for years has been partly ascribed to the inhibition of NLRP3. For example, the therapeutic application of interferon- (IFN) is effective for most but not all patients with multiple sclerosis. IFN-mediated downregulation of NLRP3 inflammasome responses might be of particular relevance in this central nervous system pathology, which has been linked to inflammasome activation150. Interestingly, a recent study has documented that IFN therapy can only reduce the pathology in mouse experimental autoimmune encephalomyelitis models that are dependent on NLRP3, which indicates that IFN can actively reduce NLRP3mediated brain inflammation invivo151. In addition, a number of small-molecule compounds have been shown to inhibit NLRP3 activation. For example, glyburide, which is a pharmaceutical compound that is used in the treatment of type2 diabetes, has been shown to directly inhibit the NLRP3 inflammasome, albeit only at high doses152. Furthermore, the anti-inflammatory compound parthenolide, which is naturally present in the plant Tanacetum parthenium (also known as feverfew) that is used in herbal remedies, and the nuclear factor-B (NFB) inhibitory compound Bay 11-7082 can both interfere with the ATPase activity of NLRP3, thereby hindering inflammasome activation153. The NLRP3 inflammasome is also inhibited by the TGF-activated kinase 1 (TAK1) inhibitor 5Z7oxozeaenol and its related compounds87. Future investments in the identification of small molecules that target these important intracellular signalling platforms or their upstream regulators could generate a novel class of highly effective anti-inflammatory therapeutics.
Taken together, these studies show that the activation of inflammasomes can be regulated at different levels and that many proteins contribute to the overall response of these important signalling platforms. Elucidating the exact function of the different components seems to be challenging. Overexpression could considerably perturb the finely balanced thresholds of inflammasome activation, and it is possible that inflammasomes respond differently depending on culture conditions and/or genetic background.
Conclusion and future perspectives In this Review, we summarize our rapidly evolving understanding of how inflammasomes are activated and we highlight the many mechanisms that are involved in their regulation. Although we have gained detailed knowledge about many aspects of inflammasome activation, several important questions remain unanswered or poorly understood. One main conceptual question that remains unanswered is whether inflammasome sensors can directly interact with their triggers and, thus, whether they represent true receptor molecules. AIM2 can tightly interact with DNA and has recently been crystallized with its ligand; it therefore qualifies as a receptor molecule130. The NLR inflammasome sensors all have LRR domains that, for example, in TLRs, directly recognize lipids, nucleic acids or proteins. However, whether the NLR inflammasome sensor molecules can directly recognize their various triggers or whether they use accessory host proteins for this process remains a matter of debate. In the case of NLRC4, additional host molecules (that is, NAIPs) have been suggested to function as the direct receptors for the ligands34,35. Therefore, NLR inflamma some sensors could, in fact, function as adaptor molecules rather than receptors. In addition, post-translational modifications of NLRs, such as phosphorylation67, ubiquitylation62,63 and even proteolysis131133, have been suggested to be necessary for the activation of certain NLR sensors. Therefore, the modification of NLRs by host enzymes could be crucial for their activation and, at the same time, could represent a novel target for pharmacological intervention strategies. Future research should focus on the identification of host molecules that function upstream of the NLRs. In the case of NLRP3, the elucidation of the unknown factors that influence its activation represents a particular challenge, as these factors could influence both the priming and activation events. Therefore, combinatorial proteomic and genomic approaches, as well as overexpression systems in which priming is not required, could be instrumental. Another important challenge will be to more precisely define the contribution of inflammasomes to the inflammatory response invivo. The ability of a particular trigger to activate inflammasomes and IL1 family cytokines invitro does not necessarily indicate that inflammasomes fully control the inflammatory response to the respective trigger invivo. It is becoming increasingly evident that the activation of inflammasome-independent IL1 can substantially contribute to
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tissue inflammation. In acute inflammatory situations, in which neutrophils have important roles, inflamma somes can be functionally redundant in the activation of IL1 and thus might only partially contribute to the inflammatory tissue response. Caspase 8, which has recently been implicated in IL1 activation, could be one of the factors that contributes to inflammasomeindependent responses invivo. As many pathways can trigger caspase 8 activation, the relative contributions of caspase 1 and caspase 8 to IL1 production and inflammation invivo need to be carefully investigated in future studies. Furthermore, caspase 8 is known to mediate cell death; hence, it will be important to better understand the consequences of the different forms of cell death in the inflammatory tissue response. Moreover, in this Review we compare the activation and regulation of inflammasomes to that of the apoptosome, which is another multimolecular caspase-activating platform. We note with interest that, in addition to the way in which the effector proteins of these platforms are tightly controlled, the activity of the triggers can be modulated; for example, cytochromec can be modified to be more or less pro-apoptotic. Indeed, apoptosome activation by cytochromec is only possible after cytochromec has
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incorporated a haem group in the mitochondrion134. By contrast, nitrosylation of cytochromec inhibits its ability to induce the formation of the apoptosome135. We predict that similar regulatory mechanisms are in place for the triggers of inflammasomes. The importance of self-proteolysis or proteolysis by the trigger anthrax lethal toxin in the activation of NLRP1 is still not fully understood131133,136. It is possible that proteolysis of inflammasome sensor molecules could lead to multiple effects that have not yet been defined. The quest for the exact details of inflammasome activation is ongoing; it seems probable that signals emanating from mitochondria and lysosomes will provide important insights. In addition, we will probably learn from comparing these potential regulatory mechanisms with those that govern apoptosome and ripoptosome activation. The identification of upstream mechanisms and the discrimination between priming and activation could be instrumental in developing novel therapies on the basis of the specific inhibition of individual inflamma somes (BOX3). Given their broad roles in mediating inflammatory pathologies, inflammasome inhibitors could become effective therapies many prevalent and emerging inflammatory diseases.
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Acknowledgements
The authors would like to thank C. M. De Nardo and B. G. Monks for critical reading of the manuscript. This work was supported by grants from the US National Institutes of Health (NIH) and the Deutsche Forschungsgemeinschaft (DFG), Germany (to E.L.), and by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH, USA (to T.S.X.). E.L. is a member of the excellence cluster ImmunoSensation in Bonn, Germany.
Competing interests statement
The authors declare no competing financial interests.
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The good, the bad and the ugly TFH cells in human health and disease
Stuart G.Tangye1,2, Cindy S.Ma1,2, Robert Brink1,2 and Elissa K.Deenick1,2
Abstract | Antibody production is an important feature of the vertebrate immune system. Antibodies neutralize and clear pathogens, thereby protecting against infectious diseases. Such humoral immunity has great longevity, often persisting for the hosts lifetime. Long-lived humoral immunity depends on help provided by CD4+ Tcells, namely T follicular helper (TFH) cells, which support the differentiation of antigen-specific Bcells into memory and plasma cells. TFH cells are stringently regulated, as aberrant TFH cell activity is involved in immunopathologies such as autoimmunity, immunodeficiencies and lymphomas. The elucidation of the mechanisms that regulate TFH cell differentiation, function and fate should highlight targets for novel therapeutics.
Germinal centres
The structures that are formed by the expansion of antigen-activated B cell blasts that have migrated into the follicles of lymph nodes. The Bcells in these structures proliferate and the immunoglobulin genes undergo somatic hypermutation before the cells leave as plasma cells or memory cells.
Immunology Research Program, Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales 2010, Australia. 2 St Vincents Clinical School, University of New South Wales, Darlinghurst, Sydney, New South Wales 2010, Australia. Correspondence to S.G.T. e-mail: s.tangye@garvan.org.au doi:10.1038/nri3447 Published online 17 May 2013
1
Following infection or vaccination, the induction of pro tective immunity against invading pathogens depends on the generation of an appropriate type of immune response. This relies on the flexibility of naive CD4 + Tcells, which differentiate into diverse subsets with spe cialized effector functions to protect against infection by distinct pathogens1 (TABLE1). The importance of hav ing distinct subsets of CD4+ Tcells is evident in disease states that arise from the perturbed differentiation or function of specific effector T cell populations2 (TABLE1). The generation of these effector T cell subsets depends on the stimulatory cytokines that are present in the microenvironment during activation; these cytokines induce transcription factors that prime naive precursor cells for differentiation. Interleukin12 (IL12) induces the T-box transcription factor Tbet (also known as TBX21) in the case of T helper 1 (TH1) cells, IL4 induces GATA-binding protein 3 (GATA3) in the case of TH2 cells and IL6 or IL23 induce retinoic acid receptor-related orphan receptort (RORt) in the case of TH17 cells1 (TABLE1). However, the concept of master regulators for lymphocyte differentiation is an overly simplified idea, as numerous transcription factors are required for the commitment of CD4+ Tcells to specific effector lineages (TABLE1). Thus, the ultimate outcome of TH cell differen tiation depends on the coordinated functions of several important molecular regulators that operate to control gene expression and effector function. TH cell subsets have been identified and character ized for immunity against specific pathogenic threats (TABLE1), but the production of neutralizing antibodies is required for the development of protective immunity
to most infectious diseases. In this case, the differentia tion of B cells to antibody-secreting cells is dependent on instructive signals that arise from T follicular helper (TFH) cells. The importance of antibody production by antigenspecific Bcells is exemplified not only by our ability to establish serological memory that provides long-lasting protection against pathogen infection but also by the suc cess of most vaccines, which rely on antibody responses for their efficacy. The fundamental role of Tcells in Bcell differentiation was first reported nearly 50years ago3. However, it is only in the past decade that we have gained a clear understanding of the biology of TFHcells. TFH cells were first described in humans as CD4+ Tcells in secondary lymphoid tissues that expressed the Bcell zone-homing chemokine receptor CXC-chemokine receptor 5 (CXCR5) and therefore localized to Bcell fol licles, including germinal centres (GCs). CD4+CXCR5+ Tcells were more efficient than CD4+CXCR5 Tcells at inducing class switching and antibody secretion in Bcells47. Further studies showed remarkable similarities between human and murine TFH cells810, which allowed elucidation of the mechanisms by which TFH cells drive affinity maturation as well as antibody and autoantibody production in GCs (reviewed in REFS1113) (BOXES1,2). The most accurate definition of TFH cells relates to their function as cells that migrate to follicles and interact with antigen-specific Bcells to support B cell differentiation. However, using this empirical definition to isolate and study TFH cells makes detailed analysis of this subset of cells challenging owing to the inherent difficulty in isolating cells from anatomically discrete regions of lymphoid tis sues. For this reason, TFH cells are more commonly defined
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Table 1 | Effector subsets of CD4+ Tcells: ontogenic and functional requirements, and roles in disease
CD4+ Tcell subset
TH1 cells
Inducing cytokines
IL12 IFN
Activated Transcription Suppressing STATs factors cytokines

STAT4 STAT1 T-bet IL4 and IL10
Canonical Roles in host cytokines protection produced

IFN Antiviral and antimicrobial immunity Cell-mediated immunity
Associated pathologies
Mendelian susceptibility to mycobacterial disease (decrease in TH1 cells) Multiple sclerosis (increase in TH1 cells)
Refs
1,121, 122
TH2 cells TH17 cells
IL4
STAT6
GATA3 and MAF RORt and ROR
IFN IL4, IFN, IL27 and IL2 TGF (suppresses IL22 expression)
IL4, IL5 and IL13
Immunity to Allergy, asthma or extracellular parasites eczema (increase in TH2 cells) Inflammatory bowel disease (increase in TH17 cells) Susceptibility to fungal infections (decrease in TH17 cells)
1,123
IL23 and IL1 IL-6 and IL1 TGF
STAT3
IL17A, Protection at IL17F, mucocutaneous sites IL21, IL22 Antimicrobial and IL26* immunity (for example, against Candida spp. and Staphylococcus spp.) Inflammatory bowel disease IL9 Protection against helminth infections
1,121, 122,124
TH9 cells
TGF IL4
STAT6
PU1 and IRF4
IFN and IL27
Allergy (atopic dermatitis) and asthma (increase in TH9 cells) Allergy (atopic dermatitis) (increase in TH22 cells) Inflammation at joints and barriers (increase in TH22 cells in mice) IPEX syndrome (decrease in TReg cells) Humoral immunodeficiency (decrease in TFH cells) Autoimmunity (increase in TFH cells) Tcell lymphoma (increase in TFH cells) (see TABLE2)
125
TH22 cells
TNF IL6
STAT1 STAT3 STAT5
RORt and AHR
High doses of TGF
IL22
Barrier immunity (skin, intestines and airways) Enhancement of innate immunity Tissue regeneration Immune suppression
126
TReg cells TFH cells
TGF and IL2 IL6, IL21 and/or IL27 IL12
STAT5
FOXP3
IL6
TGF and IL10 IL21, IL4 and IL10
75
STAT3 STAT4 STAT1
BCL6, IRF4, MAF and BATF
IL2 and IL10
Help for B cell activation or differentiation Generation of long-lived antibody responses
2325, 45
AHR, aryl hydrocarbon receptor; BATF, basic leukine zipper transcriptional factor ATF-like; BCL6, B cell lymphoma 6; FOXP3, forkhead box P3; GATA3, GATA-binding protein 3; IFN, interferon; IL, interleukin; IPEX, immunodysregulation, polyendocrinopathy and enteropathy Xlinked; IRF4, interferon-regulatory factor 4; ROR, retinoid-related orphan receptor; STAT, signal transducer and activator of transcription; TGF, transforming growth factor; TFH, T follicular helper; TH, T helper; TNF, tumour necrosis factor; TReg, T regulatory. *Human-specific cytokine. Reported in mice.
on the basis of their surface phenotype. Consequently, in both humans and mice810,1417, TFH cells are considered to be CD4+ Tcells that express the highest levels of CXCR5, together with the surface receptors inducible Tcell costimulator (ICOS) and programmed cell death protein 1 (PD1; also known as PDCD1), the transcriptional repres sor Bcell lymphoma6 (BCL6) and the cytokine IL21 (BOX3), and that have downregulated the T cell zonehoming receptor CC-chemokine receptor 7 (CCR7) and IL7 receptor (IL7R) (TABLE1). This phenotypic approach to defining TFH cells has facilitated their molecular and cellular characterization. However, it needs to be appreciated that, just as Bcells undergo important differentiation events at the Tcell Bcell border (such as the formation of extrafollicular
plasmablasts) and in GCs (such as the formation of memory and plasma cells), CD4+ Tcells can differentiate into TFH cell subsets that are strategically located at these regions to facilitate distinct phases of a Tcell-dependent Bcell response6,7,18,19. These TFH cell subsets probably pro vide early B cell help at the TcellBcell border, and/or they represent precursor cells that differentiate into GC TFH cells following receipt of appropriate signals from inside the active Bcell follicle and that guide the dif ferentiation of GC Bcells into memory or plasma cells. In this Review, we discuss recent developments in the investigation of the mechanisms that underlie TFH cell development and function, the discovery of specialized subsets of TFH cells and how perturbations to TFH cells potentially contribute to numerous human diseases.
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Box 1 | TFH cells and affinity maturation of GC Bcells
Affinity maturation of the antibody Germinal centre response is based on the selective perpetuation (known as positive Follicular selection) of germinal centre (GC) Bcells dendritic cell that have acquired increased affinity for foreign antigens through somatic CR2 or FcRII hypermutation (SHM) of their Foreign antigen immunoglobulin variable-region genes. Whereas SHM itself takes place in Bcells Adhesion Ig that occupy the GC dark zone, molecules high-affinity GC Bcells only undergo BCR CD40L positive selection after returning to the CD40 light zone where they gain preferential BCR access to foreign antigens that are TFH cell B cell expressed on the surface of follicular dendritic cells (see the figure). However, TCR B cell the precise mechanisms by which High anity MHC class II peptide complex preferential access to antigens translates into positive selection of high-affinity GC Low anity Bcells have not been clearly established. The specific provision of T follicular helper (TFH) cell-derived helper signals to high-affinity GC Bcells is proposed to be Anityone of, if not the only, major drivers of matured antibody affinity maturation12,13. In Cell death antibodies theory, the greater propensity of high-affinity GC Bcells to access foreign antigens should augment their ability to internalize, process and present foreign peptides to the TFH cells that reside in the light zone of GCs (see the figure). Nature Reviews | Immunology However, although it is clear that TFH cells are required to support the GC response, it has been difficult to establish their precise role in driving affinity maturation. In a recent study, a GC response was manipulated in mice such that antigen presentation by GC Bcells to TFH cells was decoupled from Bcell receptor (BCR)-mediated antigen recognition. This resulted in the delivery of TFH cell help to all GC Bcells and the proliferative expansion of the population of TFH cells regardless of their affinity for antigen12. This result does not prove that preferential delivery of TFH cell help is the fundamental driver of affinity maturation but it clearly demonstrates that TFH cells have the potential to perform this role if high-affinity GC Bcells are indeed superior at presenting antigen to TFH cells.
CD40L, CD40 ligand; CR2, complement receptor 2; FcRII, low affinity Fc receptor for immunoglobulin; Ig, immunoglobulin; TCR, T cell receptor.
B cell lymphoma 6
(BCL6). A transcriptional repressor identified as being crucial for the formation of T follicular helper (TFH) cells. Several mechanisms have been proposed for the role of BCL6 in TFH cell commitment, including suppression of the expression of transcription factors that are required for the generation of alternative TH fates, suppression of microRNAs and cooperation with other transcriptional regulators to induce the expression of important TFH cell-related genes.
Requirements for TFH cell formation TFH cell differentiation requires input from several surface receptors (including CD28, ICOS, CD40 ligand (CD40L) and signalling lymphocytic activation molecule (SLAM) family members), as well as from cytokines and their associated signalling pathways (for example, signal trans ducer and activator of transcription 3 (STAT3) or SLAMassociated protein (SAP; also known as SH2D1A)), which all culminate in the induction of BCL6, an important regulator of the TFH cell lineage2022. However, additional transcription factors (including interferon-regulatory factor 4 (IRF4), basic leucine zipper transcriptional factor ATF-like (BATF) and MAF) and microRNAs also have important regulatory functions during TFH cell develop ment2225 (FIG.1; TABLE1; Supplementary information S1 (table)). The requirements for TFH cell formation have been extensively reviewed in the literature2326 and are summarized in Supplementary information S1 (table). In this Review, we discuss the most recent studies that have identified pathways that positively and negatively influ ence TFH cell generation and function, and that clarify previous inconsistencies.
The TNF receptor superfamily. Bcell-activating factor (BAFF; also known as TNFSF13B) and a proliferationinducing ligand (APRIL; also known as TNFSF13) are ligands of the tumour necrosis factor (TNF) superfamily that regulate Bcell survival and differentiation11. Both ligands bind to the common receptors transmembrane activator and CAML interactor (TACI; also known as TNFRSF13B) and Bcell maturation antigen (BCMA; also known as TNFRSF17), and BAFF also binds to the BAFF receptor (BAFFR; also known as TNFRSF13C); receptors which are predominantly expressed on Bcells11. NFB-inducing kinase (NIK; also known as MAP3K14) is a component of the BAFFR signalling pathway 11. Expression of NIK and BAFFR by Bcells was found to be required for their constitutive expression of ICOS ligand (ICOSL). The significance of this is that NIK deficiency in Bcells compromises TFH cell induc tion27. Thus, sustained BAFFBAFFRNIK signalling in Bcells maintains ICOSL expression, thereby facilitating cognate ICOSICOSL interactions between activated CD4+ Tcells and Bcells, which result in optimal TFH cell differentiation.
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Box 2 | TFH cells and autoantibody production at the germinal centre
Although the generation of high-affinity antibodies directed against foreign pathogens is the raison dtre of the germinal centre (GC), the random nature of the somatic hypermutation (SHM) process can lead to the generation of antibodies that recognize self antigens with high affinity. Given the fact that many pathogenic autoantibodies are both somatically hypermutated and of high affinity13, it has long been presumed that Tfollicular helper (TFH) cell-driven selection of self-reactive GC Bcells can make an important contribution to autoimmune disease. Indeed, it has recently been demonstrated that in some circumstances TFH cells are required to drive systemic autoimmunity34. A failure of Tcell self-tolerance in the TFH cell repertoire is no doubt an important driver of autoimmunity (see the figure, panel a), but recent data indicate that TFH cell responses that are directed against foreign antigens can also support an autoantibody response under certain circumstances70. Thus, if a somatically mutating GC Bcell acquires self-reactivity but also maintains its cross-reactivity with foreign antigens, then foreign antigen-specific TFH cells can support Bcell maturation and drive autoantibody production (see the figure, panel b). This mechanism selectively operates against peripheral tissue-specific self antigen targets, as cross-reactivity with self antigens that are present in the GC microenvironment results in the deletion of the self-reactive GC Bcells70. Thus, these data indicate that pathogen-specific TFH cells might drive the production of peripheral tissue-specific cross-reactive autoantibodies that are found in post-infectious autoimmune diseases such as rheumatic fever and GuillainBarre syndrome13.
a
Follicular dendritic cell CR2 or FcRII Ig BCR Self antigen Adhesion molecules CD40 B cell CD40L TFH cell
Foreign antigen
MHC TCR class II Self antigenSelf antigenspecic specic
Foreign antigen-specic and cross-reactive to self antigen
Foreign antigen-specic
Self antigen
Distal tissue Autoantibodies Cross-reactive autoantibodies
BCR, B cell receptor; CD40L, CD40 ligand; CR2, complement receptor 2; FcRII, low affinity Fc receptor for immunoglobulin; Nature Reviews | Immunology Ig, immunoglobulin; TCR, T cell receptor.
Xlinked lymphoproliferative disease

A rare, often fatal, primary immunodeficiency disease that is characterized by an inability to mount an effective immune response against EpsteinBarr virus, as well as a susceptibility to developing lymphoma and/or hypogammaglobulinaemia.
In contrast to BAFFR, TACI negatively regulates Bcell function11. In keeping with this, Taci/ mice have expanded populations of TFH cells and GC Bcells28. In the absence of TACI, greater ICOSL expression on B cells results in an increase in the number of TFH cells28. These observations demonstrate that an important extrinsic determinant of TFH cell formation is the balance between positive and negative signalling that is provided by BAFF and APRIL through BAFFR and TACI, respectively. These interactions cooperatively regulate ICOSL expres sion on antigen-presenting Bcells (FIG.1). These studies highlight the crucial role for ICOSICOSL signalling in TFH cell formation, particularly as the phenotype of NIK-deficient mice recapitulates that of mice that are globally deficient in ICOS29 or mice with Bcells that lack expression of ICOSL30.
The SLAM family of surface receptors. The SLAM family of receptors includes SLAM (also known as SLAMF1), CD84 (also known as SLAMF5), natural killer cell recep tor 2B4 (also known as CD244), T lymphocyte surface antigen LY9 and NTBA (natural killer, T and B cell anti gen; also known as SLAMF6 and LY108 in mice). These receptors recruit SAP, thereby allowing CD4+ Tcells to activate signalling intermediates, including protein kinase C (PKC), BCL10, nuclear factor-B (NFB) and FYN, that are important for receptor function31. Naive SAP-deficient CD4+ Tcells fail to form stable conjugates with cognate Bcells32,33, and are therefore unable to differentiate into TFH cells and to help T celldependent Bcell responses17,18,23,34. This explains why individuals with Xlinked lymphoproliferative disease (XLP) caused by mutations in SH2D1A (the gene encoding
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Box 3 | IL21 is a potent differentiation factor for human Bcells
Interleukin21 (IL21) was discovered in 2000 as a pleiotropic cytokine that is capable of activating most lymphocyte populations116. The initial description reported that IL21 strongly induced the proliferation of CD40stimulated human Bcells, but that it inhibited IL4induced B cell proliferation116. Since then, many studies have established the potency of IL21 as a growth and differentiation factor for human Bcells. When human Bcells are primed with T cell help in the form of a CD40specific monoclonal antibody or CD40 ligand (CD40L), IL21 induces robust B cell proliferation as well as the expression of activation-induced cytidine deaminase (AICDA; required for immunoglobulin class switching), B lymphocyte-induced maturation protein 1 (BLIMP1; also known as PRDM1) and X box-binding protein 1 (XBP1)83,117,118, all of which mediate the differentiation of Bcells into plasma cells. Consequently, at least invitro, IL21 efficiently induces subsets of activated Bcells to undergo class switching either to become IgG- or IgA-expressing cells, or to become plasmablasts secreting IgM, IgG, IgA or IgE83,117120. IL21 predominantly induces switching to IgG3, IgG1 and IgA1 subclasses117,119,120, whereas IL21stimulated naive, GC or memory Bcells produce large quantities of immunoglobulins83,117,119. IL21 can also induce the expression of B cell lymphoma 6 (BCL6) in human naive Bcells117, which is consistent with IL21 having a role in establishing GCs. Before the discovery of IL21, it was well recognized that class switching by human Bcells was regulated by IL4 (which promotes class switching to IgG4 and IgE), IL10 (which promotes class switching to IgG1 and IgG3), IL13 (which promotes class switching to IgG4 and IgE) and transforming growth factor- (TGF; which promotes class switching to IgA), and IL10 was also considered to be a strong inducer of immunoglobulin secretion. Furthermore, Bcell survival was shown to be positively regulated by IL4 or IL10 (reviewed in REF.82). Studies over the past decade have highlighted the importance of IL21 in humoral immunity in humans by demonstrating that it has the remarkable ability to exert all of these functions on human Bcells.
SH2 domain-containing protein tyrosine phosphatase 1

(SHP1). A protein tyrosine phosphatase that is involved in suppressing intracellular signals delivered via numerous activating receptors, including T cell and B cell antigen receptors, as well as members of the signalling lymphocytic activation molecule (SLAM) family of surface receptors. One proposed mechanism of action is the direct or indirect dephosphorylation of components of the T cell receptor signalling pathway, such as CD3, LCK, -chain-associated protein kinase of 70 kDa (ZAP70) and phosphoinositide 3kinase.
Follicular T regulatory cells

A subset of T regulatory (TReg) cells that coopts the transcriptional machinery of T follicular helper (TFH) cells to facilitate their migration to germinal centres, where they can appropriately restrain humoral immune responses, thereby potentially preventing overzealous antibody responses. Follicular TReg cells can be identified by the expression of typical TFH cell surface markers (CXC-chemokine receptor 5 (CXCR5), inducible T cell co-stimulator (ICOS) and programmed cell death protein1 (PD1)) along with the TReg transcription factor forkhead box P3. Their mechanism of action remains to be completely elucidated.
SAP) have poor humoral immunity31. A role for CD84 in SAP-dependent TFH cell generation following immuniza tion with protein antigen was recently demonstrated33. However, the TFH cell deficiency in Cd84/ mice was less severe than in SAP-deficient mice33, and TFH cell formation following viral infection was unaffected by the absence of CD84 (REF.35). Analysis of gene-targeted mice has failed to show a requirement for SLAM family receptors other than CD84 in T FH cell formation (reviewed in REF. 23 ), although SLAM has been shown to be required for IL4 expression by GC TFH cells18. One interpretation of these observations is that the severe effect of SAP deficiency in TFH cells reflects a requirement for numerous SLAM receptors during the differentiation of T FH cells. Alternatively, as SLAM receptors can also recruit inhibitors of signalling (such as lipid and tyrosine phos phatases), SAP deficiency might exacerbate negative signals that are delivered through one or more of the SLAM receptors. Consistent with this idea, loss of LY108 reversed the inability of SAP-deficient CD4+ Tcells to form TFH cells and to support Bcell responses35. This was due to a reduced recruitment of SH2 domain-containing protein tyrosine phosphatase 1 (SHP1; also known as PTPN6) to the immune synapse by LY108 (REF.35). Thus, LY108 functions as a rheostat that is capable of delivering positive SAP-dependent and negative SHP1dependent signals that dynamically regulate TFH cells (FIG.1). The role of PD1. An important phenotypic deter minant of TFH cells is their high expression of PD1 (REF.14). PD1 has an inhibitory role in TFH cell differen tiation, as mice with impaired PD1 function have more TFH cells (CCR7lowICOShi cells inPd1/ mice) as a result of increased proliferation and reduced apoptosis3639. Expression of PD1 ligand 1 (PDL1; also known as CD274), rather than PDL2, on Bcells constrains TFH cell formation via the PD1 pathway 38 (FIG.1). Although these studies showed that ablating PD1 signalling increased TFH cell numbers, they yielded conflicting results about
how this affected the outcome of the GC response3639. For instance, some groups reported increased antigenspecific antibody responses in PDL1deficient mice38 or in Plasmodium-infected mice that had been treated with a PDL1specific mAb39. These findings in mice are consistent with data demonstrating that ligation of PD1 suppresses the proliferation, activation and func tion of human TFH cells invitro40. However, other groups have reported impaired plasma cell and GC responses in the absence of PD1 signalling 36,37. These differences might reflect nuances in the experimental systems that have been used, but they might also result from dis tinct functions of PD1 in the development and function of not only TFH cells but also follicular T regulatory cells (follicular TReg cells), which express higher levels of PD1 than TFH cells41. Cytokines. Numerous cytokines have been shown to be important for TFH cell generation invivo and invitro. The initial cytokines that were identified to induce TFH cell-like features in cultured CD4+ Tcells were IL6 and IL21 (TABLE1). However, subsequent analyses of IL6- and IL21- or IL21Rdeficient mice yielded conflicting results regarding the necessity of these cytokines in regulating TFH cell formation invivo (reviewed in REFS23,24). Furthermore, recent studies have provided greater insights into the roles of these cytokines in TFH cell commitment. Using several mouse models of viral infection, investigators found varying and transient degrees of impairment in TFH cell numbers in the absence of IL6, but they observed consistently reduced levels of virus-specific IgG 4245. A more severe decrease in TFH cell formation and pro tective IgG production occurred in the absence of both IL6 and IL21 (REFS42,44) (FIG.1). This requirement for IL6 and IL21 is consistent with reductions in the number of TFH cells in the absence of functional STAT3 (REFS30,46), which acts downstream of both cytokines (TABLE1). Interestingly, the early reduction in the num ber of mouse TFH cells in the absence of STAT3 was
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IL-6 IL-27 IL-12 IL-12R STAT3 IL-27R IL-21R IL-21 BCR IL-6R IL-6
DC ICOSL ICOS ? IL-10R IL-10 STAT5
CXCR5
BLIMP1
BATF MAF BCL-6 IRF4
CD28 IL-21 CXCR5 CD40L ICOS PD1 ICOS T cell PD1 activity SAP CD84 SHP1 NTBA
CD86 CD40 ICOSL PD1L CD84 NIK NTBA B cell
CD4+ T cell IL-2R
BAFFR BAFF
TACI BAFF or APRIL
Figure 1 | Cellular and molecular regulation of TFH cell formation. Naive CD4+ Tcells interact with antigen-presenting dendritic cells (DCs) in the interfollicular or T cell zones; DCprimed CD4+ Tcells acquire expression of CXC-chemokine Nature Reviews | Immunology receptor 5 (CXCR5) and Bcell lymphoma 6 (BCL6) to become early T follicular helper (TFH) cells. These cells then migrate to the TcellBcell border and following interactions with cognate Bcells differentiate into germinal centre TFH cells. This differentiation process is governed by signals provided by signal transducer and activator of transcription 3 (STAT3)-activating cytokines, including interleukin6 (IL6), IL12, IL21 and IL27. These cytokines are secreted by DCs (which produce IL6, IL12 and IL27), Bcells (which produce IL6 and possibly IL27) and CD4+ Tcells (which produce IL21). Cytokine-mediated activation of STAT1 might also contribute to this process (not shown). These cytokines operate individually or collectively to induce or enhance expression of the transcription factors BCL6, MAF, basic leucine zipper transcriptional factor ATF-like (BATF) and interferon-regulatory factor 4 (IRF4), which then imprint the TFH cell fate on a Tcell by inducing the transcription of signature genes, including CXCR5, inducible Tcell co-stimulator (ICOS), IL21 and programmed cell death protein 1 (PD1). Cellcell interactions among activated CD4+ Tcells, antigen-presenting DCs and Bcells also promote TFH cell formation. CD28CD86, CD40 ligand (CD40L)CD40 and ICOSICOS ligand (ICOSL) interactions are central to this process. Notably, ICOSL expression on Bcells is controlled through the opposing effects of Bcell-activating factor (BAFF) signalling via the distinct receptors BAFF receptor (BAFFR) and transmembrane activator and CAML interactor (TACI). In addition, signalling lymphocytic activation molecule (SLAM) family receptors have a dual role in TFH cell generation: the recruitment of SLAM-associated protein (SAP) to these receptors facilitates and maintains conjugate formation between T and Bcells, whereas recruitment of inhibitory phosphatases (such as SH2 domaincontaining protein tyrosine phosphatase 1 (SHP1)) suppresses these interactions, thereby influencing the ability of CD4+ Tcells to form TFH cells and to support Bcell responses. TFH cell generation is restricted by B lymphocyte-induced maturation protein 1 (BLIMP1), which is induced by IL2 in a STAT5dependent manner; BLIMP1 functions by repressing the expression of BCL6. IL10 also suppresses TFH cell formation, but it is unknown whether this is also mediated via BLIMP1. APRIL, a proliferation-inducing ligand; BCR, B cell receptor; IL10R, IL10 receptor; NIK, NF-B-inducing kinase; NTBA, natural killer, T and B cell antigen (also known as LY108 in mice); PDL1, PD1 ligand 1.
also transient and not as severe as that observed in IL6deficient mice, which indicates that there might be a requirement for an alternative signalling pathway downstream of IL6R45. Indeed, the TFH cell deficit in IL6deficient mice was recapitulated when STAT1 and STAT3 were deleted from CD4+ Tcells, which indicates that IL6mediated activation of both STATs is required for full TFH cell development 45. These results suggest that significant functional redundancy exists between IL6 and IL21, and that the relative importance of either cytokine is related to the level of its production at a given time and is inversely proportional to the levels of other compensatory cytokines.
Whereas IL21 is likely to be produced by CD4+ Tcells themselves, IL6 could be derived from activated Bcells44, from dendritic cells (DCs)47 or from follicular dendritic cells43. IL6 expression by Bcells or DCs has been shown to promote IL21 production by CD4+ Tcells that have been activated invitro44,47, and B cell-derived IL6 restored TFH cell numbers following transfer into mice that were deficient in both IL6 and IL21 (REF.44). Thus, although interactions between DCs and naive CD4+ Tcells are important in initiating the TFH cell programme23, subse quent cognate interactions between these early TFH cells and antigen-specific Bcells are required to ensure their progression to a TFH cell fate (FIG.1).
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Although it is known that numerous cytokines can promote TFH cell formation (reviewed in REFS23,24) (Supplementary information S1 (table)), much less is known about the factors that restrain this process. An important attribute of all TH cell lineages is their ability to suppress the generation of cells with alternative effec torfates1 (TABLE1). Thus, it is not surprising that TFH cell formation can also be suppressed by several immunoreg ulatory cytokines. Impaired IL10 signalling was shown to promote TFH cell formation through at least two sep arate mechanisms. First, IL10R deficiency enhanced TFH cell function, as was shown by the increased anti body responses of cognate Bcells following interactions with Il10rb/ TFH cells. Second, the inability of DCs to respond to IL10 resulted in greater production of IL6, IL23 and IL12; this, in turn, contributed to an increased frequency of TFH cells in Il10rb/ mice (FIG.1). Furthermore, Il10rb/ TFH cells expressed higher levels of IL17 and IL21 than wild-type TFH cells, which indi cates that IL10 regulates TFH cells both quantitatively and qualitatively 48. Importantly, the increased production of both IL17 and IL21 substantially shaped humoral immune responses in Il10rb/ mice, as blockade of these cytokines not only impeded TFH cell formation but also reduced the augmented antigen-specific antibody response48. Thus, consistent with its ability to suppress TH1 and TH17 cells1 (TABLE1), IL10 also negatively regulates TFH cells. However, the molecular mechanism through which IL10 exerts its repressive effect is unknown. TFH cell formation can also be limited by IL2STAT5 signal ling, which induces B lymphocyte-induced maturation protein 1 (BLIMP1; also known as PRDM1) to suppress BCL6 function49 (FIG.1); therefore, IL10mediated BLIMP1induction might also contribute to the inhibitory effect of IL10 on TFH cells. Overall, positive and negative signals from numerous receptors and cytokines fine-tune TFH cell formation, homeostasis and function. Consequently, perturbations in this balance might deregulate TFH cell differentiation, thereby accelerating disease development in genetically susceptible hosts. The exact role of specific cytokines in the regulation of human TFH cells remains unknown, but the identification of individuals with inactivating mutations in important cytokines and cytokine recep tors will shed light on these requirements. rather than as a result of apoptosis. Thus, these for mer TFH cells most probably differentiate into memory cells that downregulate CXCR5, PD1 and BCL6, that reexpress CCR7, IL7R and CD62L (also known as L-selectin), and that persist for a long time. To add further support to the idea that TFH cells can generate memory cells, it was recently shown that gene expression profiles of early TFH cells share many simi larities with precursor memory CD8+ Tcells52. Upon subsequent antigenic challenge, these memory-like cells form TFH cells more quickly and promote GC forma tion and antibody production more effectively than naive or memory cells that originated from non-TFH cells50,51,5355. Interestingly, it was shown that not all of these cells adopted a TFH cell phenotype, which indicates that cellular plasticity might allow them to differentiate into other TH cell subsets. A caveat to these studies is the reliance on identifying TFH cells by phenotype alone. In the future it would be useful to more precisely track TFH cells in GCs and to determine their fate. However, taken together, these data demonstrate that, at least in mice, some TFH cells can enter the memory pool and can provide long-term protection following reinfection. Antigen-specific T cells have also been identified within the population of TFH-like cells that is detected in the peripheral blood of humans. These cells seem to be more readily detected following recent antigen exposure, which indicates that they exist as TFH-like cells or that their precursors adopt a TFH cell phenotype in response to antigenic stimulation24,56,57, as discussedbelow.
The expanding universe of TFH-like cells TFH cells that provide B cell help during responses to conventional T cell-dependent antigens have been well characterized, but unconventional subsets of TFH cells have also been identified, and these are discussedbelow.
Extrafollicular TH cells. Humoral immune responses are initiated in extrafollicular areas of lymphoid tissues where Bcells differentiate into short-lived plasmablasts following interactions with TH cells11,13,26. Similar to T FH cells, the formation of extrafollicular T H cells requires BCL6, and their function is mediated by CD40L, ICOS and IL21 (REF.26). However, extrafollicu lar TH cells do not express high levels of CXCR5; rather, they are attracted to these sites by the CXCR4 CXCL12 (CXC-chemokine ligand 12) axis 26. Extrafollicular TH cells might also give rise to GC TFH cells following interactions with cognate Bcells19,23,24. NKTFH cells. Natural killer T (NKT) cells are innatelike Tcells expressing a semi-invariant Tcell receptor (TCR) that recognizes lipid antigens presented by the non-polymorphic MHC molecule CD1d. The ability of the NKT cell population to rapidly expand and to produce effector cytokines following encounter with an antigen typically galactosyl ceramide (GalCer) links the innate and adaptive immune responses. NKT cells can initiate or enhance antibody responses to lipid and protein antigens via cognate5860 and non-cognate61 mechanisms, respectively.
Follicular dendritic cells

Specialized non-haematopoietic stromal cells that reside in lymphoid follicles and germinal centres. These cells possess long dendrites and carry intact antigen on their surface. They are crucial for the optimal selection of Bcells that produce antigen-binding antibodies.
TFH cell memory Although TFH cells are unquestionably important in establishing B cell memory, the fate of TFH cells that are generated during GC reactions is unclear. Several recent studies in mice have investigated whether TFH cells are short-lived effector cells or whether they differentiate into long-lived memory-type cells that can resume a TFH cell state following reexposure to the same initiating antigen. Although both CXCR5+PD1low TFH cells and CXCR5hiPD1hi GC TFH cells are gener ated following vaccination or infection, the number of GC TFH cells decreases more rapidly than that of CXCR5+PD1low TFH cells50,51. The decrease in GC TFH cells is partly due to their loss of the TFH phenotype
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Recent studies have reassessed the nature of NKT cells that provide Bcell help. It was shown that follow ing immunization with GalCerantigen conjugates, a small proportion of mouse NKT cells acquired a TFH-like phenotype62,63. These NKT follicular helper (NKTFH) cells were detected in human tonsils62 and their development depended on the same factors that are important for conventional TFH cells62. By produc ing IL21, NKTFH cells supported the rapid formation of GCs, yielding detectable levels of antigen-specific IgG, with some evidence of affinity maturation6264 (FIG.2). Although NKT FH cells resembled T FH cells, the most striking difference was their inability to invoke long-lived memory responses to lipid antigens (FIG.2). Furthermore, the magnitude of the NKTFH cell-induced antibody responses to lipid antigens was inferior to those driven by conventional TFH cells6264. Despite this, administering GalCer as an adjuvant or as a conju gated component of immunizing antigens increased the production of antigen-specific antibodies. This probably reflects the direct actions of NKTFH cells on antigen-specific CD1d+ Bcells, as well as the indirect actions of NKTFH cells, such as cytokine production, on other cell types5861. It might also indicate that there is synergy between NKTFH and TFH cells. Taken together, these findings support the inclusion of GalCer in vaccine adjuvants. TFHcells. The finding that mice and humans lack ing the conventional TCR have TH cells that elicit humoral responses against T cell-dependent antigens led to the realization that Tcells can provide help to Bcells to generate GCs65. Similar to conventional TFH cells, some human TFHcells express CXCR5 and localize to follicles and GCs6668. Tcells express a semi-invariant TCR repertoire and recognize nonpeptidic phosphoantigens that are derived from micro bial metabolites 65. These antigens rapidly activate Tcells, which can subsequently acquire TFH cell fea tures66,68,69; the differentiation of Tcells to TFH-like cells is increased by exogenous IL21 (REFS68,69) (FIG.2). As Tcells do not produce IL21 (REFS68,69), they are dependent on extrinsic sources of this cytokine to differentiate into TFH-likecells. As conventional CD4+ Tcells, Tcells and NKT cells recognize different repertoires of microbial anti gens, their ability to differentiate into TFH-like effec tor cells provides a mechanism whereby humoral immunity can be generated against a broad range of pathogen-associated antigens (FIG.2). This expands the number of molecular targets that initiate protec tive immunity, but it might also contribute to postinfection autoimmunity by generating cross-reactive autoantibodies13,70 (BOX2). Follicular TReg cells. Recent studies have proposed that TFH cells are controlled by follicular TReg cells a spe cialized subset of TReg cells that colocalize within Bcell follicles. Follicular TReg-like cells were first described in human tonsils in 2004 (REF.71), but it took another 78years for them to be examined in greaterdetail. Follicular TReg cells comprise approximately 1015% of the TFH cell population in human and murine lym phoid tissues7274. They show characteristics of both TFH cells and TReg cells, but they lack expression of CD40L, IL4 and IL21 (REF.73). Abrogating either follicular TReg cell development or their follicular localization was shown to enhance GC responses and subsequent anti body production72,74. Although follicular TReg cells show similar requirements to conventional TFH cells for their development 73, they actually originate from thymusderived TReg cells, rather than from peripherally derived TReg or TFH cells. Similar to other specialized TReg cell sub sets that have evolved to selectively control TH1, TH2 and TH17 cells75, follicular TReg cells seem to have co-opted the transcriptional machinery of TFH cells to migrate into GCs to exert their regulatory effects7274. PD1 is more highly expressed on follicular TReg cells than on TFH cells. Interestingly, PD1 or PDL1 deficiency was shown to favour the development of follicular TReg cells but not TFH cells41. Furthermore, PD1deficient follicular TReg cells were more effective than normal follicular TReg cells at impeding T FH cell-mediated Bcell differentiation41. Thus, PD1 seems to negatively regulate not only the development and maintenance of follicular TReg cells but also their suppressive func tion. The increase in the number of follicular TReg cells compared with TFH cells, together with the increased suppressive function of PD1deficient follicular TReg cells41, might explain some of the discrepancies in the findings regarding the consequences of PD1 deficiency on TFH cell-dependent B cell responses3639. The mechanisms by which follicular TReg cells attenu ate humoral immunity remain unknown. Follicular TReg cells express IL10 mRNA73 (TABLE1), and IL10 can attenuate TFH cell formation in normal48 and autoim mune76 settings. Thus, follicular TReg cell-derived IL10 might be one means by which TFH cell-mediated B cell responses are regulated. However, as follicular TReg and TFH cells express similar levels of IL10, it is difficult to predict how follicular TReg-derived IL10 would affect TFH cells in a different manner to their own endogenous IL10. Although it is known that follicular TReg cells are likely to limit GC reactions by impeding both TFH cells and GC Bcells, therapeutically exploiting the immuno regulatory function of follicular TReg cells will require further delineation of their mechanism ofaction.
Tcells
Tcells that express the T cell receptor. These Tcells are present in the skin, vagina and intestinal epithelium as intraepithelial lymphocytes. Although the exact function of T cells is unknown, it has been suggested that mucosal T cells are involved in innate immune responses.
Human circulating TFH-like cells As access to human lymphoid tissues is limited, stud ies of human T FH cells have exploited the fact that CD4+CXCR5+ Tcells comprise a small subset of circu lating lymphocytes4,5. However, there are clear differ ences between CD4+CXCR5+ Tcells in the blood and those in the tonsils. For instance, CD4+CXCR5+ Tcells in the blood do not express BCL6 and their expres sion of ICOS and PD1 is substantially lower than that of TFH cells4,5,56,77,78. Despite this, invitro-cultured bloodderived CD4+CXCR5+ Tcells produce more IL21, IL10 and CXCL13 which are all features of TFH cells8,9 and are more efficient at inducing B cell differentiation
VOLUME 13 | JUNE 2013 | 419
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CXCR4 IL-21R IL-21 Foreign antigen or self antigen In vitro and in vivo
IgG
Extrafollicular TCR TH cell CD40L BCL-6
ICOS
ICOSL MHC class II CD40 B cell
Plasma cell
CXCR5
IL-21R IL-21 ICOS ICOSL CD1d CD40 SLAM B cell
Glycolipid and lipid antigen
Low-magnitude antibody response IgG IgM IgA
NKTFH cell
TCR
In vitro and in vivo
CD40L BCL-6 SLAM
Plasma cell
SAP IL-21
DC
Naive T cell
IL-4 IL-4R T cell-dependent protein antigen
CXCR5
IL-10 IL-10R ICOS TCR ICOSL IL-21R
Long-lived serological memory IgG IgM IgA
TFH cell
CD40L BCL-6 SLAM
MHC class II B cell CD40 SAP SLAM
In vitro and in vivo
Plasma cell
IL-21 CXCR5 IL-10 IL-10R ICOS TFH cell TCR
IL-4
IL-4R
Non-peptide phosphoantigen
IgM
IgG IgA
ICOSL In vitro B cell CD40 Plasma cell
BCL-6 CD40L
Figure 2 | TFH cell subsets with specialized effector functions. Naive CD4+ Tcells, natural killer TReviews (NKT) cells and Nature | Immunology Tcells can all differentiate into T follicular helper (TFH) cells following instructive signals that are derived from antigen-presenting cells. These cells share many features, including their requirements for differentiation, their expression of transcription factors and their surface phenotype, which underlies their ability to induce B cell differentiation. Extrafollicular T helper (TH) cells, NKT follicular helper (NKTFH) cells and TFH cells require inducible Tcell co-stimulator (ICOS), B cell lymphoma 6 (BCL6), SLAM-associated protein (SAP) and CD28 signalling for their differentiation, which occurs invitro and invivo in the presence of Bcells. TFH cells also express ICOS, but the molecular requirements for their generation remain unknown. Extrafollicular TH cells provide help to Bcells outside the follicles; unlike other TFH-type cell populations, these cells do not express CXC-chemokine receptor 5 (CXCR5). Rather, their localization to extrafollicular areas seems to be mediated by CXCR4. NKTFH cells and TFH cells support the rapid formation of GCs and the generation of antigen-specific antibodies in an interleukin21 (IL21)dependent manner. TFH and TFH cells might also support GC responses by secreting IL4 and IL10. TFH cells do not express IL21 but might rely on exogenous IL21 expression for their TH cell function. TFH cells respond to protein antigens and induce long-lived serological memory. Thus, they have a central role in pathogen-specific responses as well as in several immunopathologies. NKTFH cells respond to glycolipid antigens and induce antibody responses of a lower magnitude than those induced by TFH cells. NKTFH cells might have a role in antibody responses against pathogens that express glycolipid antigens such as Borrelia hermsii, Streptococcus pneumonia and Plasmodium falciparum6264. As Tcells recognize microbial metabolites, TFH cells might have an adjuvant role in responses to most infectious pathogens. Extrafollicular TH cells have been most effectively characterized for their role in disease pathogenesis in murine models of human autoimmunity26. These cells might also represent precursors of TFH cells19. CD40L, CD40 ligand; DC, dendritic cell; ICOSL, ICOS ligand; IL21R, IL21 receptor; SLAM, signalling lymphocytic activation molecule; TCR, T cell receptor.
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than CD4+CXCR5 Tcells56,78. Thus, human circulating CD4+CXCR5+ Tcells have some features of TFH cells, which indicates that precursors of circulating CD4+CXCR5+ Tcells might have experienced some aspects of a TFH cell differentiation programme invivo. Interestingly, subsets of circulating CD4 +CXCR5+ Tcells that have distinct effector functions also exist. By assessing the expression of CXCR3 and CCR6 chemokine receptors that are associated with TH1 and TH17 cells, respectively CD4+CXCR5+ Tcells could be classified into T H1-like, T H17-like and T H2-like (CXCR3CCR6) TFH cell subsets56. Interestingly, the TH2- and TH17like TFH cell subsets were shown to produce higher levels of IL21 and to induce naive Bcell differentiation more efficiently than the TH1like TFH cell subset. This showed that there is substantial heterogeneity among blood-derived CD4+CXCR5 + T cells and potentially clarified previous findings indicating that circulating CD4+CXCR5+ Tcells might be inefficient helpers for Bcell differentiation5. Despite the improved characterization of circu lating CD4+CXCR5+ Tcells, their exact relationship with GC TFH cells remains incompletely defined. It is also unclear what functional role BCL6 has in bloodderived CD4+CXCR5+ Tcells, as it does not seem to be required for their persistence, expression of CXCR5, production of IL21 or for their ability to induce Bcell differentiation. Kinetic analysis of TH cell activation in mice showed that although BCL6 is expressed by developing TFH cells, it is downregulated at later time points79. Apart from BCL6 expression, there seemed to be few differences between BCL6 + and BCL6 TFH cells with respect to their expression of important TFH cell-related genes79. This indicates that although BCL6 is required for TFH cell formation, it might be dispensable for their maintenance following antigen clearance and the resolution of an immune response. Furthermore, BCL6 TFH cells upregulated Il7r and Ccr7 and downregulated the cell cycle machinery 79. Thus, T FH cells probably yield a population of cells that exit the GC and lymphoid tissues, and that return to the circulation as a population of quiescent memorytype CD4+CXCR5+ Tcells. growing interest in identifying appropriate biomarkers of successful vaccination. Several publications reported the outcomes of immunization against influenza dur ing the 2009 H1N1 epidemic. These studies included healthy individuals and a cohort of HIV-infected indi viduals. Whereas all healthy individuals generated protective levels of H1N1specific IgG 4weeks after vaccination, such a response was observed in only 50% of HIV-positive individuals. Successful induction of H1N1specific IgG coincided with significant increases in both serum levels of IL21 and frequencies of cir culating CD4+CXCR5+ Tcells and memory Bcells80,81; this correlated with titres of serum H1N1specific antibodies81. Thus, the ability of the H1N1 vaccine to induce protective antibody responses correlated with the induction of detectable features of TFH cell-mediated immunity 80,81. Another study found that the emergence of CD4 +CXCR5 +CXCR3 +ICOS + T cells in influenzavaccinated individuals correlated with increased lev els of neutralizing antibodies and in adults with the appearance of circulating plasmablasts57. This was unexpected, as it was previously found that circulat ing CXCR3+ TFH-like cells were poor inducers of naive B cell differentiation and that they did not produce IL21 (REF.56). However, CD4+CXCR5+CXCR3+ICOS+ Tcells did promote the differentiation of memory, but not naive, B cells into plasmablasts invitro through the production of IL10 and IL21 (REF.57). This suggests that increased numbers of CD4+CXCR5+CXCR3+ICOS+ Tcells could be used as an indicator of the develop ment of protective antibody responses from pre-exist ing memory Bcells, rather than of primary responses of naive Bcells57. Despite this conclusion, it remains unknown which signals induce IL21 expression in CD4+CXCR5+CXCR3+ ICOS+ Tcells and why these cells fail to activate naive Bcells even though they express CD40L, IL10 and IL21, which, in combination, can strongly induce the differentiation of naive B cells to plasmablasts82,83. Taken together, these studies show that quantifying the frequencies of circulating CD4+CXCR5+ Tcells, or of their subsets, is a reliable predictor of vaccine suc cess and of the magnitude of the induced response57,80,81. It also allowed for the separation of vaccine respond ers from non-responders80,81. Understanding why some individuals failed to elicit a substantial vaccine-specific TFH cell-dependent antibody response might facilitate the development of improved vaccination strategies, particularly in individuals with suboptimal humoral immunity. Autoimmune diseases. The detection of CD4+CXCR5+ Tcells and CXCL13 in organs that are affected by autoimmune disorders, such as the salivary glands in Sjgrens syndrome, suggested that aberrant TFH cell development can drive autoimmunity 84. As a result of this finding, several studies have assessed the possibil ity of enumerating circulating CD4+CXCR5+ Tcells as a potential biomarker of autoimmune diseases, espe cially given the difficulty of accessing and analysing
VOLUME 13 | JUNE 2013 | 421 2013 Macmillan Publishers Limited. All rights reserved
Sjgrens syndrome
A systemic autoimmune disease in which autoantibodies target and destroy exocrine glands such as the tear ducts and the salivary glands.
Human TFH cells in immunity and disease Dysregulated behaviour of TFH cells and extrafolli cular TH cells has been found to contribute to auto immune or immune-deficient states in several mouse models of human disease 10,24,26. Consequently, there is great interest in determining the role of TFH cells in human disease. Although the exact nature of circulat ing CD4+CXCR5+ Tcells in humans remains unclear, investigation of this population of cells in various contexts has nonetheless provided important insights into their potential functions during normal immune responses and in immunopathologies (TABLE2).
Circulating CD4+CXCR5+ Tcells as biomarkers of effective humoral immunity. Long-lived T cell-dependent antibody responses underlie the success of most vac cines that are currently in use. For this reason, there is
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Table 2 | Human diseases associated with aberrant TFH cell function
Human disease Autoimmunity
Systemic lupus erythematosus
TFH cell-related phenotype

Increased frequencies of circulating CD4+CXCR5+PD1hi cells, CXCR5+ICOShi cells or ICOShi Tcells Increased serum levels of IL21 and CXCL13 Increased frequencies of circulating CD4+PD1hi T cells, CD4+CXCR5+ICOShi T cells or CD4+CXCR5+CCR6+ (TH17type) Tcells Increased frequencies of circulating CD4+CXCR5+PD1hi cells or CD4+CXCR5+ICOShi Tcells Increased serum levels of IL21 Increased TH2 and TH17like subsets among circulating CD4+CD45RO+CXCR5+ Tcells Increased frequencies of circulating CD4+CXCR5+PD1hi T cells or CD4+CXCR5+ICOShi Tcells Increased frequencies of circulating CD4+CXCR5+ T cells or CXCR5+CD57+ Tcells Normal frequency of circulating CD4+CXCR5+ Tcells but impaired helper function invitro Reduced frequency of circulating CD4+CD45RO+CXCR5+ Tcells Reduced frequency of circulating CD4+CD45RO+CXCR5+ Tcells Impaired generation of TFH-like cells from naive precursors invitro Increased frequency of TFH cells in lymph nodes of HIV-infected individuals Enrichment of HIV-specific CD4+ Tcells in the TFH cell compartment Increased number of CXCR5+PD1hiICOShiOX40+S APhiBCL6+MAF+ malignant cells Detectable expression of CXCL13 and IL21
Correlated pathology
Increased severity of end-organ damage Higher serum levels of dsDNA-specific autoantibodies Higher serum levels of autoantibodies
Refs
77,85,94
Sjgrens syndrome
77,93
Rheumatoid arthritis
Higher serum levels of CCP-specific autoantibodies Higher disease score and serum levels of IL21and CCP-specific autoantibodies Higher disease score Increased number of circulating plasmablasts Increased titres of serum autoantibodies against thyroid stimulating hormone receptor and thyroglobulin Increased disease severity
90,92
Juvenile dermatomyositis Autoimmune thyroid disease (Graves disease or Hashimotos thyroiditis) Myasthenia gravis
56 91
86,87
Immunodeficiency
X-linked lymphoproliferative disease
Impaired cytokine production, ICOS expression and B cell help invitro Impaired development of TFH cells owing to an inabilty to form GCs Inability of naive CD4+ Tcells to express IL21 and to provide help to Bcells in response to TFH cell-inducing cytokines (for example, IL12) Positive correlations with plasma viraemia, numbers of GC Bcells and plasma cells, hypergammaglobulinaemia and virus-specific antibodies Might be correlated with aberrant humoral features such as follicular hyperplasia, hypergammaglobulinaemia and autoantibody production in these PTCLs
17,96 29
CVID (ICOS deficiency) and hyper-IgM syndrome (CD40 or CD40L deficiency) ADHIES (STAT3 deficiency)
46
HIV
99, 101
Lymphoma
AITL and FTCL
104111
ADHIES, autosomal dominant hyper-IgE syndrome; AITL, angioimmunoblastic Tcell lymphoma; BCL6, B cell lymphoma 6; CCP, cyclic citrullinated peptide; CCR6, CC-chemokine receptor 6; CD40L, CD40 ligand; CVID, common variable immunodeficiency syndrome; CXCL13, CXC-chemokine ligand 13; CXCR5, CXC-chemokine receptor 5; dsDNA, double-stranded DNA; FTCL, follicular Tcell lymphoma; GC, germinal centre; ICOS, inducible Tcell co-stimulator; IL, interleukin; PD1, programmed cell death protein 1; PTCL: peripheral Tcell lymphoma; SAP, SLAM-associated protein; STAT3, signal transducer and activator of transcription 3; TFH, T follicular helper; TH; T helper.
Systemic lupus erythematosus

(SLE). An autoimmune disease in which autoantibodies that are specific for DNA, RNA or proteins associated with nucleic acids form immune complexes. These complexes damage small blood vessels, especially in the kidneys. Patients with SLE generally have abnormal B and T cell function.
secondary lymphoid organs in affected individuals. Early studies showed increases in the frequency of CD4+ICOS+ Tcells in the peripheral blood of patients with systemic lupus erythematosus (SLE)85 and in the frequency of CD4+CXCR5+ Tcells in the peripheral blood of patients with myasthenia gravis. Furthermore, in myasthenia gravis, the frequency of CD4+CXCR5+ T cells in the circulation positively correlated with clinical disease scores86,87. Subsequent studies reported increases in circulating CD4 +CXCR5 +ICOS hiPD1 hi Tcells in some patients with SLE or Sjgrens syndrome, and these were shown to correlate with autoantibody titres, frequencies of circulating GC Bcells, plasma cells and disease severity 77,88,89. Increased frequencies of cir culating CXCR5+ICOShi or CD4+CXCR5+PD1+Tcells
have also been observed in patients with rheumatoid arthritis and autoimmune thyroid diseases ( Graves disease and Hashimotos thyroiditis ), and have been shown to be accompanied by elevated levels of serum autoantibodies9092 (TABLE 2). Furthermore, it was found that the TH2- and TH17 (but not TH1)-like CD4+CXCR5+ Tcell subsets that provide B cell help are increased in juvenile dermatomyositis 56. In light of this observation, circulating CD4+CXCR5+ Tcells from patients with Sjgrens syn drome were re-examined with respect to their THlike phenotypes93. A positive correlation was found between the levels of serum autoantibodies and the numbers of circulating CD4+CXCR5+ Tcells, particularly with regard to those that also expressed CCR6 (TABLE 2).
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As these CD4+CXCR5+CCR6+ Tcells did not produce IL17 (REF.93), this study highlighted the possibility that CD4+CXCR5+CCR6+ Tcells are not strictly TH17like TFH cells, but that they acquire CCR6 expression to facilitate migration to inflamed tissues. Although some studies did not detect increases in T FH-like cells in SLE 94 and rheumatoid arthri tis95, the concept has emerged that levels of circulat ing CD4+CXCR5+ Tcells are generally increased in humoral autoimmune conditions. As this increase is usually correlated with clinical disease, the frequency of circulating CD4+CXCR5+ Tcells or the serum levels of IL21, which are both indicative of T FH cell activ ity, seem to be useful biomarkers for predicting dis ease outcome. Furthermore, in patients with SLE, both the increased frequency of circulating CD4+CXCR5+ Tcells and the clinical manifestations that are associ ated with this disease were reduced following cortico steroid treatment 88. This indicates that T FH cells are likely to contribute to disease pathogenesis, and it highlights the possibility of targeting TFH cells to effec tively treat antibody-mediated autoimmune conditions. However, it remains to be determined whether the association between autoimmunity and the expansion of the population of TFH cells might reflect the activa tion of self-reactive TFH cells or whether might reflect the increased stimulation of GC Bcells producing cross-reactive autoantibodies as a result of increased TFH cell help13,70 (BOX2). Primary immunodeficiencies. Several primary immu nodeficiencies have been associated with genetic defects that might affect TFH cell differentiation and function. Mutations in the gene encoding SAP cause XLP31, whereas mutations that affect the genes encod ing CD40L and ICOS cause hyper-IgM syndrome and common variable immunodeficiency, respectively 2. Important features of these immunodeficiencies include impaired humoral immune responses and a paucity of well-formed GCs in secondary lymphoid tissues 2. The numbers of circulating CD4 +CXCR5 + Tcells are considerably decreased in CD40L- or ICOSdeficient individuals29. Although patients with XLP have normal numbers of CD4+CXCR5+ Tcells, possibly as a result of persistent activation with viral antigens17, their CD4+ Tcells fail to acquire features of TFH cells invitro17,96 (TABLE 2). Patients with autosomal dominant hyper-IgE syn drome a primary immunodeficiency that is caused by mutations in STAT3 also show impaired func tional antibody responses2. These patients have a par tial deficiency in numbers of circulating CD4+CXCR5+ Tcells, and their naive CD4+ Tcells fail to differentiate into TFH-like cells invitro 46. STAT3 is likely to func tion downstream of several cytokines including IL6, IL12, IL21 and IL27 which indicates that signal integration among multiple cytokine receptors is required for TFH cell formation46 (TABLE1). Patients with mutations in IL12RB1, which encodes IL12R1, also have fewer circulating CD4+CXCR5+ Tcells97. This correlates with reduced numbers of memory Bcells, abnormal GCs and low-avidity antibody responses to tetanus. Interestingly, antibody responses follow ing natural infections or vaccination have been shown to be unaffected or even enhanced by IL12R1 deficiency, and the defect in circulating CD4+CXCR5+ Tcells improved with age97. Thus, IL12 signalling may only be required for TFH cell formation and function early in life and for responses to only some antigens (for example, antigens that are derived from non-replicating pathogens)97. Taken together, these analyses of monogenic immu nodeficiencies showed that CD40L, ICOS, SH2D1A, STAT3 and possibly IL12RB1 are required for TFH cell formation, function and/or maintenance. These stud ies also provided further correlative evidence that cir culating CD4+CXCR5+ Tcells are related to bonafide TFH cells, as a deficit in CD4+CXCR5+ T cells mimicked a deficiency of GCs and presumably lymphoid organresident TFH cells. Importantly, the impaired formation and/or function of circulating CD4 +CXCR5+ Tcells is associated with compromised humoral immune responses, which highlights the contribution of TFH cells to successful T cell-dependent antibody responses. As some immunodeficiencies are associated with mutations in TACI and BAFFR2, and as studies in mice indicate that these receptors modulate TFH cell formation27,28, it will be interesting to assess the formation and function of circulating TFH-type cells in individuals with these genetic lesions. Acquired immunodeficiencies. CD4+ Tcells are targets of HIV infection and are therefore depleted in infected individuals. Paradoxically, several aspects of humoral immunity are augmented following HIV infection, including polyclonal Bcell activation, the appearance of peripheral blood plasmablasts and hypergammaglob ulinaemia98. To investigate the basis for these contrast ing phenomena, TFH cells have recently been assessed in individuals with HIV and in simian immuno deficiency virus (SIV)-infected macaques. A marked accumulation of TFH cells was observed in the lymph nodes both of individuals with chronic HIV infection and of macaques that were chronically infected with SIV40,99102. Interestingly, a substantially higher pro portion of TFH cells were specific for HIV compared with effector and memory CD4 + Tcell subsets 99,101 (TABLE 2) . T FH cells were found to be infected with SIV or HIV 100102, and human T FH cells were shown to contain substantially more copies of HIV DNA than naive, memory or effector T cell populations101. Strikingly, human T FH cells were also better at sup porting viral replication and infection of susceptible host cells101. Furthermore, TFH cells that were present during chronic infection seemed to be persistently activated 99101, unlike those in uninfected donors. Thus, ongoing antigenic stimulation of activated CD4+ Tcells in HIV-infected individuals might cooperatively drive these cells to a TFH cell fate. Sustained signalling through IL6R which is highly expressed on TFH cells derived from both normal donors and those with
Myasthenia gravis
A chronic autoimmune disease that involves the generation of T cell-dependent autoantibodies that are specific for the acetylcholine receptor. These antibodies interfere with the transmission of signals at neuromuscular junctions.
Rheumatoid arthritis
An immunological disorder that is characterized by symmetrical polyarthritis, often progressing to crippling deformation after years of synovitis. It is associated with systemic immune activation and the presence of acute-phase reactants in the peripheral blood, as well as rheumatoid factor (immunoglobulins that are specific for IgG), which forms immune complexes that are deposited in many tissues.
Graves disease
A type of autoimmune disease, and the most common form of hyperthyroidism in humans. It results from activating antibodies that are specific for the thyroid stimulating hormone receptor (TSHR). In mouse models of Graves thyroiditis, the disease is induced by immunization with the TSHR.
Hashimotos thyroiditis
An autoimmune disease in which self-reactive Bcells and Tcells target the thyroid, resulting in hypothyroidism.
Juvenile dermatomyositis
A chronic, multisystem autoimmune and inflammatory disease involving muscle, skin, blood vessels, the gastrointestinal tract and other organs. Autoantibodies are often detected in these patients, but their specificities have not yet been completely defined.
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chronic HIV or SIV infection16,100,102 in response to IL6, which is abundantly produced during HIV infec tion98, might also cooperatively drive these cells to a TFH cell fate24. As IL2 can suppress TFH cell differen tiation49, it is possible that the loss of IL2producing Tcells following HIV infection102 also contributes to the increased numbers of T FH cells in HIV-infected individuals. The increase in TFH cell numbers has functional consequences for humoral immunity during HIV infection. Thus, the frequency of TFH cells positively correlates with plasma viraemia, the numbers of GCs and plasma cells, the levels of virus-specific IgG and the onset of hypergammaglogulinaemia40,99101 (TABLE2). Furthermore, antiretroviral therapy has been shown to reduce the frequencies of TFH cells, GC Bcells and plasma cells, as well as the numbers of HIV-specific and HIV-infected TFH cells101. These findings strongly indi cate that the expansion of the TFH cell population and the subsequent dysregulated antibody response might be driven by chronic viral infection. It is possible that increases in HIV-specific TFH cells would result in protective HIV-specific anti body responses; however, these are usually not broadly neutralizing 98. It was recently found that although TFH cells from uninfected and HIV-infected individu als show similar phenotypes and express comparable levels of cytokines, the T FH cells from HIV-infected individuals were unable to promote B cell differentia tion invitro 40 as a result of heightened PDL1 expres sion on GC Bcells, which inhibits TFH cell function via PD1 (REF.40). This extrinsically mediated functional impairment of TFH cells might contribute to the inabil ity of HIV-infected individuals to generate neutralizing HIV-specific antibodies. Elucidating mechanisms to promote the effector function of HIV-specific TFHcells for example, through PD1PDL1 blockade might facilitate the development of improved HIV vaccines. Malignancies. Peripheral Tcell lymphomas (PTCLs) are rare haematological malignancies constituting approximately 510% of all non-Hodgkins lympho mas. PTCLs include angioimmunoblastic T cell lym phoma (AITL), follicular Tcell lymphoma (FTCL) and PTCL-not otherwise specified (PTCL-NOS)103,104. Morphological, phenotypic and molecular character ization of malignant cells in AITL has shown that these cells have many of the features of TFH cells105109 (TABLE 2). In a substantial proportion of cases of FTCL, which was previously considered to be a follicular variant of PTCLNOS, the malignant cells were also found to show TFH cell traits104,108111. Importantly, the assessment of a set of TFH cell-related genes in different malignancies led to the reclassification of several cases of PTCL-NOS as AITL109, which demonstrates the robustness of molecu lar diagnoses of PTCLs. These studies established that numerous PTCLs probably arise from normal TFH cells. Some of the cardinal features of AITL and FTCL include Bcell activation, follicular hyperplasia, hypergamma globulinaemia and autoantibody production103,104,111. These humoral immunity-associated features of AITL and FTCL probably reflect the dysregulated activity of malignant TFH cells, which parallels the aberrant func tion of TFH cells in autoimmunity. Thus, it might be possible to treat these features of AITL and FTCL by targeting TFH cell-related molecules such as PD1, CXCL13 and IL21 that are abundantly expressed in these diseases. It is unknown why TFH cells give rise to several types of PTCLs. Interestingly, TFH cells undergo prompt apo ptosis, at least invitro9, which indicates that the mecha nisms that control their survival might be stringently regulated. Thus, the molecular lesions that override TFH cell apoptosis may contribute to the malignant transformation of thesecells. Cytogenetic abnormalities occur infrequently in PTCLs103,104. However, mutations in isocitrate dehydrogenase 2 (IDH2) and TET2 were recently detected in approximately 25% and 45% of cases of AITL, respec tively, and TET2 was also shown to be mutated in approximately 30% of cases of PTCL-NOS 112114. In AITL, there was no difference in clinical outcome between patients with IDH2 mutations and those without IDH2 mutations113. However, patients with TET2 mutations were associated with more advanced disease and poorer clinical outcome than patients with out TET2 mutations114. How TET2 mutations lead to the transformation of TFH cells remains to be deter mined. Interestingly, a novel mouse model of AITL was recently reported in which 50% of mice that were heterozygous for the Roquin (also known as Rc3h1) allele developed AITL-like disease115. This model might provide important insights into the pathophysiology of AITL in humans and might facilitate preclinical test ing of potential therapeutics. It also raises the possibil ity that heterozygous mutations in ROQUIN underlie AITL in some individuals.
Isocitrate dehydrogenase 2
(IDH2). An enzyme that catalyses the oxidative decarboxylation of isocitrate to 2oxoglutarate and is a component of the tricarboxylic acid cycle. Mutations in IDH1 and IDH3 have been detected in glioma, glioblastomas and acute myeloid leukaemia.
TET2
The TET2 gene encodes an oxygenase that catalyses the oxidation of 5methylcytosine to 5hydroxymethylcytosine to alter the epigenetic status of DNA. It is frequently mutated in human lymphomas. Roquin A RING-type ubiquitin ligase that represses the expression of inducible T cell co-stimulator (ICOS), thereby restraining the development and function of T follicular helper cells. A mutation in the Roquin (also known as Rc3h1) gene results in lupus-like disease in mice.
Conclusions Nearly half a century has passed since Jacques Miller first described the fundamental requirement for thy mus-derived cells to support antigen-specific anti body production3. It has taken a substantial period of time to identify the Tcell subset that is responsible for mediating Bcell responses but in the past decade there have been important advances in our knowledge and understanding of the molecular and cellular biol ogy as well as the function and regulation of TFH cells. Indeed, we now have an appreciation of how these cells operate during normal immune responses and, perhaps more importantly, how they might underlie immunological diseases as diverse as autoimmunity, immunodeficiency and lymphomas. These rapid dis coveries in TFH cell biology should allow us to exploit these cells (as well as their associated receptors, cytokines, chemokines and biochemical pathways) for the development of novel therapeutics to treat these conditions, as well as for the development of nextgeneration vaccines to induce sustained protection against infection. Hopefully it will not take another 50years before TFH cell-targeted therapies are available to improve human health.
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Acknowledgements
Studies in the authors laboratories into T follicular helper cells are supported by grants and fellowships that have been awarded by the National Health and Medical Research Council of Australia. The authors thank S. Crotty and H. Ueno for providing papers before publication.
FURTHER INFORMATION
Stuart Tangyes homepage: http://www.garvan.org.au/ research/research-groups/immunobiology.html
SUPPLEMENTARY INFORMATION
See online article: S1 (table)
REVIEWS
Regulation of innate and adaptive immunity by Notch

Freddy Radtke1, H.Robson MacDonald2 and Fabienne Tacchini-Cottier3
Abstract | Coordinated function of the innate and adaptive arms of the immune system in vertebrates is essential to promote protective immunity and to avoid immunopathology. The Notch signalling pathway, which was originally identified as a pleiotropic mediator of cell fate in invertebrates, has recently emerged as an important regulator of immune cell development and function. Notch was initially shown to be a key determinant of cell-lineage commitment in developing lymphocytes, but it is now known to control the homeostasis of several innate cell populations. Moreover, the roles of Notch in adaptive immunity have expanded to include the regulation of Tcell differentiation and function. The aim of this Review is to summarize the current status of immune regulation by Notch. A better understanding of Notch function in both innate and adaptive immunity will hopefully provide multiple avenues for therapeutic intervention in disease.
Notch signalling is an evolutionarily conserved celltocell communication cascade that was originally identified in flies1. Signalling is mediated by Notch ligandreceptor interactions between neighbouring cells. Flies have a single typeI transmembrane-bound receptor that can be activated by two transmembranebound ligands named Serrate and Delta. Mammals possess four receptors (Notch14) that are bound by five ligands of the Jagged family and Delta-like family (Jagged1 and Jagged2, and Delta-like ligand 1 (DLL1), DLL3 and DLL4). The biochemical details of the canoni cal Notch signalling cascade have been comprehensively reviewed2,3 (BOX1). In recent years, evidence has been found of non-canonical Notch signalling that does not require the RBPJ transcriptional mediator complex. These non-canonical signal transduction pathways may occur in the absence of receptor cleavage or through crosstalk with other signalling pathways (including the nuclear factorB (NF-B), transforming growth factor- (TGF) and hypoxia-induced signalling pathways)46. Genome-wide expression and chromatin immuno precipitation (ChIP) studies suggest the existence of a large number of genes that can be regulated by Notch7,8. Despite the large number of potential Notch target genes, the best-characterized are the basic helixloop helix (bHLH) transcriptional repressors of the hairy enhancer of split (HES) and hairy-related (HRT) protein families9. As Notch signalling is recurrently either used for the generation and development of diverse blood cell lineages or used during peripheral immune responses following pathogenic infections, one of the major chal lenges is to identify the crucial driver target genes in these different settings in order to better understand how Notch exerts its pleiotropic functions. The best-studied functions of Notch signalling in haematopoiesis are its essential roles during lympho cyte development, in particular during Tcell lineage commitment and maturation in the thymus, and during marginal zone B (MZB) cell development in the spleen. More recently, Notch has also emerged as a key player in dendritic cell (DC) homeostasis and in the development of several lymphocyte subsets belonging to the innate immune system. In this Review, we discuss the role of Notch in the development of these specific blood line ages. Moreover, we highlight recent advances pertaining to Notch signalling in subsets of mature CD4+ and CD8+ Tcells in peripheral lymphoid tissues.
Ecole Polytechnique Fdrale de Lausanne, School of Life Sciences, Swiss Institute for Experimental Cancer Research, Lausanne, Vaud 1015, Switzerland. 2 Ludwig Center for Cancer Research of the University of Lausanne, Epalinges 1066, Switzerland. 3 Department of Biochemistry, WHO Immunology Research and Training Center, University of Lausanne, Epalinges 1066, Switzerland. e-mails: freddy.radtke@epfl.ch; HughRobson.Macdonald@ unil.ch; Fabienne.Tacchini-Cottier@ unil.ch doi:10.1038/nri3445 Published online 13 May 2013
1
Developmental roles for Notch Notch signalling in T cell and MZB cell development. Bone marrow progenitors seed the thymus via the bloodstream, where they are instructed to adopt a Tcell fate and further differentiate into Tcells or Tcells before emigrating to the periphery. The first insights of Notch function in this context were derived from complementary genetic lossoffunction and gainoffunction studies. Inducible inactivation of Notch1 or recombination signal binding protein for
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Box 1 | A brief overview of Notch signalling
Mammals possess four Notch receptors (Notch14) that are bound by five ligands of the Jagged, and Delta-like family (Jagged1 and Jagged2, and Delta-like ligand 1 (DLL1), DLL3 and DLL4). Newly synthesized receptors are proteolytically processed in the Golgi during their transport to the cell surface by a furin-like convertase. This results in the generation of heterodimeric receptors present at the cell surface (see the figure). Signalling is initiated by ligand binding to the receptors, which subsequently undergo two successive proteolytic cleavages; the first is mediated by disintegrin and metalloproteinase domain-containing protein (ADAM) family metalloproteinases at the extracellular S2 cleavage site close to the transmembrane domain. This results in the shedding of the extracellular part of the receptors, which are endocytosed by the ligand-expressing cell. This process requires monoubiquitylation of the cytoplasmic tail of the ligands by E3 ubiquitin ligases of the Mindbomb and Neuralized families. After a successful S2 cleavage and shedding of the extracellular domain, a last cleavage within the transmembrane domain is triggered by the secretase activity of a presenilin multi-protein complex, thus liberating the Notch intracellular domain (NICD). This is a rate-limiting step during Notch activation, which can be pharmacologically blocked by small-molecule -secretase inhibitors112. Once the NICD is liberated, it translocates to the nucleus and binds to the transcription factors of the recombination signal binding protein for immunoglobulin J region (RBPJ) family (also known as CSL in humans, Suppressor of hairless in Drosophila melanogaster, and LAG1 in Caenorhabditis elegans). When bound to RBPJ, the NICD recruits additional coactivators, including mastermind proteins (MAML13) and p300 in order to induce transcriptional expression of downstream target genes. Furthermore, Notch signalling is regulated at multiple levels. For example, Notch receptors undergo post-translational modifications by Fringe family glycosyltransferases. These transferases add Nacetylglucosamine to Ofucose residues present in certain epidermal growth factor (EGF) repeats of the extracellular domain of Notch receptors113. This influences the relative binding avidity of ligandreceptor pairs, which translates into different efficiencies or signalling strength of Notch receptors114116.
Mindbomb and Neuralized Notch 1 Notch 2 Notch 3 Notch 4 Cell membrane Fringe ADAM family protease S2 S3 -secretase complex Jagged 1 Jagged 2 DLL1 DLL3 DLL4
S1 cleavage site Furin-like convertase Cytoplasm RBPJ
Nucleus No transcription RBPJ Target gene transcription
Proteins shown in red are co-repressors of RBPJ; those shown in green are cofactors for RBPJ. Nature Reviews | Immunology
immunoglobulin J region (RBPJ; also known as CSL in humans) in bone marrow progenitors results in a complete block of Tcell development, accompanied by the accumulation of ectopic Bcells in the thymus10,11. By contrast, the constitutive expression of active forms of Notch induced ectopic Tcell development and sup pressed Bcell development in the bone marrow 12. Since then, multiple studies, including studies interfering with Notch signalling by transgenic expression of Notch
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modulators (such as Fringe proteins, Deltex1 and Notchregulated ankyrin repeat-containing protein (NRARP)) or studies expressing dominant-negative forms of the transcriptional co-activator Mastermind-like protein1 (MAML1), have confirmed the original findings1316. This led to a model in which Notch1 ensures Tcell lineage commitment by inhibiting the other multiple cell-fate potentials of thymus-seeding cells, including myeloid cell and Bcell potential, as well as conventional DC and plasmacytoid DC potential1720 (FIG. 1). Although the transcription factor HES1 was recently shown to be an important Notch mediator for Tcell lineage commit ment, conditional inactivation of HES1 does not lead to the accumulation of Bcells or DCs in the thymus21. This result suggests that Notch signalling specifies the Tcell lineage through the activation of additional downstream targetgenes. Although DLL1 and DLL4 can both instruct bone marrow cells to adopt a Tcell fate invitro, genetic ablation studies showed that invivo the instructive signal is triggered through the interaction of Notch1express ing thymus-seeding cells with DLL4expressing thymic epithelial cells2226. Notch signalling is highest in imma ture Tcells (including in early thymic progenitors (ETPs), double-negative 2 (DN2) thymocytes and DN3a thymocytes) up to the DN3 stage, at which cells have to pass a critical checkpoint known as -selection27. In these immature thymocytes, Notch1, but not HES1, is con tinuously required to restrict developing Tcells to the Tcell lineage21. However, once specified, Tcells are less dependent on Notch signalling, at least in the mouse. For human thymocytes the situation seems to be different, as Tcells require higher levels of Notch signalling compared with developing Tcells28,29. After thymocytes successfully pass selection, they immediately downregulate Notch 1 expression, a process that is triggered by the pre-Tcell receptor (preTCR)-mediated induction of the HLH transcription factor inhibitor of DNA binding 3 (ID3). ID3 inhibits E2Ainduced transcription of Notch1 (REF.30). As a consequence, double-positive (CD4 +CD8+) thymo cytes have very low levels of Notch signalling, which is presumably required to avoid interfering with positive and negative selection in double-positive thymocytes, as well as to avoid the oncogenic properties of Notch signalling and its targets8 (FIG.1). Another well-established role for Notch signalling dur ing lymphocyte development is its role in the specification of two different major subsets of splenic Bcells, namely MZB cells and follicular zone Bcells. Follicular zone Bcells participate in Tcell-mediated immune responses. These circulating Bcells represent the majority of Bcells within the spleen, where they localize to Bcell follicles, hence the name follicular zone Bcells. By contrast, MZB cells are found in the outer region of the splenic white pulp between the marginal sinus and the red pulp31. They are important in driving fast and vigorous Tcellindependent antibody responses to blood-borne patho gens32. Moreover, they express high levels of CD1d, thus allowing them to capture and present lipid antigens to invariant natural killer Tcells. Both MZB cells and follicular
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HSC Bone marrow MPP
TEC Notch 1 ETP DLL4 -selection
Thymus
DN2
DN3
DN4 Notch?
DP
CD4+ naive T cell
CMP
CLP
B cell DC
Myeloid cell
CD8+ naive T cell Thymus-derived TReg cell
Notch signalling switched o MDP B cell T1 B cell T2 B cell
Follicular B cell Marginal zone B cell
RBPJ transcriptional mediator complex

This is the assembly of proteins including RBPJ (known as CSL in humans), the Notch intracellular domain (NICD) and transcriptional coactivators such as Mastermind-like proteins (MAMLs), histone acetyltransferases and the mediator complex in order to generate an active transcriptional complex on target promoters.
CDP
Notch 2 RBPJ Pre-DC Intestinal lamina propria Notch signalling Notch 2
DLL1 Notch 2 RBPJ CD8ESAM+ DC
Pre-DC
cDC
pDC CD103 DC CD103+CD11b+ DC Spleen
-selection
During development, immature double-negative 3 thymocytes have to pass a critical checkpoint known as -selection, or the pre-Tcell receptor (pre-TCR) checkpoint, at which they have to signal via the pre-TCR to continue their development.
Pre-Tcell receptor
(Pre-TCR). The pre-TCR consists of a productively rearranged TCR chain associated with CD3 components and an invariant pre-TCR chain.
Invariant natural killer Tcells

These are a specialized subset of innate-like lymphocytes that share properties of both natural killer (NK) cells and Tcells. They express NK-related molecules and Tcell receptors (TCRs), and their TCRs recognize self and foreign lipids presented on CD1d molecules.
Figure 1 | Notch signalling in immune cell development. Bone marrow haematopoietic stem cells (HSCs) give rise to Nature Reviews | Immunology multipotent progenitors (MPPs) before differentiating into common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs). CLPs migrate from the bone marrow to the thymus, where thymic epithelial cells that express Delta-like ligand 4 (DLL4) trigger canonical Notch1 signalling in early thymic progenitors (ETPs). This Notch1 signal is essential for Tcell lineage commitment and is further required during early phases of thymocyte differentiation up to the double-negative 3 (DN3) stage. Active Notch signalling during these early stages of Tcell development inhibits other lineage potentials, such as Bcell and myeloid cell (including dendritic cell (DC)) potential. During selection, Notch signalling is turned off as a consequence of pre-T cell receptor signalling. Thus subsequent stages of Tcell development exhibit very low levels of Notch signalling. Notch was also suggested to influence the development of regulatory T (TReg) cells (specifically, thymic TReg cells). In bone marrow-residing CLPs, Notch signalling must be switched off to allow proper Bcell development. After migration of immature Bcells to the spleen, interaction of DLL1 with Notch2 (mediated by recombination signal binding protein for immunoglobulin J region (RBPJ)) induces Notch signalling in transitional B(T2) cells to specify marginal zone B cells as opposed to follicular Bcells. The vast majority of DCs are derived from CMPs in the bone marrow, which give rise to macrophageDC progenitors (MDPs). Subsequently, common DC progenitors (CDPs) develop into pre-DCs, seeding lymphoid and non-lymphoid organs via the bloodstream. In the spleen these pre-DCs are specified into multiple DC subsets, including classical DCs (cDCs) and plasmacytoid DCs (pDCs). Splenic CD8 endothelial cell-selective adhesion molecule (ESAM)+ DCs and CD103+CD11b+ DCs in the lamina propria of the intestine require Notch signalling mediated by the Notch2 receptor. DP, double-positive; TEC, thymic epithelial cell.
Bcells originate from Bcell progenitors in the bone mar row. When Bcells migrate out of the bone marrow, they colonize secondary lymphoid organs, including the spleen, where they further mature through transitional stages (known as T1 and T2), before ultimately giving rise to mature follicular B cells or MZB cells in thespleen.
The specification process of T2 Bcells into either of the two mature Bcell fates is influenced by multiple factors31. However, conditional gene-targeting experiments of mul tiple Notch components revealed that the specification of MZB cells is strictly dependent on DLL1mediated Notch2 signalling (FIG.1). Mice with conditional inactivation of
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DLL1 or Notch2 have severely decreased numbers of MZB cells33,34, a phenotype that was also observed in RBPJmutant35, MAML1-mutant^^36,37, Mind bomb1-mutant^^38 and disintegrin and metalloproteinase domain-containing protein 10 (ADAM10)39-mutant mice. Taken together, these genetic lossoffunction experiments strongly sug gest that this process is mediated via canonical Notch signalling, although, surprisingly, HES1 is not required21. Inactivation of Msx2interacting protein (MINT; also known as SPEN), which is a negative regulator of Notch signalling, resulted in increased MZB cell numbers40, which is an additional confirmation of Notch being an important regulator of MZB cell specification. Moreover, the cooperative action of two Fringe family members (Lunatic fringe and Manic fringe) seems to be necessary to strengthen the presumably weak interaction between MZB cell precursors and DLL1expressing splenic niche cells41. Although it is clear that DLL1 is the non-redundant ligand that triggers a Notch2 signal to specify MZB cells in the spleen, the nature of the DLL1expressing cells is still unclear. Although non-haematopoietic cells33 and, in particular, endothelial cells have been shown to express DLL1 in the red pulp of the spleen41, it remains to be shown whether DLL1expressing endothelial cells are indeed the splenic niche cells that support MZB cell development. Notch signalling in DC development. DCs are a subset of haematopoietic cells that are specialized in antigen presentation. Until recently the only evidence for Notch involvement in DC differentiation was based on invitro studies in which DC development could be influenced by the overexpression of Notch receptors or ligands and pharmacological manipulation by Notch inhibi tors. More recently, lossoffunction studies have pro vided conclusive evidence that Notch signalling plays an important part in DC development and homeosta sis. Conditional inactivation of RBPJ or Notch2 specifi cally in DCs led to a selective reduction of a subset of DCs in the spleen but not in other lymphoid tissues42,43. Splenic DCs comprise a plasmacytoid DC compartment as well as several subsets of conventional DCs that can be identified by their differential expression of CD8 and CD11b, together with their expression of other markers, such as the adhesion molecule endothelial cell-selective adhesion molecule (ESAM). In the absence of Notch2, only the CD8CD11b+ESAM+ DC subset was missing, whereas other DC subsets (both conventional and plas macytoid) were not affected43 (FIG.1). The requirement for Notch2 in the maintenance of splenic DCs seems to be non-redundant, as conditional inactivation of Notch1 either in DCs43 or in all haematopoietic cells44,45 does not affect DC numbers. The Notch ligand responsible for directing splenic DC development remains to be identified. However, it is noteworthy that the CD8CD11b+ESAM+ DC subset that depends on Notch2 signalling is localized to the splenic marginal zone in close proximity to stromal cells expressing DLL1 (REF.42). As MZB cell development in the spleen is strictly dependent on DLL1expressing stromal cells33 it is tempting to speculate that splenic DC development is likewise dependent on DLL1.
In addition to splenic DCs, a subset of DCs located in the lamina propria of the intestine is strongly reduced in the absence of Notch2. These DCs have a CD103+CD11b+ phenotype and are believed to be specialized in antigen capture and transport to the mesenteric lymph nodes, where they can activate CD4+ helper Tcells, and in par ticular those secreting interleukin17 (IL17): that is, T helper 17 (TH17) cells. Consistent with this model, TH17 cell numbers are also decreased in the intestine fol lowing DCspecific conditional inactivation of Notch2 (REF.43). Taken together, these recent studies reveal a novel and tissue-specific role for Notch2 in the homeo stasis of distinct DC subsets and consequent control of Tcell priming (FIG.1). Notch signalling in innate lymphoid cell development. More recently, the development of another set of inter esting immune cells, the innate lymphoid cells (ILCs), was reported to be influenced by Notch signalling. ILCs encompass a novel family of haematopoietic effector cells that have important functions in innate immune responses to infectious microorganisms, in the genera tion of secondary lymphoid organs and in tissue remod elling after tissue injury or infection. ILCs develop from common lymphoid progenitors, but unlike Bcells and Tcells they do not rearrange immunoglobulin genes or express antigen-specific receptors. In general they can be subdivided in three major subclasses (group1, group 2 and group 3 ILCs), depending on whether they express TH1type, TH2type or TH17type cytokines, respectively 46,47. Whereas natural killer (NK) cells, which belong to the group 1 ILCs, have cytotoxic activ ity and functions, lymphoid tissue-inducer (LTi) cells (a type of group 3 ILC) are essential for the development and generation of secondary lymphoid organs. LTi cell development is dependent on retinoid-related orphan receptor-t (RORt)48. In this context, Notch signalling was shown to be transiently required for the generation of fetal 47+ LTi cell progenitors before upregulation of RORt. Subsequently, Notch signalling must be down regulated again to allow the expression of RORt and the final maturation of LTi cells49. More recent additions to the RORt-dependent group3 ILC subclass are IL22producing ILCs, which have been referred to as ILC22s and which share hall marks of LTi cells and NK cells. The ILC22s are noncytotoxic but express the NK marker NKp46 and produce high levels of IL22, and are therefore also known as NK22 cells. They are mostly found in mucosal tissues (such as the lamina propria of the intestine, Peyers patches or ton sils) in humans and mice, where they induce early protec tive immune responses to colitis-inducing pathogens50,51. Both human and mouse ILC22s express the transcription factor aryl hydrocarbon receptor (AHR), which becomes activated by xenobiotics (that is, chemical compounds that are found in an organism but that are not normally produced by it, such as polycyclic aromatic compounds). On activation and ligand binding, AHR translocates from the cytoplasm to the nucleus and binds to pro moter regions containing so-called xeno biotic response elements in order to induce target gene expression.
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Group 1 ILC ILC1 (e.g. NK cells)
Group 2 ILC HSC Notch ROR+ Group 3 ILC CLP RORt+ IL-17-producing ILC RORt+ AHR Notch Notch Notch NKp46+ IL-22-producing ILC ILC2 (e.g. nuocytes)
B cell
T cell
47+ RORt
RORt+
LTi cell
Figure 2 | The role of Notch signalling in the development of innate lymphoid cells. Haematopoietic stem cell (HSC)-derived common lymphoid progenitors (CLPs) give rise Nature Reviews | Immunology to adaptive immune cells, such as T cells and Bcells, as well as to innate lymphoid cells (ILCs) that do not express antigen receptors. ILCs fulfil important functions in innate immune responses through their ability to generate and secrete different cytokines and/or to exhibit cytotoxic activity. They can be grouped into three major classes: group 1, group 2 and group 3 (REF.47). ILCs diverge in their requirement for Notch (as indicated). AHR, aryl hydrocarbon receptor; IL, interleukin; LTi, lymphoid tissue-inducer; NK, natural killer; ROR, retinoid-related orphan receptor.
progenitor (CLP) reconstitution assays revealed that nuocytes are derived from CLPs in the bone marrow. However, they can also be generated invitro under appropriate culture conditions. CLPs, along with DN1 and DN2 thymocytes, develop into nuocytes when cul tured on DLL1expressing OP9 cells in the presence of IL7 and IL33. In the absence of DLL1mediated Notch signalling, these progenitors develop into Bcells, sug gesting that Notch signalling is required at least for the invitro generation of nuocytes. It remains to be investi gated whether the invivo development or expansion of these cells is also dependent on or influenced by Notch signalling. Although DN1 and DN2 thymocytes retain the potential to develop into nuocytes invitro, forkhead box N1 (FOXN1)nu/nu mice (which lack a thymus) have normal nuocyte numbers; this indicates that invivo these cells are likely to develop at extrathymic sites, such as the bone marrow 58. Taken together, these studies reveal that Notch signalling can influence the development and/or expan sion of certain subsets of ILCs, which is probably also microenvironment dependent (FIG.2). Future studies will be necessary to more specifically address the invivo requirements and specific receptorligand interactions that are necessary for the development and function of certain ILC subsets.
Interestingly, both NOTCH1 and NOTCH2 promoters contain such xenobiotic response elements, and expres sion of their transcripts is induced invivo by the admin istration of an AHR ligand. Conditional inactivation of the transcription factor RBPJ, which mediates signalling downstream of all Notch receptors, resulted in a substan tial reduction of NKp46+ ILCs in the lamina propria of the intestine but not in Peyers patches. Similar results were obtained with Ahr-mutant mice52,53, suggesting that the development and/or expansion of NKp46+ ILCs in certain microenvironments is mediated by AHRinduced Notch signalling 54. Another RORt-dependent but NKp46negative sub set of ILCs is mostly found in the colon of mice during inflammation. These cells express high levels of IL17 in response to IL23, which is responsible for gut pathol ogy 55. Whether this particular subset of ILCs is also dependent on Notch is currently unknown. Nuocytes, which are also known as ILC2s because of their ability to secrete high levels of type2 cytokines, including IL5 and IL13, are ROR-dependent cells. They proliferate in response to IL25 and IL33 admin istration or in response to pathogens, including parasitic helminths, viruses and fungi5658. They have an impor tant role in type 2-mediated immunity. For example, activation of these cells by the administration of IL25 is sufficient to clear parasitic worms even in the absence of adaptive immunity 57. Invivo, common lymphoid
Notch and helper Tcell functions During immune responses, naive CD4+ Tcells encounter antigens in peripheral lymphoid organs. Recognition of cognate peptide antigens presented by antigen-presenting cells (APCs) triggers clonal Tcell expansion and their dif ferentiation into several functionally distinct CD4+ TH cell subsets. Each TH cell subset secretes a specific pattern of effector cytokines that coordinates immune responses against various types of pathogens and also has an impor tant role in autoimmune inflammatory diseases. The most well-characterized TH cell subsets include TH1 cells, TH2 cells59, TH17 cells60,61 and regulatory T (TReg) cells62, as well as the more recently described TH9 cells63,64 and follicu lar T helper (TFH) cells65,66. Although these TH cell subsets were long thought to be fixed lineages, some plasticity in the pattern of cytokines that they secrete may occur dur ing the course of infection, a process that would allow a specific TH cell subset to react to changing environmental conditions67,68. Among the several factors contributing to the differentiation of naive CD4+ Tcells towards a given TH cell subset, accumulating data indicate a crucial role for Notch signalling. However, the mechanisms involved have been somewhat controversial6971. We discuss below recent advances in our understanding of the role for Notch in the differentiation and/or function of CD4+ THcells.
Role of Notch ligands in TH cell differentiation and function. The induction of specific Notch ligands by pathogen-derived signals has a profound impact on the differentiation or function of CD4+ T helper cells. A cor relation between the type of Notch ligand expressed on APCs and the development of TH1 and TH2 cells was first reported by Flavell and colleagues72 and further extended by different groups to these and other TH cell
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subsets. Studies carried out both invitro and invivo convincingly showed that the engagement of Delta-like Notch ligands favours the development of interferon- (IFN)-secreting TH1 cells, whereas the engagement of Jagged ligands preferentially induces the development of TH2 cells and TReg cells (reviewed in detail in REFS71,73). The type of Notch ligands involved in the differentiation of other TH cell populations awaits further studies. The impact of Notch on TH1 cell function invivo. The impact of Notch receptor triggering on TH cell dif ferentiation or function was first investigated using welldefined invitro TH cell polarization conditions. The numerous studies performed did not provide conclusive evidence for a role for Notch signalling during TH cell differentiation69,71. The importance of Notch signalling on TH cell differentiation or function is better documented by an increasing number of studies carried out invivo. The role of Notch in the differentiation of IFNsecreting TH1 cells invivo has been investigated using several approaches designed to inhibit Notch signalling. Treatment of mice with secretase inhibitors impeded disease progression in TH1 cell-mediated experimental autoimmune encephalomyelytis (EAE)74,75. It was hypoth esized that Notch1 could bind to an RBPJ-binding sequence on the T-box 21 (Tbx21) gene promoter, which encodes Tbet, the master regulator of TH1 cell differen tiation74; however, such binding of Notch to the Tbx21 gene was not confirmed in another study 76. Targeting secretase may not only affect Notch cleavage but may also affect the cleavage of many other targets that could affect TH cell differentiation independently of Notch signalling, emphasizing the importance of using vari ous experimental approaches to block Notch signalling. In contrast to the results obtained following secretase treatment, functional TH1 cells could develop in response to infection in mice that do not activate RBPJ because of dominant-negative MAML expression77. In the same line, mice deficient for RBPJ expression in their Tcells were also able to mount a protective TH1 cell response following infection with the parasite Leishmania major 78. Altogether, these studies suggest that TH1 cell differentia tion does not involve canonical Notch signalling, defined as the association of the Notch intracellular domain (NICD) with RBPJ in the nucleus. Several partners could interact with Notch during non-canonical signalling in peripheral Tcells. Subunits of the NFB transcription factor were identified as potential Notch partners79,80, and further research is needed to characterize the interactions of Notch with NFB (FIG.3a). To further study the impact of Notch on TH cell dif ferentiation, Notch receptors were inactivated by genetic deletion in Tcells or were blocked using monoclonal antibodies directed against Notch1 or Notch3. In a model of passively transferred EAE, the adoptive transfer of lymph node cells treated with Notch 3specific neutralizing antibodies, but not with Notch1specific neutralizing antibodies, reduced the release of IFN and IL17by myelin-reactive cells, with a correspond ing decrease in the EAE disease scores75. Of note, it was not possible to determine in this study whether the lower levels of IFN produced resulted from impaired TH1 cell differentiation and/or from impaired TH1 cell function. Expression of Notch1 (and compensatory expression of Notch2 in the absence of Notch1) on Tcells is crucial for the differentiation of functional IFN-secreting TH1 cells during infection with L.major. Following parasite inoculation, Notch-deficient naive CD4+ Tcells dif ferentiated into TH1 cells expressing Tbx21 mRNA and IFN proteins, but these cells were unable to secrete IFN78 (FIG.3a). These data suggest that Notch signalling is involved in the control of TH1 cell effector functions, rather than in the differentiation of TH1 cells. Thus, Notch1, Notch2 and Notch3 have each been individu ally shown to affect the functions of TH1 cells invivo, and it is possible that different Notch receptors may be involved in different TH1type contexts. The impact of Notch on TH2 cell differentiation invivo. Regulation of TH2 cell differentiation invivo by Notch was documented in several experimental models81,82. In contrast to what was reported in TH1 cell differentiation, Notch signalling in TH2 cell differentiation was shown to be dependent on RBPJ (canonical Notch signalling). Several RBPJ-binding sites were identified on the Il4 enhancer (conserved non-coding sequence 2 (CNS2); also known as HS5), suggesting a direct role of Notch in IL4 transcription in TH2 cells, NKT cells and possibly other IL4secreting cells72,82. As IL4 is a master regulator of TH2 cell differentiation, Notch signalling may thereby promote TH2 cell differentiation by both cell-intrinsic and cell-extrinsic mechanisms. In addition to its direct regu lation of Il4, Notch was reported to bind to the promoter of GATA-binding factor 3 (GATA3), a master regulator of TH2 cell differentiation83, thus inducing the expression of the Gata3 exon 1a transcript 83. Both Notch1 and RBPJ were shown to bind close to the Gata3 promoter, and GATA3 activity was required for the Notch-dependent induction of IL4, suggesting a synergy between both pathways76,81. Expression of GATA3 from the exon 1b transcript was shown to be independent of Notch. The conditions determining the selective usage of either GATA3 exon 1a or exon 1b transcripts and the need for Notch in TH2 cell differentiation remain to be defined and are likely to be context dependent (FIG.3b). The impact of Notch on other TH cell subsets invivo. The Notch signalling pathway was also shown to cooperate with TGF to induce TH9 cells84. Interaction between SMAD3, a TGF target protein, and RBPJ resulted in the induction of the IL9 promoter. Mice deficient in Notch1 and Notch2 in their Tcells developed milder EAE compared with control mice, and both IL9 and IL17 cytokine production was decreased upon anti genic re-stimulation invitro. This suggested that Notch signalling is regulating TH9 cell function during EAE. However, although the impact of Notch signalling on EAE is well documented, it is difficult to evaluate the impact of Notch on individual TH subsets during EAE owing to the plasticity among TH subsets (TH17, TH1, TH9 and TReg cells) that develops during the disease85.
secretase inhibitors
These are small-molecule inhibitors that block the S3 cleavage of Notch receptors, thereby inhibiting the liberation of the Notch intracellular domain (NICD) and the activation of the Notch signalling cascade.
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a TH1 cell dierentiation
APC TH1-inducing stimuli DLL1 or DLL4 Notch 1 Notch 2 S2 S3 IFN IL-4 IL-4R TH2-inducing stimuli Jagged 1 or Jagged 2 Notch 1 Notch 2
b TH2 cell dierentiation

APC
Cytoplasm ? STAT6
NF-B Nucleus p50 p65 Ifng IFN RBPJ CoA Gata3 (exon 1a) GATA3 RBPJ CoA Il4 IL-4 IL-5 IL-13 Il5, Il13
GATA3 Gata3 (exon 1b)
Figure 3 | Role of Notch in T helper 1 and T helper 2 cell differentiation and function. T helper (TH) cell-promoting signals induce the expression of Notch ligands (Delta-like ligands (DLLs) or Jagged) on antigen-presenting cells (APCs). Nature Reviews | Immunology a|TH1 cell-promoting signals induce the expression of DLLs and the release of the Notch intracellular domain (NICD), which can bind to the nuclear factor-B (NF- B) family proteins p50 and p65. In addition, the NICD can control the release of interferon- (IFN) either directly or indirectly. b | TH2 cell-promoting signals induce the expression of Jagged ligands and the release of the NICD, which interacts with recombination signal-binding protein for immunoglobulin J region (RBPJ), converting it to a transcriptional activator. RBPJ recruits co-activators (CoAs) and the complex binds and transactivates the promoter of GATA binding protein 3 (GATA3), transcribing exon 1a. Interleukin4 (IL4) can also initiate TH2 cell differentiation by triggering signal transducer and activator of transcription 6 (STAT6), which induces the transcription of Gata3. Transcription of Gata3 exon 1b is Notch independent. Gata3 and Il4 expression reinforce GATA3 expression. GATA3 modifies the conformation of the Il4, Il5 and Il13 loci, allowing their transcription. Notch 1 is the predominant pathway used (indicated in bold). Dashed arrows represent hypothetical pathways. IL4R, IL4 receptor.
Altogether, increasing evidence reveals that Notch signalling considerably influences the development of immune responses by acting on the differentiation or function of different TH subsets (FIG.3). Better under standing of the mechanisms involved in these processes will allow the design of appropriate strategies to favour or prevent the development of a TH cell subset during pathologies in which these Tcells have importantroles.
Notch and regulatory Tcell functions TReg cells have important functions in the maintenance of peripheral self-tolerance and in the modulation of vari ous polarized TH cell immune responses. There are two main types of TReg cell: TReg cells that develop in the thymus (thymic TReg cells) and TReg cells that develop in the periph ery from naive CD4+ Tcells (peripherally induced TReg cells). Common features of TReg cells are their expression of the transcription factor FOXP3 and their suppression or control of pro-inflammatory immune responses86.
The first indications that Notch signalling could be involved in TReg cell function originated from studies showing that CD4+ splenic Tcells positive for CD25, a molecule expressed by TReg cells, express higher levels of Notch3 receptor than do CD4+CD25 wild-type cells, and that transgenic expression of intracellular, constitu tively active Notch3 in Tcells induced the accumula tion of TReg cells in the thymus and periphery, leading to protection against experimentally induced autoimmune diabetes in mice87. Further studies by this group reported that Notch3 promoted the development of TReg cells and improved their suppressive activity by upregulating FOXP3 expression88,89. In a more physiological setting, TReg cell exposure to Jagged2expressing haematopoietic progenitors induced the activation of Notch3 signal ling and promoted the expansion of TReg cells that could prevent disease onset in an experimental type1 diabetes model90. Collectively, these studies indicate an important role for Notch signalling in the expansion of TRegcells.
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Neutralization of DLL4 during the induction of EAE resulted in an increase in the number of peripherally induced TReg cells with a corresponding decrease in dis ease severity 91. The effect was specific to DLL4, as DLL1 blockade had no effect on TH17 and TReg cell differen tiation during EAE92. In addition, a more recent study showed that antiDLL4 treatment prevented the devel opment of, and even reverted, type1 diabetes in nonobese diabetic (NOD) mice by inducing an increase in the number of DCdependent denovo thymic TReg cells93. Taken together, these results demonstrate an essential role for DLL4mediated immune regulation in the con trol of both thymic TReg cell and peripherally induced TReg cell homeostasis, although mechanistic details remain to be elucidated. Blocking Notch ligands could be a strategy to selectively regulate TReg cell prolifera tion. Increasing data suggest that the selective blocking of DLL4mediated Notch signalling ameliorates multiple forms of experimental autoimmunity via the regulation of TRegcells (BOX2). Jagged-mediated Notch signalling is also important in the control of peripherally induced TReg cell differen tiation. TGF induces FOXP3 expression in peripheral naive Tcells, allowing their differentiation into periph erally induced TReg cells62,94. A role for Notch signalling in peripherally induced TReg cell differentiation was first suggested by several experiments showing that over expression of the Notch ligand Jagged1 on APCs led to peripherally induced TReg cell differentiation both invitro and invivo95,96. The peripheral induction of TReg cells by Notch signalling was shown to involve the activation of FOXP3, the master transcription factor of TReg cells. Notch1 co operates with SMAD3 (a mediator of TGF signalling) and RBPJ to activate FOXP3 transcription5,97. Accordingly, invivo treatment of mice with secretase inhibitors reduced the TGF-mediated induction of FOXP3, decreased peripherally induced TReg cell devel opment and maintenance, and led to the development of autoimmune hepatitis5. The importance of Notch signal ling in ensuring sustained FOXP3 expression and main tenance of peripherally induced TReg cells was recently confirmed in human cells98. Pluripotent stem cells trans duced with FOXP3 and cocultured on Notch ligandexpressing stromal cells generated stable TReg cells invitro99, suggesting that this could be a strategy for producing stable TReg cells for therapeutic adoptive transfer. Collectively, increasing experimental evidence demonstrates a crucial role for Notch signalling in the expansion of thymic TReg cells, in the differentiation of peripherally induced TReg cells and in the maintenance of both of these TReg cell populations. However, more direct lossoffunction experiments will be required to validate these conclusions.
Box 2 | Notch and autoimmune disease

Autoimmune diseases such as multiple sclerosis and type1 diabetes arise from inappropriate immune responses against self antigens, leading to the selective destruction of particular cell types or tissues. These inappropriate immune responses involve both adaptive and innate immune cells. As Notch signalling is involved in the regulation of peripheral immune responses, multiple groups have investigated the outcome of manipulating Notch signalling in experimental autoimmune disorders. Experimental autoimmune encephalomyelitis (EAE) is a frequently used mouse model for multiple sclerosis117. This inflammation-mediated demyelination disease of the central nervous system (CNS) can be induced by immunization with myelin antigens, viral infection or transfer of autoreactive Tcells. Genetic and pharmacological interference with Notch signalling, as well as administration of blocking antibodies against Notch receptors or Delta-like ligand 4 (DLL4), impeded progression of the disease, resulting in reduced clinical severity74,75,84,91,92,118119. The mechanisms by which blockage of Notch signalling ameliorates EAE are not fully understood and require further investigation. Possibilities include impaired T helper 1 (TH1)-type and TH17-type immune responses91; impaired migration of antigen-specific CD4+ Tcells to the CNS as a consequence of downregulation of chemokine receptors119; and/or promotion of regulatory Tcell development91. Similar observations have been reported in a more recent study using non-obese diabetic (NOD) mice as a model for type1 diabetes. In these mice, pancreatic cells are selectively destroyed through autoreactive Tcells. Administration of DLL4specific antibodies prevented the development of type1 diabetes and in some cases even caused the reversion of already established disease by inducing denovo expansion of regulatory Tcells93. Taken together, these preclinical studies indicate that selective blocking of DLL4mediated Notch signalling may ameliorate multiple forms of autoimmunity.
Notch, cytotoxic Tcells and GVHD Notch signalling promotes cytotoxic T lymphocyte differentiation. Both CD4+ and CD8+ Tcells are required to eliminate many intracellular pathogens. Upon recog nition of antigens presented by MHC classI-expressing cells, naive CD8+ Tcells multiply and differentiate into cytotoxic T lymphocytes (CTLs). Effector functions of CTLs include the secretion of IFN, the lysis of target cells using perforins and granzymes, and the induction of target cell apoptosis through FASFAS ligand (FASL) interactions. A role for Notch in the differentiation of CD8+ Tcells is supported by several studies. The ligation of Notch ligands was shown to affect the differentiation of CTLs. DLL1 ligation by Notch expressed on splenic CD8+ Tcells changed their patterns of cytokine secretion, decreasing their production of IFN and increasing their production of IL10 (REF.100). In addition, CD8+ Tcells exposed to allogenic DCs transduced with DLL1 showed increased granzymeB production and lysed target cells more efficiently invitro, suggesting that DLL1induced signalling contributes to CTL differentiation invivo101. DLL1 blockade similarly decreased the frequency of granzymeBproducing CTLs, and lower cytotoxic activity of CD8+ Tcells was observed in a transplantation model102. Notch2 expression on CD8+ Tcells was reported to promote CTL differentiation and to directly regulate granzyme B and perforin expression both invitro and invivo, demonstrating a crucial role for Notch signal ling in CD8+ Tcell cytotoxic responses. Notch2 formed a complex with phosphorylated cyclic AMP-responsive element-binding protein 1 (pCREB1) and the transcrip tional co-activator p300 on the granzyme B (Gzmb) pro moter 101. The same group later reported that Notch2 signalling, but not Notch1 signalling, was required for the generation of antitumour CTL responses 103. Further indication for a role of Notch signalling in CTL effector functions was reported using secretase inhibitor treatment, which blocked the expression of the Tbox eomesodermin (EOMES) transcriptional factor, thus reducing perforin and granzyme B expression.
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Treatment with secretase inhibitors did not affect the FASFASL interactions of CTLs. Notch1 antisense (Notch1 AS) mice, which have reduced expression of Notch1, also showed reduced EOMES, perforin and granzyme B expression, suggesting that Notch1 signal ling is involved in CTL differentiation104. The nature of the Notch receptor involved (Notch1 or Notch2) may vary depending on the experimental context. A recent study reported a role for Notch also in the generation and function of human CD8+ Tcells, suggesting that its role in this particular subset of Tcells is conserved105. Collectively, these experiments show an important role for Notch signalling in the differentiation of func tional CTLs. The implication of Notch signalling in the generation of memory CD8+ Tcells has not been investigated yet and should be of interest. Notch signalling in GVHD. More recent evidence also suggests a role for the Notch cascade in regulating graftversus-host disease (GVHD) following allogeneic bone marrow transplantation (allo-BMT). Patients with leu kaemia and patients with lymphoma have to undergo allo-BMT in cases in which tumour cells cannot be eradi cated by chemotherapy. The goal of allo-BMT is that transplanted donor Tcells mediate graft-versus-tumour (GVT) activity and thereby kill the cancer cells. However, GVT activity is often associated with GVHD because donor Tcells also react against normal host tissue. This represents one of the major complications of allo-BMT in patients with cancer. Although these patients can subsequently be treated with immunosuppressive drugs to decrease the risk of GVHD, it comes at the price of impairing GVT activity. Such patients have a higher risk of undergoing tumour relapse, which compromises their overall survival106. In this context, preclinical studies were used to evalu ate the function of Notch signalling in Tcells in mouse models of allo-BMT settings using genetic lossof function approaches. Expression of a dominant-neg ative form of MAML1 (which blocks canonical Notch signalling of all receptors) in donor Tcells resulted in near complete protection from acute GVHD in multi ple models of allo-BMT (including major antigen mis matched models, such as transplantation of C57Bl/6 bone marrow into BALB/c hosts). More importantly, these Notch signalling-incompetent Tcells retained cyto toxic and anti-leukaemic activity, leading to substantially improved overall survival of host animals challenged with a Bcell-lineage lymphoma. The protection against GVHD in this experimental setting was not mediated by an overall immunosuppression, as the Notch-deficient alloreactive Tcells exhibited normal invivo proliferative responses107. However, Tcells expressing the dominantnegativeMAML1 mutant or lacking RBPJ produced reduced levels of multiple inflammatory cytokines, including IFN, IL4, IL17, tumour necrosis factor and IL2, compared with wild-type Tcells. Interestingly, the levels of Tbet and EOMES, which are, respectively, master regulators of TH1 cell and effector CD8+ Tcell differen tiation, were unchanged in Notch signalling-deficient Tcells107. This argues against a simple differentiation
defect of helper or effector Tcells as a consequence of loss of Notch signalling. A more recent study from the same group in which blocking antibodies were used revealed that the beneficial effect of inhibiting Notch signalling in models of GVHD is mediated via the specific blockade of Notch1 and Notch2 on the receptor side and DLL1 and DLL4 on the ligand side. Blockage of individual recep tors and ligands revealed dominant effects for Notch1 and DLL4. Importantly, the combined administration of DLL1specific and DLL4specific antibodies pro vided long-lasting protection against GVHD without any apparent gut toxicities, such as those observed using blocking antibodies against the Notch receptors. The pro tection correlated with the persistent expansion of TReg cells108. Although the elucidation of the precise mecha nism requires further investigation, the impressive effects of blocking Notch signalling in mouse models of GVHD indicate a strong potential for clinical translation. In addition to the role of Notch in alloreactive Tcells, recent work suggests that Notch signalling in DCs can also influence GVHD109. Ikaros is a transcriptional repressor that in some contexts functions as a nega tive regulator of Notch signalling 110,111. Ikaros-deficient bone marrow chimaeras revealed an enhanced GVHD in multiple models of allo-BMT compared with con trol animals. Ikaros deficiency resulted in upregulation of multiple Notch receptors, ligands and Notch target genes in DCs. Allogeneic Tcells proliferated more when cocultured with Ikaros-deficient DCs compared with when they were cocultured with wild-type DCs, but this increased proliferation was reverted following the treat ment of Tcells with secretase inhibitors to block Notch signalling. More importantly, allogeneic Ikaros-deficient bone marrow chimaeras treated with secretase inhibitors invivo showed a significantly diminished GVHD pathology compared with vehicle-treated con trol chimaeras109. It remains to be investigated whether Notch signalling in wild-type DCs influences GVHD. However, taken together, these reports show that block ing Notch signalling ameliorates GVHD in allo-BMT models, whereas GVT activity is preserved.
Conclusion and perspectives During the past decade the Notch cascade has emerged as an important regulator of multiple cell fate decisions and differentiation processes during the development and function of the haematopoietic system. Among the most well-established functions of Notch are its essential roles in the specification and maturation of Tcells, as well as of MZB cells. For these two lymphoid lineages, the relevant receptorligand pairs have been identified by conditional genetic lossoffunction approaches. T cell lineage com mitment and maturation is mediated by DLL4Notch1 interactions, and MZB cell development is mediated by DLL1Notch2 interactions. Both of these processes use canonical (that is, RBPJdependent) Notch signal ling. More recently, a role for Notch during development and/or expansion of ILCs has been identified. The role of Notch in ILCs has been inferred from multiple stud ies through the requirement of DLL-expressing feeder cells to generate ILCs invitro. However, conditional
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inactivation of RBPJ has clearly demonstrated that Notch signalling is required invivo for the development and/or expansion of NKp46+ ILCs within the lamina propria of the intestine. Additional lossoffunction studies will be required in the future to establish the invivo relevance of Notch in ILCs, as well as for elucidating the ligandreceptor interactions that are involved. Genetic lossoffunction experiments also show an important role of Notch signalling in both TH2 cell dif ferentiation and TH1 cell function. Experimentally, Jagged or DLL expression on APCs has been associated with TH2 cell and TH1 cell differentiation, respectively. Genetic, pharmacological and antibody-based block age of specific ligands and receptors confirmed the role of DLL-mediated Notch signalling in TH1 cell function. Interestingly, this is a process that does not require canoni cal RBPJ-mediated signalling. By contrast, TH2 cell differ entiation is dependent on canonical Notch signalling. It is
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Immunol. 25, 139170 (2007). 107. Zhang,Y. etal. Notch signaling is a critical regulator of allogeneic CD4+ Tcell responses mediating graftversus-host disease. Blood 117, 299308 (2011). 108. Tran,I.T. etal. Blockade of individual Notch ligands and receptors controls graft-versus-host disease. J.Clin. Invest. 123, 15901604 (2013). This study shows that blocking Notch signalling in donor Tcells significantly reduces GVHD in mouse models of allo-BMT while preserving GVT activity. 109. Toubai,T. etal. Ikaros-Notch axis in host hematopoietic cells regulates experimental graftversus-host disease. Blood 118, 192204 (2011). 110. Dumortier,A. etal. Notch activation is an early and critical event during Tcell leukemogenesis in Ikarosdeficient mice. Mol. Cell. Biol. 26, 209220 (2006). 111. Gomez-del Arco,P. etal. Alternative promoter usage at the Notch1 locus supports ligand-independent signaling in T cell development and leukemogenesis. Immunity 33, 685698 (2010). 112. Wolfe,M. S. -secretase inhibitors as molecular probes of presenilin function. J.Mol. Neurosci. 17, 199204 (2001). 113. Haltiwanger,R.S. & Stanley,P. Modulation of receptor signaling by glycosylation: fringe is an Ofucose1,3 Nacetylglucosaminyltransferase. Biochim. Biophys. Acta 1573, 328335 (2002). 114. Bruckner,K., Perez,L., Clausen,H. & Cohen,S. Glycosyltransferase activity of Fringe modulates Notch- interactions. Nature 406, 411415 (2000). 115. Yang,L.T. etal. Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by 1 and Jagged1. Mol. Biol. Cell 16, 927942 (2005). 116. Besseyrias,V. etal. Hierarchy of Notch- interactions promoting T cell lineage commitment and maturation. J.Exp. Med. 204, 331343 (2007). 117. Baxter,A.G. The origin and application of experimental autoimmune encephalomyelitis. Nature Rev. Immunol. 7, 904912 (2007). 118. Takeichi,N. etal. Ameliorating effects of antiDll4 mAb on Theilers murine encephalomyelitis virusinduced demyelinating disease. Int. Immunol. 22, 729738 (2010). 119. Reynolds,N.D., Lukacs,N.W., Long,N. & Karpus,W.J. -like ligand 4 regulates central nervous system T cell accumulation during experimental autoimmune encephalomyelitis. J.Immunol. 187, 28032813 (2011).
Acknowledgements
The work in the authors laboratory is supported in part by the Swiss National Science foundation, the Swiss Cancer League and OptiStem.
FURTHER INFORMATION
Freddy Radtkes homepage: http://radtke-lab.epfl.ch Fabienne Tacchini-Cottiers homepage: http://www.unil.ch/ib/page9491_en.html
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Immunology of age-related macular degeneration

Jayakrishna Ambati1,2, John P.Atkinson3 and Bradley D.Gelfand1,4,5
Abstract | Age-related macular degeneration (AMD) is a leading cause of blindness in aged individuals. Recent advances have highlighted the essential role of immune processes in the development, progression and treatment of AMD. In this Review we discuss recent discoveries related to the immunological aspects of AMD pathogenesis. We outline the diverse immune cell types, inflammatory activators and pathways that are involved. Finally, we discuss the future of inflammation-directed therapeutics to treat AMD in the growing aged population.
Retina
A highly organized and specialized neural network where light is converted into electrical impulses. Diseases of the retina such as age-related macular degeneration are leading causes of blindness in the developed world.
Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, Kentucky 40506, USA. 2 Department of Physiology, University of Kentucky, Lexington, Kentucky 40506, USA. 3 Division of Rheumatology, Department of Medicine, Washington University, St. Louis, Missouri 63110, USA. 4 Department of Biomedical Engineering, University of Kentucky, Lexington, Kentucky 40506, USA. 5 Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40506, USA. Correspondence to J.A. email: jamba2@email.uky.edu doi:10.1038/nri3459
1
Optimal vision requires a high-functioning central retina, in particular the photoreceptor-dense macula (FIG.1), which is responsible for the fine visual acuity that is required for tasks such as reading, facial recognition and driving. Age-related macular degeneration (AMD) refers to the progressive degeneration of the macula that commonly occurs in people over 60years of age. More than 30 million individuals worldwide suffer from visual impairment as a result of AMD, which is estimated to account for more than US$300billion in annual economiccosts1. The study of AMD has, in many respects, been at the forefront of research into complex diseases. The discovery of the Tyr402His polymorphism of complement factorH (CFH) that confers increased statistical risk of developing AMD was the first of its kind for a complex disease25. Furthermore, multiple biological therapies that target vascular endothelial growth factor A (VEGFA) in neovascular AMD (also known as exudative or wet AMD) have recently revolutionized the clinical management of this disease. However, despite advances in the understanding, diagnosis and treatment of AMD in the past decade, disease prevalence increases with the ageing human population. The next decade will see a steady increase in the incidence and economic cost ofAMD1. Despite the fact that AMD is presumed to have a multifaceted aetiology, immune dysfunction is a recurring theme in its pathogenesis. In this Review, we discuss the fundamental concepts and current ideas of AMD pathogenesis, with a particular focus on several recent advances in the immune aspects of the disease. We begin by describing the normal structure of the retina and the characteristic features of AMD. Next, we focus on the major immunological processes that have
important roles in AMD development and we discuss the inflammatory component of neovascular AMD. We then present an integrated model of the immune modulation of AMD pathogenesis. Finally, we summarize the possibility of using immune-based therapeutics to prevent and treat AMD development. The aim of this Review is to present an uptodate discussion of recent immunological findings in AMD. Consequently, we have condensed the discussion of the complement pathway, which is the most thoroughly studied immune pathway in AMD; for a more comprehensive summary of complement biology in AMD, the reader is referred to other reviews69.
Structure of the retina The sensory retina is organized into alternating layers of neuronal extensions and cell nuclei. The conversion of light into chemical signals occurs in the outer segments of the cone and rod photoreceptors in the posterior of the neural retina (FIG. 1). Encoded visual information is integrated through retinal networks and ultimately travels to the optic nerve. Blood is supplied to the neural retina by retinal blood vessels that originate from the central retinal artery. Transport across retinal blood vessels is controlled by endothelial tight junctions, which constitute the inner bloodretinal barrier. Next to the photoreceptor layer is a highly organized monolayer of retinal pigmented epithelium (RPE), which has several roles in supporting the metabolically active photoreceptor layer. These include recycling biochemical by-products of photoreception through the phagocytosis of photoreceptor outer segments, supplying trophic factors such as VEGFA10 and maintaining the integrity of the outer bloodretinal barrier through tight junctions. Thus, the integrity of the RPE is essential for homeostasis
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a Normal retina
Ganglion cell
Macula
A specialized region of the retina densely populated with cone photoreceptors, which is responsible for fine visual acuity. Degeneration of the macular photoreceptors following either atrophy of the retinal pigmented epithelium (geographic atrophy) or fluid leakage from choroidal neovessels (neovascular age-related macular degeneration (AMD)) is the cause of vision loss in AMD.
c Early or intermediate dry AMD
Microglial cell Bipolar cell Horizontal cell
Photoreceptor Retinal pigmented epithelium Bruch membrane Choroidal vasculature
Drusen Macrophage
Complement factor H
(CFH). A negative regulator of alternative complement pathway activation. Single nucleotide polymorphisms in CFH that reduce its inhibitory potential are responsible for a substantial proportion of the genetic risk for the development of age-related macular degeneration.
b Cross-section of human eye d Geographic atrophy

Cornea Lens Pupil
e Neovascular AMD
Neovascular AMD
(also known as exudative or wet age-related macular degeneration). Characterized by degeneration of the macula following fluid leakage from choroidal neovessels that have invaded the retina. The use of vascular endothelial growth factor Atargeted therapies has revolutionized the management of this disease, which accounts for the majority of blindness that results from AMD.
Optic nerve
Retina Macula New blood vessels
Photoreceptors
Specialized neurons that are responsible for the conversion of light into biochemical signals.
Bloodretinal barrier
A tightly controlled transport barrier that is maintained by tight junctions in the retinal capillary endothelium (comprising the inner blood retinal barrier), and by the Bruch membrane and the retinal pigmented epithelium monolayer (comprising the outer bloodretinal barrier). Integrity of the bloodretinal barrier is important for control of fluid leakage, solute transport and immune quiescence, all of which support the functional homeostasis of the retina.
Figure 1 | Retinal anatomy in health and disease. The retinal anatomy is composed of several layers (part a); and a cross-section of the human eye (part b) shows focusing of light into the macular area, whichNature is a dense collection of Reviews | Immunology retinal photoreceptors. Normal retinal architecture (part a) is comprised of (from anterior to posterior) a ganglion cell layer, resident retinal microglia, bipolar cells, horizontal cells, the photoreceptor layer, the retinal pigmented epithelium (RPE), the Bruch membrane and a choroidal vascular network. Normal microglia migrate into and out of the subretinal space (as shown by the dashed arrow). Early or intermediate dry age-related macular degeneration (AMD) (part c) is associated with the accumulation of subretinal drusen and microglia and of choroidal macrophages and a thickened Bruch membrane. Geographic atrophy (part d) is the advanced form of dry AMD, which is characterized by confluent regions of RPE and photoreceptor degeneration as well as constriction of choroidal blood vessels. Neovascular AMD (part e) is characterized by the invasion of abnormal, leaky choroidal blood vessels and accompanying macrophages into the retina through breaks in the Bruch membrane, which leads to photoreceptor degeneration.
in the retina. Posterior to the RPE is the Bruch membrane a thick, elasto-collagenous extracellular matrix. Together, the RPE and the Bruch membrane form the outer bloodretinal barrier, which prevents the entrance of macromolecules and immune cells from the underlying choroid into the photoreceptor layer. Located behind the Bruch membrane, the choroid contains a
dense network of blood vessels (choriocapillaris), which supplies oxygen and nutrients to the RPE, outer retina and optic nerve. The choroid endothelium is fenestrated, which enables the transport of molecules to the metabolically demanding RPE11. The choroid also contains tissue-resident melanocytes, fibroblasts, macrophages, mast cells and dendriticcells.
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Box 1 | The pro-inflammatory components of drusen
Drusen are extracellular deposits that accumulate between the retinal pigmented epithelium (RPE) and the Bruch membrane, or sometimes between the RPE and the photoreceptors117. The RPE is a major source of drusen components, and extracellular and serum-derived factors are also highly abundant12. RPE monocultures can be made to produce drusen-like deposits and to release factors that are abundant in drusen, such as vitronectin, clusterin, serum amyloid P, complement proteins and apolipoprotein E (APOE)118. Local drusen biogenesis probably occurs as a result of a failure of lysosome- and autophagy-mediated digestion of cellular components119. The accumulation of waste material is eventually extruded in exosomes and might function as nucleating sites for the deposition of other extracellular factors51. Data on the genetic influence of drusen formation have not conclusively determined whether complement dysregulation has a role in this process 22,120,121. Furthermore, drusen also contain components of dendritic cell processes, which supports a role for local immunity in their biogenesis122. Drusen consist of aggregated intracellular, extracellular and secreted proteins, and lipids and cellular components. Proteomic, histological and biochemical analyses show that the major components of human drusen include albumin, APOE, complement factors and related proteins (including complement components C1q, C3, C5 and C5b9, and vitronectin), immunogloblulins and amyloid123125. Interestingly, the protein content of drusen from the eyes of individuals who do not have age-related macular degeneration (AMD) is similar to that from the eyes of individuals with AMD123. The proteins in drusen commonly contain carboxyethylpyrrole modifications123. Drusen also contain double-stranded RNAs60. Drusen extracts can induce inflammasome activation58, and individual components of drusen can experimentally induce profound cellular effects: for example, C1q induces inflammasome activation58 and amyloid induces retinal degeneration43. However, because drusen are solid, insoluble deposits, it is difficult to infer the insitu activity of drusen constituents which may have abnormal conformation and low biological availability from cell culture treatments of soluble factors. Thus, the relationship between drusen formation and AMD is entirely correlative increased numbers and size of drusen correlate with increased risk of developing advanced AMD. Whether drusen are a cause or a symptom of AMD or some combination of the two is unknown. Although AMD almost never occurs in the absence of prior drusen formation, the transition from intermediate AMD to geographic atrophy is associated with drusen regression126. Indeed, drusen formation could be an adaptive process to prevent the widespread dispersal of activated complement and other toxic constituents in the retina. One might speculate that the spontaneous release of drusen material might be an immediate cause of RPE loss in geographic atrophy.
Retinal pigmented epithelium

(RPE). A monolayer of epithelial cells that has multiple essential roles in visual function, including recycling components of the visual cycle, secreting trophic factors and maintaining the outer blood retinal barrier. The RPE is widely considered to be the focal point of age-related macular degeneration pathogenesis, in which breakdown of the RPE leads to secondary photoreceptor degeneration.
Drusen
Discrete extracellular deposits that commonly precede the development of age-related macular degeneration, and that are comprised of numerous cellular and inflammatory factors.
Geographic atrophy
(also known as end-stage dry age-related macular degeneration (AMD) or atrophic AMD). A disease affecting the macula in which the retinal pigmented epithelium can no longer support photoreceptor function owing to spontaneous degeneration of large confluent regions. Although geographic atrophy occurs less frequently than neovascular AMD (approximately 50% as common), there are no currently approved therapies.
A brief description of AMD The first clinical feature of early dry AMD is the presence of drusen (BOX1), which are extracellular deposits below the RPE that comprise lipid- and protein-rich debris12. Small, isolated drusen do not affect visual function but their expansion and coalescence are hallmarks of AMD progression. The two main advanced forms of AMD are: neovascular AMD, which is characterized by the invasion of abnormal choroidal (or occasionally retinal) blood vessels and fluid leakage into the retina; and geographic atrophy (also known as end-stage dry AMD or atrophic AMD). Neovascular AMD is the leading cause of blindness among the elderly in industrialized nations, affecting more than 1 million individuals over the age of 40 in the United States alone13. Although the fundamental aetiology of neovascular AMD remains unclear, therapies that target VEGFA, which is a potent stimulator of angio genesis and vasopermeability, have been successful in substantially improving central vision in approximately 30% of patients and in arresting vision loss in 94% of patients (compared with 62% of sham-treated patients)14,15. Geographic atrophy is characterized by large, confluent regions of atrophied RPE in the macular area. There are no approved therapies for geographic atrophy, which is mainly due to the lack of suitable molecular targets. Unlike VEGFA in neovascular AMD, a single crucial factor has not been identified as promoting RPE degeneration in geographic atrophy. As geographic atrophy
is considered to be the default end point of dry AMD16, further investigation into the initiating factors that promote the early development of this disease should be useful in the search for new therapies. Importantly, these advanced stages of AMD are not mutually exclusive neovascular lesions are often present in the periphery of eyes with geographic atrophy17. In addition, long-term treatment of neovascular AMD with VEGFAtargeted therapy is associated with the development of geographic atrophy18, possibly as a result of atrophy of the choriocapillaris19 and loss of the neurotrophic activity of VEGFA in the retina10. Genetic polymorphisms that are associated with AMD including those in the genes encoding CFH2023 and high-temperature requirement A serine peptidase 1 (HTRA1)24,25 confer similar statistical risk of developing both forms of AMD, which indicates that there are many shared underlying pathological mechanisms. The effect of polymorphisms in the HTRA1 or age-related maculopathy susceptibility2 (ARMS2) gene locus on gene activity or expression, as well as the functional consequences for AMD, are unclear. AMD is associated with marked changes in normal retinal anatomy. Early AMD is associated with thickening of the Bruch membrane, deposition of drusen, RPE hypertrophy and pigment extrusion26. Advanced AMD is associated with photoreceptor degeneration following either invasion of choroidal blood vessels through the Bruch membrane into the retina in neovascular AMD or degeneration of the choroidal vasculature and RPE cells in geographic atrophy27.
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Immune surveillance in maintaining retinal health Maintenance of the bloodretinal barrier confers a degree of immune privilege to the mammalian eye. This immune privilege is characterized by tolerance of foreign antigens owing to the expression of endogenous immunosuppressive factors and the absence of functional intraocular lymphatics2830. However, the introduction of specific foreign or endogenous inflammatory signals in the retina evokes innate immune responses. The resident inflammatory cells of the retina are the microglia, which are similar to tissue macrophages and central nervous system microglia. The retinal microglia normally reside in proximity to retinal blood vessels in the inner layers of the neural retina. The number of resident retinal microglial cells increases with age in mice31. Aged microglia have a branched morphology, which is indicative of a resting phenotype, and they show decreased responsiveness to tissue injury 31. Evidence indicates that the subretinal migration of microglia is necessary to eliminate visual by-products and to maintain vision31,32. The impairment of microglial migration into or out of the subretinal space promotes the death of photoreceptor cells33,34. In AMD, microglia accumulate in the subretinal space35; this is probably both a symptom of inflammatory damage and a beneficial response to injury, and the impairment of this accumulation in the subretinal space exacerbates retinal degeneration. However, infiltration of microglia and macrophages to sites of retinal injury can also promote the growth of neovascular lesions32,36,37. Macrophages, together with RPE cells, are a major source of pro-angiogenic factors such as VEGFA38. Macrophages and dendritic cells are not normally present in the retina but reside in the underlying choroid. In cases of breakdown of the bloodretinal barrier, these cells are recruited from the underlying choroid or from the systemic circulation into the retina where they modulate disease. The use of aged animals in models of impaired immune cell trafficking, such as those with CC-chemokine ligand 2 (CCL2), CC-chemokine receptor 2 (CCR2) and/or CX3C-chemokine receptor 1 (CX3CR1) deficiency, results in animals that have AMDlike features, including changes to the Bruch membrane, RPE swelling and retinal thinning 3941. Aged mice that are deficient in CCL2 or its receptor CCR2 develop spontaneous hypopigmented subretinal lesions that are visible using fundus photography33,39,42, as well as developing photoreceptor and RPE damage33,39; they also infrequently develop spontaneous choroidal neovascular lesions that are similar to those in neovascular AMD. Subretinal lesions of CCL2- or CCR2deficient mice are comprised of bloated outer segment-laden macrophages42 (which are similar to foam cells in atherosclerosis), and CCL2or CCR2deficient macrophages in the periphery show impaired chemotaxis and phagocytosis33. Importantly, CCL2CCR2dependent immune cell function prevents photoreceptor apoptosis following amyloid treatment43, which adds further support to the idea that immune cell trafficking to sites of local tissue damage is an essential component of retinal health. Similarly, aged
mice that are deficient in CX3CR1 show subretinal microglial accumulation and retinal degeneration, which have both been attributed to impaired trafficking of immune cells in the retina40. As in the CCR2- and CCL2-deficient mouse models that accumulate subretinal macrophages, CX3CR1-deficient mice acquire large bloated subretinal microglia44. Genetic studies also support a role for impaired repair of tissue damage in AMD, as polymorphisms in CX3CR1 that cause macrophage migratory defects38,43 are associated withAMD45. Whether increased or impaired CCL2CCR2 signalling is a cause or a symptom of AMD pathogenesis remains to be shown. CCL2 levels are increased in the RPE and the choroid of aged mice46, as well as in the serum of elderly individuals47; it is possible that this is related to the increased burden of tissue damage in the aged retina. The presence of increased systemic chemokine levels in patients with AMD indicates that systemic inflammation manifests itself locally in the retina. In addition, CCL2 expression is upregulated early in laser-induced choroidal neovascularization48, and inhibition of CCR2 decreases neovascular growth49. At first glance, these observations of increased CCL2CCR2 signalling in AMD are not consistent with the idea that deficiencies in CCL2CCR2mediated immune cell trafficking contribute to AMD. However, the distinction between local and systemic chemokine signalling is important; local signalling probably has a protective role in the immune-mediated repair of damaged retinal tissue in early AMD, whereas systemic signalling contributes to the recruitment of pro-angiogenic immune cells to sites of neovascularization in late-stage AMD. It is also important to consider the distinction between immune responses to acute events and those to long-term chronic para-inflammation or to persistent low-level inflammation (for example, involving complement activation and cytokine expression) owing to sustained tissue stress in the ageing retina35. This distinction probably accounts for some of the discrepancies that have been observed between age-related animal models of retinal degeneration, which rarely involve choroidal neovascularization, and acute injury models (such as light-induced retinal damage), in which retinal degeneration occurs within days. CCL2CCR2 signalling might modulate AMD development in different directions depending on the disease context; this would be in line with current models of immune cell involvement in AMD development that suggest that immune cells have dual, opposing roles in preventing and promoting disease.
Immune privilege
The property of a tissue being tolerant to antigen. Retinal immune privilege is maintained by bloodretinal barrier integrity and the absence of functional lymphatic circulation.
Fundus photography
A common method for visualizing the retinal pigmented epithelium, retina and retinal circulation photographically. Funduscopy is used by ophthalmologists to diagnose retinal disorders such as age-related macular degeneration.
Para-inflammation
A state in which tissue homeostasis is maintained by low-grade inflammatory-based clearance of noxious stimuli. In the retina, para-inflammation may persist and ultimately contribute to age-related macular degeneration through increased immune cell infiltration and activation at sites of tissue damage.
Dysregulated immune activation in AMD Although a functioning retinal immune system is crucial for visual homeostasis, a substantial amount of evidence also indicates that the overactivation of specific immune processes is important in AMD pathogenesis. Over a lifetime, the retina and RPE are in contact with a large number of innate immune activators that could potentially contribute to vision loss in AMD. Data from a decade of research strongly indicate that improper immune activation is involved in inducing and advancing AMD pathology. Among these immune activation pathways,
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Box 2 | Genetics of complement in AMD
The risk of developing age-related macular degeneration (AMD) is influenced not only by the common complement factor H (CFH) polymorphic variant 402His (the minor allele) but also by multiple other variants (common, uncommon and rare)69,50. The complement factor Hlike protein (CFHL1) is generated by alternative splicing of the CFH gene127. The complement factor Hrelated proteins 15 (CFHR15) arose by gene duplication events and are encoded just downstream of CFH on the long arm of chromosome 1. Their function is poorly understood. A simple concept is that CFHR1 competes with CFH for binding to substrates and thereby reduces the effectiveness of CFH. This hypothesis is consistent with a deletion of CFHR1 and CFHR3 being protective against AMD128. These studies of CFH and related proteins, combined with the identification of risk and protective variants in alternative pathway complement activators and other regulators, provide powerful evidence implicating overactivation of the alternative complement pathway in AMD. A finding that is consistent with this idea is that the protective variants result in less alternative pathway activity, whereas risk variants result in more activity129. In summary, common and rare variants in multiple members of a pro-inflammatory pathway of innate immunity are associated with the same disease; those that decrease the function of the pathway are protective and those that increase the function create risk.
Genome-wide association studies

(GWASs). The process by which genetic variations and disease phenotypes are statistically correlated. Linkage studies carried out with markers located across the entire genome that were traditionally carried out with approximately 300 markers of simple sequence-length repeats but that have been more recently carried out with approximately 15 million single nucleotide polymorphisms.
the complement pathway is the most well-established and widely accepted as contributing to AMD. Genetic evidence from genome-wide association studies (GWASs) and rare variant analyses50 strongly indicates that the alternative pathway of complement activation is overactive in AMD (BOX2). This evidence has been discussed in several excellent reviews69 and, therefore, these data are only summarized in this Review in relation to the general idea that excessive inflammation contributes to AMD pathogenesis. Complement. In 2005, GWASs showed that approximately 50% of the heritability of AMD could be accounted for by a single nucleotide polymorphism (SNP) in an exon encoding CFH25. However, it should be noted that the complement system had been implicated in AMD pathogenesis before this discovery 12,51,52. The risk variant of CFH (402His) does not regulate the alternative pathway of complement activation as efficiently as the main allele (Tyr402) does. The 402His variant binds with lower affinity to numerous constituents of the damaged retina6,5356; therefore, the inhibitory effect of CFH on the complement pathway is decreased, which results in a greater degree of complement activation following retinalinjury. Thus, one hypothesis for the aetiopathogenesis of AMD is that in individuals carrying a complement hyperinflammatory phenotype the complement pathway overreacts to cellular damage and debris in the retina69, 160. In addition to common alleles of CFH that confer greater risk of, or protection from, AMD as well as rare variants of CFH with high penetrance50, similar findings have been shown for other members of the alternative complement pathway, including activating components C3 and factor B (gain-of-function mutations associated with AMD) and the regulator factorI (loss-offunction mutations associated with AMD). Carrying the 402His CFH variant or these other risk factors enables the alternative pathway to generate undesirable quantities of complement components C3b and C3a, as well as of the downstream effectors C5a and C5bC9.
Alternative pathway of complement activation

An evolutionarily ancient innate immune process by which microorganisms are destroyed through opsonization and activation of the membrane attack complex. According to the complement hypothesis, misactivation of, and/or the inability to appropriately inhibit, the alternative pathway results in retinal tissue damage and drives age-related macular degeneration pathology.
Carboxyethylpyrroleadducted proteins
Proteins that are modified through the oxidation of the fatty acid docosahexaenoic acid. These adducts are abundant in the retina. They can have direct pro-inflammatory effects through pattern recognition, and autoantibodies that recognize them are abundant in the circulation of patients with age-related macular degeneration.
Inflammasome activation. The inflammasome is a protein complex that is activated by foreign or endogenous danger signals, which culminate in caspase 1mediated maturation of the cytokines interleukin1 (IL1) and IL18 and, ultimately, in pyroptosis or apoptosis. The inflammasome complex is comprised of three protein constituents: a NOD-like receptor family member (the best studied of which is NLRP3 (NOD-, LRR- and pyrin domain-containing 3)), the adaptor protein ASC and the protease caspase 1. Inflammasome activation has recently been implicated in AMD pathogenesis. One group found that carboxyethylpyrrole-adducted proteins, which accumulate in the retina with age57, prime the NLRP3 inflammasome58. They also found that drusen extracts that had been isolated from AMD donor tissue, as well as the complement component C1q (a component of human drusen), activate the NLRP3 inflammasome in macrophages. In a separate study, we identified another endogenous activator of the inflammasome that is present in the RPE of patients with geographic atrophy 59. Repetitive element-derived Alu RNA transcripts, which are non-canonical targets of DICER1-mediated enzymatic degradation, accumulate in human geographic atrophy following the loss of DICER1 expression (which might result from oxidative stress in the RPE)60. These transcripts function as both priming and activating signals to stimulate NLRP3 inflammasome signalling. Importantly, we found evidence of inflammasome activation in the eyes of patients with AMD; this included increased levels of NLRP3, IL18 and activated caspase 1 in the RPE. Two recent studies have confirmed evidence of inflammasome activation in the eyes of patients with AMD61 (C. Chan, personal communication). An important inflammasome effector is IL1. Human drusen extracts induce NLRP3dependent IL1 secretion from lipopolysaccharide-primed peripheral blood mononuclear cells58. In our study we did not detect significant release of mature IL1 from RPE cells in response to Alu RNA59, although inflammasome-mediated IL1 release from RPE cells has been more recently found in response to 4hydroxynonenal62, amyloid-63, A2E (N-retinyl-N-retinylidene ethanolamine) (D.Shima, personal communication) and lysosomal destabilization61, which indicates that RPE cells are capable of inflammasome-mediated IL1 release. In our study we also found increased levels of IL1B mRNA in the RPE of donor eyes from individuals with geographic atrophy59, which indicates that IL1 might be involved in the development ofAMD. Taken together, these studies show that the retina can respond to diverse danger signals that are abundant in AMD through the activation of the NLRP3 inflammasome. However, the consequences of inflammasome activation in AMD pathogenesis are poorly understood. Both IL1 and IL18 signal through the adaptor protein myeloid differentiation primaryresponse protein 88 (MYD88) to induce inflammatory and apoptotic effects. However, although we have found
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potent cytotoxic effects of IL18 and IL1 on the RPE59, in another study, beneficial effects of inflammasomemediated IL18 release were reported to occur through the inhibition of neovascularization in an acute laserinduced injury model of neovascular AMD58. These contrasting findings might imply that a single factor (IL18) or pathway (NLRP3 inflammasome activation) can be simultaneously anti-angiogenic and destructive to the RPE. This model mirrors our findings that Tolllike receptor 3 (TLR3) activation is beneficial in terms of decreasing choroidal neovascularization64, but that it also promotes RPE degeneration65 (see below). In contrast to the reported anti-angiogenic effects of IL18 on laser-induced choroidal neovascularization58, IL1 promotes neovascularization66. In summary, the extent, the underlying mechanisms and the cumulative effect of inflammasome activation and the secretion of IL1 and IL18 in human AMD remain uncertain. Therefore, we caution against pursuing IL1- or IL18based therapeutic strategies for neovascularAMD. TLR signalling. Activation of pathogen- or dangerassociated molecular pattern-mediated signalling in the retina often results in rapid and vigorous inflammatory responses. Retinal cells express multiple TLR family members, and the RPE in particular expresses most TLRs 67. The contribution of TLR signalling to AMD is still under intense investigation. Initial reports of the association of polymorphisms in TLR3 (REF.68) and TLR4 (REF.69) with AMD susceptibility were controversial70. TLR activation in the retina by synthetic or endogenous ligands can have positive or negative effects in AMD pathogenesis and this probably depends on context- and ligand-specific factors. Activation of TLR3 signalling by administration of double-stranded RNAs (dsRNAs) of 21 nucleotides or longer inhibits the development of choroidal neovascularization after acute laser-induced injury 64. However, the benefits of TLR3 signalling in terms of preventing neovascular lesions are counteracted by RPE cytotoxicity 65. Genetic ablation of TLR3 prevents disease development in a mouse model of retinal injury that is induced by impairment of all-trans-retinal clearance71, which indicates that endogenous TLR3 agonists promote retinal degeneration. Our investigations identified strong immunoreactivity with a dsRNA-specific antibody in human drusen, which supports the idea that the TLR3 signalling pathway is potentially involved in the progression of geographic atrophy60. The findings of TLR3induced RPE degeneration probably limit the use of nonspecific TLR3 agonist-based therapies for neovascularAMD. Unlike experimental TLR3 activation that suppresses the development of choroidal neovascularization, TLR2 ligands (carboxyethylpyrrole-adducted proteins and Chlamydia pneumoniae antigen) promote experimental choroidal neovascularization72,73. We speculate that TLR activation broadly contributes to AMD pathologies; however, the full range of agonists and outcomes is unknown.
Adaptive immunity. Although AMD research has mostly focused on innate immune processes, a growing body of evidence supports the idea that there is some contribution of adaptive immunity to AMD. The mammalian eye lacks functional intraocular lymphatic vessels, and B cells or Tcells have not been widely reported to be present in proximity to either neovascular or geographic atrophy lesions. As B cells and Tcells do not have a directly defined role in mediating angiogenesis or tissue damage in human AMD, any contribution of adaptive immunity to AMD development probably involves retinal antigen presentation and indirect autoantibody-mediated retinal degeneration. Increased titres of autoantibodies recognizing components that are enriched in the retina have been measured in the serum of patients with AMD7476 and have led to the hypothesis that AMD might be an autoimmune disorder. Indeed, the Ccl2/Cx3cr1/ mouse model of retinal degeneration involves retina-specific autoantibodies41, which indicates that there might be at least a circumstantial relationship between AMD and adaptive immune processes. Immunization of mice with carboxyethylpyrrolemodified albumin induces retinal degeneration that has features similar to those of AMD, including drusen-like deposits, sub-RPE complement deposition and photoreceptor dysfunction57, and that requires B cells and Tcells. Importantly, the authors of this study observed macrophage infiltration into retinal lesions, which indicates that antibody-mediated complement activation can induce a primary retinal injury to which macrophages respond and potentially further modulate the disease. The mechanisms involved in antigen presentation and antibody-mediated RPE degeneration (presumably by activation of the classical complement cascade) have not been resolved; they may provide important and interesting insights into AMD pathogenesis. The lack of functional lymphatic vessels and immune cells in the normal mammalian retina indicates that antigen presentation involves either a direct breakdown of the bloodretinal barrier and could be mediated by local dendritic cells, or the leakage of retinal antigens into the circulation. Importantly, some retinal antigens such as docosahexaenoic acid-derived carboxyethylpyrrole protein modifications are not strictly unique to the retina. Thus, the antigen need not be derived from the retina for a damaging antibody-mediated immune response to be mounted against the retina. However, whether lymphocytes have a causal role in AMD pathogenesis is uncertain. Other immune stimulators. In addition to the pathways highlighted above, several other factors that are linked to immune activation in AMD merit discussion. One of the most important of these factors is A2E, which is a principal component of retinal lipofuscin. Accumulation of A2E owing to mutations in the retinal-specific ATPbinding cassette A4 transporter (ABCA4) causes a form of Stargardts disease, which is an early-onset, inherited disease that results in retinal degeneration and that has many aetiological similarities to AMD. A2E has multiple damaging effects on RPE cells, including complement
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activation77, inflammasome activation (D. Shima, personal communication; J.A. and N. Kerur unpublished observations), cytokine expression78,79 and RPE degeneration80,81. ABCA4deficient mice also have increased complement activation82, and subretinal injection of A2E exacerbates experimental neovascularization83. Other important immune activators in the retina that might have a role in AMD are lipid oxidation byproducts, which are formed from the oxidation of polyunsaturated fatty acids. The retina is prone to lipid peroxidation because it has an abundance of lipid material and a high metabolic demand84. The major by-products of these reactions are malondialdehyde (MDA), carboxy ethylpyrrole and 4hydroxynonenal (4HNE) protein modifications. Lipid oxidation products have potent degenerative and inflammatory effects: both MDA and 4HNE induce lysosomal dysregulation85 and lipofuscin generation86, which prevent the photo receptor maintenance function of the RPE; MDA-modified proteins induce VEGFA expression in RPE cells and pro-inflammatory gene expression in macrophages87, which is prevented by the binding of CFH to MDA; 4HNE induces inflammasome activation62 and RPE apoptosis88; and carboxyethylpyrrole (as discussed above) directly activates TLR2 signalling 72 and might function as an antigen for retina-specific autoantibody production57. Amyloid is also abundant in drusen89; it promotes RPE and photoreceptor damage 90 as well as retinal inflammation63,70,91. Other environmental factors, such as excessive light exposure, hypercholesterolaemia and smoking, damage the RPE and the retina by inducing pro-inflammatory effects that are associated with AMD (reviewed in REF.92). In summary, clear evidence supports a role for a range of pro-inflammatory factors that are abundant in the ageing retina as driving forces in AMD pathogenesis. We discuss below the special case of neovascular AMD, in which the breakdown of the bloodretinal barrier allows systemic immune cells unusual access to the retina. In this way, immune regulation of neovascular AMD represents an outsidein phenomenon, in contrast to early dry AMD and geographic atrophy, in which immune activation is generally self-contained and self-inflicted. geographic atrophy is less impressive. Histological evidence showing relatively few immune cells in the atrophic area of geographic atrophy98 indicates that local immune cells may not have a substantial role in this form of AMD. However, this does not rule out immune-mediated tissue damage being involved in geographic atrophy, as emerging evidence highlights RPE-specific inflammasomemediated degeneration as a driving force for RPE atrophy 59,61,62 (D. Shima and C. Chen, personal communication). In this respect, the role of innate immunity in neovascular AMD compared with geographic atrophy pathogenesis probably diverges at the local tissue level, and might represent both a consequence and a cause of the ultimate pathogenic outcome. The development of neovascular AMD is accompanied by the recruitment of inflammatory cells to the site of vascularization, the consequence of which is the expression of numerous cytokines and chemokines that induce angiogenesis. Human choroidal neovascular lesions contain inflammatory cell infiltrates9396,99, and experimental depletion of macrophages decreases choroidal neovascularization in laser-injured mice36,37. Subsequent analyses have also shown that macrophages can have antiangiogenic properties in the context of experimental choroidal neovascularization; indeed, intraocular injection of macrophages before injury decreases laser-induced choroidal neovascularization100. Furthermore, mice that are deficient in the anti-inflammatory cytokine IL10 have both an increased accumulation of macrophages in neovascular lesions and decreased choroidal neo vascularization100. Indeed, our initial finding of spontaneous choroidal neovascularization in Ccl2- or Ccr2deficient mice 39 can be generally interpreted as a systemic impairment of macrophage chemotaxis enhancing the development of choroidal neovascularization. The contradictory contributions of macrophages that have been observed in choroidal neovascularization might be explained by differential macrophage polarization with respect to M1 macrophages (pro-inflammatory) and M2macrophages (pro-angiogenic). So far, the role of macro phage polarization in AMD development has not been well characterized. One report identified an increased expression of the M1 marker CCL22 compared with an M2 marker, CXC-chemokine ligand11 (CXCL11), in macrophages from individuals with AMD compared with those from the eyes of unaffected individuals101, which indicates that inflammatory macrophage polarization might contribute to or be symptomatic of AMD development. Furthermore, choroidal M1 (inducible nitric oxide synthase (iNOS)+) macrophages have been measured in proximity to subclinical neovascular AMD lesions98. Whether M1 macrophages contribute to pathological angiogenesis and whether increased numbers of M2 polarized macrophages are also present in choroidal or retinal neovascular membranes are not yet known but further studies will probably provide insights into the role of macrophage polarization in neovascular AMD. In mice, IL10 was found to be a crucial factor driving M2 polarization, and inhibition of IL10 decreased angio genesis100. Although the role of IL10 remains unclear (two studies show that IL10 suppresses choroidal angiogenesis
M1 macrophages
A pro-inflammatory or classically activated subset of macrophages, which are characterized by phagocytic activity and expression of particular inflammatory cytokines (such as tumour necrosis factor) and inflammatory mediators (such as inducible nitric oxide synthase).
M2 macrophages
A pro-angiogenic or alternatively activated subset of macrophages, which are characterized by the expression of particular angiogenic cytokines (such as vascular endothelial growth factor A) and anti-inflammatory mediators (such as arginase).
Immune cell recruitment in neovascular AMD Clear evidence from animal models supports dual roles for resident and invading macrophages/microglia in preventing and promoting neovascular AMD. These paradoxical effects have been attributed to two different functions of innate immune cells. First, as discussed above, the surveillance and phagocytosis of damaged cellular components is a necessary function of resident microglia for the maintenance of retinal health. Second, invading macrophages provide potent pro-angiogenic signals that exacerbate choroidal vessel invasion and pathogenesis. Importantly, whereas ample evidence supports the involvement of immune cells in both human neovascular AMD9396 and experimental choroidal neovascularization36,37,97, the evidence for immune cell involvement in early and intermediate dry AMD and
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in other inflammatory contexts102,103), a followup study found that both IL10 levels and M2 polarization are increased in the retinal macrophage population of aged mice104. Macrophage polarization as a directing factor in AMD is an intriguing possibility that merits furtherstudy. In addition to macrophages, other analyses have found invading neutrophils to make variable contributions to promoting choroidal neovascularization in animal models. With respect to the number of invading cells, neutrophils constitute a large fraction of the inflammatory cell infiltrate in experimental neovascular lesions of the retina, although their contribution to laser-induced choroidal neovascularization is minimal when macrophage infiltration is impaired 97. Other studies have found that antibody-mediated depletion of neutrophils prevents laser-induced choroidal neovascularization97,105, and that they produce VEGF 105 as well as matrix metalloproteinase 9 (MMP9), which promote the loss of RPE barrier integrity 106. However, neutrophils are not a major constituent of human AMD lesions107. The role of neutrophils in animal models of AMD probably represents an acute injury response and may not reflect the mechanisms that occur in human AMD, which is a progressive disease that typically spontaneously develops over decades. In addition to the roles of CCL2, CCR2 and CX3CR1 in immune cell migration and retinal neovascularization, several other cytokines and chemokines have potentially important roles in the development of neovascular AMD (BOX3).
An integrated model of immunity in AMD The metabolic and immunological demands of the macular retina are unlike any other tissue in the body. Recent findings have brought diverse immunological aspects of AMD pathogenesis into sharper focus. We propose below a generalized model of AMD progression on the basis of these observations (FIG. 2). We propose that the fundamental abnormality in AMD is a dysfunction of RPE cells; this can result from multiple types of insult but the final common pathway is damage to an important metabolically active cell type that maintains homeostasis in the neuroretina. The importance of individual insults, especially in individual patients, in mediating RPE injury is unclear. Many of these insults, which can be both local and systemic, arise as a result of inappropriate immune activation, such as dysregulated complement activation, production of inflammasome activators and autoantibodies that are specific for retinal antigens.
Box 3 | Cytokine and chemokine signalling in neovascular AMD

In addition to the main molecules and pathways involved in neovascular age-related macular degeneration (AMD), several important secreted factors might also affect disease processes and could be possible therapeutic targets.
Eotaxins Eotaxins (CC-chemokine ligand 11 (CCL11), CCL24 and CCL26) and their receptor CC-chemokine receptor 3 (CCR3) are highly expressed in neovascular lesions before the retinal invasion by new blood vessels130. CCL11 levels are increased in retinal specimens from aged humans and in choroidal endothelial cells from patients with AMD131, and both CCL11 and CCL24 levels are increased in the circulation of patients with AMD132,133. CCR3 antagonism prevents laser-induced choroidal neovascularization, and CCR3 expression may be a useful biomarker of subclinical AMD130,134. Studies of eotaxin signalling might provide therapeutic insights that enable neovascular AMD to be diagnosed and treated at early stages. IL6 Increased interleukin6 (IL6) levels are found in ocular fluids of patients with neovascular AMD and they predict AMD progression135. The pro-angiogenic effects of IL6 are well-described in the context of tumour angiogenesis and involve the upregulation of vascular endothelial growth factor A136. Genetic ablation of IL6 or its receptor decreases laser-induced choroidal neovascularization137. IL6 signalling also promotes degeneration of the retinal pigmented epithelium (RPE) following lipopolysaccharide stimulation138. Thus, IL6 might have dual pathogenic functions by promoting both angiogenesis and RPE degeneration in advanced AMD.
CXCL10 CXC-chemokine ligand 10 (CXCL10), which is a potent anti-angiogenic chemokine, is highly expressed in the retina of patients with AMD132 and laser-injured mice139. Choroidal endothelial cells express CXC-chemokine receptor 3 (CXCR3) the CXCL10 receptor and genetic ablation of CXCR3 exacerbates laser-induced choroidal neovascularization in mice139. Thus, CXCL10 might be an important endogenous negative regulator of neovascular AMD. TNF Tumour necrosis factor (TNF) is abundant in human choroidal neovascular membranes95, and genome-wide association studies have identified two polymorphisms in the TNF receptor TNFRSF10 (which encodes the protein TAILR1) that are associated with neovascular AMD140,141. Inhibition of TNF by antibodies142,143 or recombinant receptor fragments144 decreases experimental choroidal neovascularization in laser-injured rodents. However, TNF-targeted therapy was not well tolerated in human trials of neovascular AMD145. CXCL12 CXCL12 is a potent chemoattractant. It drives the recruitment of endothelial progenitor cells and haematopoietic stem cells to regions of neovascularization146; these cells constitute as much as 50% of the neovascular lesions in laser injury models. In laser-induced choroidal neovascularization, CXCL12 expression is increased, and inhibition of its receptor CXCR4 decreases angiogenesis146,147. Conversely, expression of CXCL12 is variable in neovascular human AMD (two of ten148 and zero of four149 neovascular lesions stained positive for CXCL12 expression). Thus, the contribution of CXCL12 signalling in human AMD has not been firmly established.
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a Impaired immune-mediated retinal maintenance
Ageing Photo-oxidative stress Genetics CX3CR1 polymorphism
RPE phagocytotic impairment Insucient tissue repair
Microglia dysfunction
Lipid peroxidation MDA CEP 4-HNE
DICER1 insuciency Alu RNA
Build-up of visual cycle waste Lipofuscin A2E
b Immune-mediated retinal damage

Classical complement pathway C1q TLR signalling Inammasome activation IL-1 IL-18 RPE damage Drusen Amyloid- Alternative complement pathway C1q
Bloodretina barrier breakdown
Retinal autoantibodies
c Geographic atrophy
d Neovascular AMD
Peripheral immune cell inltration Macrophages Dendritic cells iNKT cells Angiogenesis suppression IL-10 CXCL10
RPE atrophy
Choriocapillaris atrophy
Retinal neural cell atrophy
Proliferative angiogenic response Eotaxins IL-1 IL-6 TNF VEGFA
Prolonged VEGFA-specic antibody therapy
Abnormal vascular network Macular exudate
Blindness
Figure 2 | An integrated model of AMD pathogenesis. The cell types, environmental factors and immune pathways Nature Reviews | Immunology that contribute to age-related macular degeneration (AMD) are categorized as either impaired immune-mediated retinal maintenance (part a) or immune-mediated retinal damage (part b). The combination of these factors leads to either geographic atrophy (part c) or neovascular AMD (part d). The most important pathways are highlighted in bold. The earliest known steps in AMD pathogenesis (part a) reflect a reduced capacity to manage the metabolic demands of the retina. The convergence of genetic, environmental and metabolic factors leads to a state in which the accumulation of toxic elements (such as lipid peroxidation by-products, lipofuscin and Alu RNAs) ultimately tips the balance towards immune activation. Once it is replete with unwanted waste material (part b), the retina is the target of inappropriate immune activation. The toxic contents of the retina induce inappropriate activation of diverse immune pathways, including classical and alternative complement pathways, the inflammasome and Toll-like receptor (TLR) signalling. Ultimately, the sustained activation of these pro-inflammatory and damaging pathways leads to advanced AMD. In the case of geographic atrophy (part c), sustained damage to the retinal pigmented epithelium (RPE) leads to the development of degeneration of the RPE, the choriocapillaris and, finally, neural retinal cells. In neovascular AMD (part d), breakdown of the bloodretinal barrier results in immune cell trafficking into the retina, which drives vascular endothelial growth factor A (VEGFA)dependent neovascularization, causing blindness. 4HNE, 4hydroxynonenal; A2E, N-retinyl-N-retinylidene ethanolamine; C1q, complement component 1q; CEP, carboxyethylpyrrole; CX3CR1, CX3C-chemokine receptor 1; CXCL10, CXC-chemokine ligand 10; IL, interleukin; iNKT, invariant natural killer T; MDA, malondialdehyde; TNF, tumour necrosis factor.
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Under normal conditions, the RPE layer continuously maintains immune suppression through both tight junction-mediated barrier integrity and antiinflammatory cytokine production. Retinal microglia provide additional immune surveillance by clearing cell debris. Many different hazards can create initial foci of tissue damage and, importantly, also render the retina unable to removing dying, dead or stressed retinal cells. This concept is similar to that of other progressive agerelated diseases (BOX4). The inability to sufficiently cope with increased tissue damage leads to drusen accumulation, and the drusen function as concentrated immuno stimulatory punctae. As drusen accumulate, the development of AMD depends on the type of immune response. In the neovascular form of AMD, breakdown of the bloodretinal barrier provides circulating immune cells with unusual access to a highly immunogenic environment, which results in macrophage recruitment and activation. When these macrophages encounter an avascular, metabolically demanding tissue, they initiate a VEGFAdependent neovascular response. The complex mixture of pro-angiogenic cytokines that they produce results in a leaky, abnormal vascular network that is associated with fluid leakage and, if left untreated, in the eventual development of fibrosis108. In the case of geographic atrophy, circulating immune cells are denied access to the degenerating retina. Rather than a proliferative vascular response, the inability to repair the increasing tissue damage leaves large, confluent areas of degenerated RPE and photoreceptors. The degenerating area expands as diseased RPE cells damage neighbouring cells by releasing toxic inflammatory stimulators and cytokines.
Box 4 | Age-dependent degenerative diseases
Most tissues require methods to clear dead cells and intracellular and extracellular waste. Necrosis and apoptosis are two examples of these methods, which can be triggered by wear and tear (socalled normal ageing phenomena), ischaemia reperfusion injury, trauma, foreign agents (microorganisms) and toxins. The complement system is one of the first (if not the first) responder to such events and is activated by necrosis and apoptosis150. It is involved in degenerative arthritis151 and atherosclerosis152,153, including complement activation by lipid particles, and there is also a role for the complement pathway in the development of abdominal aortic aneurysm. In this regard, stressed and damaged retinal pigmented epithelium (RPE) could be seen as analogous to cartilage loss predisposing an individual to osteoarthritis151. Relative to atherosclerosis, drusen accumulation could be viewed as comparable to extracellular lipid deposits154. Indeed, a recent collaborative genome-wide association study identified age-related macular degeneration (AMD) risk variants in loci that are also involved in atherosclerosis and lipid metabolism155. Although the nature of the debris, the generation of the inflammatory response and the clearance mechanisms are likely to have unique features in specialized tissues, the innate immune response and the degradative and clearance processes will have overlapping features92. Thus, the four great diseases of ageing Alzheimers disease, atherosclerosis, AMD and degenerative arthritis can all be viewed in this perspective: they feature a generally unknown age-dependent degenerative and destructive process, as well as an accumulation of self debris: amyloid (in Alzheimers disease), lipids (in atherosclerosis), drusen (in AMD) or glycosaminoglycans (in degenerative arthritis). The debris is not adequately eliminated and the dying cells (neurons, RPE cells or chondrocytes) are not easily replaceable. The innate immune response to this damage is insufficient. Initially, the innate immune response is presumably beneficial and promotes longevity, but at later stages in the disease process it is probably detrimental.
In this model, AMD arises from both immune hypoactivity in the case of inadequate tissue repair and immune hyperactivity in the case of complementmediated tissue damage. One emerging theme from such a model is that pan-immunosuppression could potentially reduce late-stage AMD; however, by preventing endogenous immune-mediated tissue repair processes, new foci of dysfunction and disease might be created in the remaining functional retina. Another prediction from this model is that the most effective immunotherapies must be highly target- and contextspecific to inhibit specific pathogenic factors but to only minimally affect tissue repair mechanisms. As the search for new and improved AMD therapeutics continues, we must not turn a blind eye to the Janus-faced nature of ocular immunity.
Immune-based therapy for AMD Any discussion about the future potential for AMD therapeutics must be placed in the context of the current standard of care. The management of neovascular AMD has undergone a transformation over the past 10years as a result of the astonishing success of VEGFAtargeted therapies (<10% of patients treated with VEGF-targeted agents exhibit considerable vision loss14). The off-label use of bevacizumab (Avastin; Genentech/Roche) a humanized monoclonal antibody targeting VEGFA which improves or maintains vision in the majority of patients and is available in doses that cost in the order of tens of dollars18,109, has somewhat diminished future prospects for the development and commercialization of new drugs to treat neovascular AMD. Nonetheless, in cases in which VEGFAbased treatment is insufficient or ineffective, or in the case of geographic atrophy, combination therapies or immune-based therapies that are directed towards alternative targets are being explored. As our understanding of the role of immunity in AMD has advanced, we have gained an appreciation of the nuanced and conflicting roles of the immune system as a protector, an instigator and a driver of retinal degeneration. The complex cell type-, pathological context-, temporal- and pathway-specific aspects of immune regulation of retinal health demand targeted medicine. It is perhaps a lack of understanding or appreciation of these facts that has led to the numerous failures of immune-based therapies; for example, steroidal anti-inflammatory drugs have only achieved very modest success as an adjunct to VEGFAtargeted treatment 110,111.
Complement-based diagnostics and therapeutics. Despite the knowledge that complement activation is a genetic risk factor for AMD development, so far the clinical predictive or therapeutic value of these findings has not been realized. Several clinical trials using complement inhibition-based treatment of AMD have produced disappointing primary outcome data that show minimal improvement in visual acuity or reduction in disease progression (TABLE1). It is also worth noting that, despite millions of dollars having been spent and
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Table 1 | Current immune-based clinical trials for AMD
Compound
POT4 (Alcon)
Target
C3
Structure
Indications
Trial status
Phase I has been completed 93% of patients had no improvement in visual acuity (S. Bakri, personal communication) Phase II is ongoing There was no improvement in mean visual acuity or area of geographic atrophy in the first 6months of the trial (P. Rosenfeld, personal communication) Phase II is ongoing (Phase I data are unpublished) Phase I has been completed (Phase I data are unpublished) Phase I is ongoing Phase II is ongoing (Phase I data are unpublished) Phase II is ongoing (Phase I data are unpublished) Phase II and Phase III are inactive A small cohort (N=8) showed reduction in drusen size156 Phase II is ongoing (Phase I data are unpublished) Phase II has been completed It reduced the frequency of VEGFAspecific injections in four patients157 Phase II has been completed It reduced the number of VEGFAspecific injections in 16 patients but had no benefit for visual acuity158 Phase II has been completed It reduced the number of VEGFAspecific injections in three patients157 Phase II has been completed (data are unpublished) Phase III has been completed It reduced the number of VEGFA-specific injections but had no benefit for visual acuity159 Phase II (Phase I data are unpublished)
Trial number
NCT00473928
Cyclic peptide Neovascular AMD inhibitor
Eculizumab (Alexion)
C5
Monoclonal antibody
Dry AMD (presence of drusen) and geographic atrophy
NCT00935883
LFG316 (Novartis)
C5
Monoclonal antibody Aptamer
Geographic atrophy Advanced neovascular AMD Neovascular AMD Geographic atrophy
NCT01527500 NCT01535950 NCT00709527 NCT00950638 NCT01602120
ARC1905 (Ophthotech)
C5
FCFD4514S (Genentech) Sonepcizumab(Lpath)
Factor D
Monoclonal antibody (Fab fragment) Monoclonal antibody Small peptide
Geographic atrophy
Sphingosine-1 phosphate Unknown anti-inflammatory target Amyloid- IL2 receptor subunit- Cyclooxygenase
Neovascular AMD, subresponders to VEGFA-targeted therapy Early and intermediate dry AMD Geographic atrophy Neovascular AMD, coadministered with VEGFA-targeted therapy Neovascular AMD, coadministered with VEGFA-targeted therapy Neovascular AMD, coadministered with VEGFA-targeted therapy Neovascular AMD, coadministered with VEGFA-targeted therapy
NCT01414153
Glatiramer acetate (Teva) RN6G (Pfizer) Daclizumab (HoffmanLa Roche) Bromfenac(ISTA)
NCT00466076
Monoclonal antibody Monoclonal antibody NSAID
NCT01577381 NCT00304954
NCT00805233
Infliximab(Janssen)
TNF
Monoclonal antibody Monoclonal antibody
NCT00304954
Adalimumab(Abbott)
TNF
NCT01136252
Triamcinolone acetonide (Bristol-Myers Squibb) Fluocinoloneacetonide (Alimera Sciences)
Broad anti-inflammatory target
Corticosteroid Neovascular AMD, coadministered with VEGFA-targeted therapy
NCT00370370
Broad antiCorticosteroid Geographic atrophy inflammatory target
NCT00695318
AMD, age-related macular degeneration; C, complement component; IL2, interleukin2; NSAID, non-steroidal anti-inflammatory drug; TNF, tumour necrosis factor; VEGFA, vascular endothelial growth factor A.
nearly a decade of intense research, the predictive value of genetic screening for AMD is limited by the simple reality that the relatively inexpensive procedure of taking a patients history and a routine eye examination provide more relevant information with respect to current and potential future clinical AMD development. Data about whether polymorphisms in complement genes and other AMD risk factor genes can be used to
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predict the response to VEGFAtargeted therapies are conflicting 112,113; however, given the lack of alternative treatments that are available, these findings are unlikely to change the management of neovascular AMD. Similarly, genetic models fail to predict progression to geographic atrophy114,115 but without an approved therapy for geographic atrophy, once again, there is no immediate clinical use for such genetic information.
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Despite considerable interest, no complement-based therapeutic has yet progressed to a Phase III clinical trial. The early results from complement-based strategies have dampened enthusiasm for using these as targets and have highlighted our lack of basic understanding about the mechanisms by which complement factors influence AMD development. Indeed, one mechanism that is apparently involved in complement-mediated AMD development is the upregulation of VEGFA48,116 this only reminds us that VEGFAbased therapies already do an excellent job in the majority of patients. Other immune-based clinical trials. So far, immunebased therapies have exclusively evaluated antiinflammatory agents. The successes of these therapies have been measured in terms of a reduction in the number of VEGFAtargeted injections that are needed for neovascular AMD treatment; it is possible that a positive outcome results from a better response to trauma from repeated intraocular injections rather than prevention of a fundamental disease process. Although reducing the number of injections is an important goal, no trial has reported improvements in visual acuity as a result of anti-inflammatory monotherapy or adjunctive therapy. Ocular immunity is involved both in maintaining visual homeostasis and in driving AMD pathogenesis, so we support the development of new targeted
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therapies as the most promising approach to treat this disease; however, this awaits a more thorough investigation of the molecular mediators of AMD. Recent studies have identified new potential targets (such as IL1, IL18 and inflammasome components) that merit further evaluation for potential intervention. The search for new targets will also benefit from a reevaluation of current animal models of advanced AMD. Laser photocoagulation-induced neovascularization effectively models the VEGFA-dependent angiogenic component but probably does not mimic the inflammatory component of human neovascular AMD and has a timescale that differs by several orders of magnitude. Similarly, no animal model has yet been developed that mimics all of the changes that are associated with advanced geographic atrophy. We postulate that the future of AMD-based therapies lies in pathomechanism-driven discovery and caution against demanding that putative targets be validated in GWASs. VEGFA is a prime example of a validated target for AMD that arose via mechanistic biology and which would not withstand the scrutiny of statistical genetics. Immunology-based insights are likely to have an important role in the future of clinical AMD management once a more thorough understanding of friend and foe in the context of AMD pathogenesis is realized.
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Cytokines in neovascular age-related macular degeneration: fundamentals of targeted combination therapy. Br. J.Ophthalmol. 95, 16311637 (2011). 146. Sengupta,N. etal. Preventing stem cell incorporation into choroidal neovascularization by targeting homing and attachment factors. Invest. Ophthalmol. Vis. Sci. 46, 343348 (2005). 147. Lee,E. & Rewolinski,D. Evaluation of CXCR4 inhibition in the prevention and intervention model of laser-induced choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 51, 36663672 (2010). 148. Guerin,E. etal. SDF1 is associated with VEGFR2 in human choroidal neovascularisation. Microvasc. Res. 75, 302307 (2008). 149. Bhutto,I.A., McLeod,D.S., Merges,C., Hasegawa,T. & Lutty,G.A. Localisation of SDF1 and its receptor CXCR4 in retina and choroid of aged human eyes and in eyes with age related macular degeneration. Br. J.Ophthalmol. 90, 906910 (2006). 150. Mevorach,D. Clearance of dying cells and systemic lupus erythematosus: the role of C1q and the complement system. Apoptosis 15, 11141123 (2010). 151. Wang,Q. etal. Identification of a central role for complement in osteoarthritis. Nature Med. 17, 16741679 (2011). 152. Wu,G. etal. Complement regulator CD59 protects against atherosclerosis by restricting the formation of complement membrane attack complex. Circ. Res. 104, 550558 (2009). 153. Szeplaki,G., Varga,L., Fust,G. & Prohaszka,Z. Role of complement in the pathomechanism of atherosclerotic vascular diseases. Mol. Immunol. 46, 27842793 (2009). 154. Hansson,G.K. & Hermansson,A. The immune system in atherosclerosis. Nature Immunol. 12, 204212 (2011). 155. Fritsche,L.G. etal. Seven new loci associated with age-related macular degeneration. Nature Genet. 45, 433439 (2013). 156. Landa,G., Butovsky,O., Shoshani,J., Schwartz,M. & Pollack,A. Weekly vaccination with Copaxone (glatiramer acetate) as a potential therapy for dry age-related macular degeneration. Curr. Res. 33, 10111013 (2008). 157. Nussenblatt,R.B. etal. A randomized pilot study of systemic immunosuppression in the treatment of age-related macular degeneration with choroidal neovascularization. Retina 30, 15791587 (2010). 158. Gomi,F., Sawa,M., Tsujikawa,M. & Nishida,K. Topical bromfenac as an adjunctive treatment with intravitreal ranibizumab for exudative age-related macular degeneration. Retina 32, 18041810 (2012). 159. Ahmadieh,H. etal. Intravitreal bevacizumab versus combined intravitreal bevacizumab and triamcinolone for neovascular age-related macular degeneration: sixmonth results of a randomized clinical trial. Retina 31, 18191826 (2011). 160. Lachmann, P. J. The amplification loop of the complement pathways. Adv. Immunol. 104, 115149 (2009).
Acknowledgements
J.A. is supported by US National Institutes of Health (NIH) grants (R01EY018836, R01EY020672 and R01EY022238), the Doris Duke Charitable Foundation, USA, the Ellison Medical Foundation, USA, the Burroughs Wellcome Fund, USA, the Reeves Foundation, USA, a Dr. E. Vernon Smith and Eloise C. Smith Endowment and a Research to Prevent Blindness Unrestricted Grant, USA. J.P.A. is supported by NIH grants (AI041592, AR007279, AR0483335, GM099111 and HL112303), the Edward N. and Della L. T h o m e M e m o r i a l Fo u n d a t i o n , U SA , a n d A l ex i o n Pharmaceuticals, USA. B.D.G. is suported by the National Center for Advancing Translational Sciences, USA, grants UL1TR000117 and UL1TR000117. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
The authors declare competing financial interests. See Web version for details.
FURTHER INFORMATION
Jayakrishna Ambatis homepage: http://www.mc.uky.edu/angiogenesis/
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TIMELINE
The history of Toll-like receptors redefining innate immunity

Luke A.J.ONeill, Douglas Golenbock and Andrew G.Bowie
Abstract | The discovery of Toll-like receptors (TLRs) was an important event for immunology research and was recognized as such with the awarding of the 2011 Nobel Prize in Physiology or Medicine to Jules Hoffmann and Bruce Beutler, who, together with Ralph Steinman, the third winner of the 2011 Nobel Prize and the person who discovered the dendritic cell, were pioneers in the field of innate immunity. TLRs have a central role in immunity in this Timeline article, we describe the landmark findings that gave rise to this important field of research.
Before the discovery of Toll-like receptors (TLRs), innate immunity was seen as a crude and unsophisticated part of the immune system; its main purpose was considered by immunologists to be the initiation of the more sophisticated adaptive immune response, which was thought to confer protection on the infected organism. In addition, innate immunity was implicated in systemic responses such as fever. The molecular basis for innate immunity was not known, particularly with respect to how innate immune agents such as the cytokines interleukin1 (IL1), tumour necrosis factor (TNF) and IL6 were induced. In addition, the signalling events that promote the expression of the antiviral interferons (IFNs) were unclear. The characterization of TLRs has provided molecular insights into these processes and has also allowed the discovery of other families of innate immune receptors, revealing new perspectives to researchers in immunology. In this Timeline article, we describe the beginnings of this field of research, following the identification of the IL1 receptor (IL1R) in mammals and the cell surface protein Toll in Drosophila melanogaster. We discuss the identification of the mammalian Toll-like receptor 4 (TLR4) as the lipopolysaccharide (LPS) receptor, the subsequent discovery of further ligands for different TLRs and the elucidation of TLR signalling pathways (TIMELINE). Finally, we briefly
review the evolution of our understanding about the role of TLRs in disease and the therapeutic applications of TLR agonists and antagonists.
TIR domains: flies, plants and mammals TLRs are prototype pattern-recognition receptors (PRRs) that recognize pathogenassociated molecular patterns (PAMPs) from microorganisms or danger-associated molecular patterns (DAMPs) from damaged tissue. The idea that such innate immune receptors exist dates back to 1989, when Charles Janeway 1 predicted, in an important monograph, that PRRs recognizing microbial products link innate and adaptive immunity 1. The question of how research into TLRs began can be answered in several ways, but the first molecule of relevance to be identified was IL1R type 1 (IL1R1). IL1 is a pleiotropic proinflammatory cytokine, and it was reported by several laboratories in the 1980s to be involved in Tcell activation, pyrogenicity, the promotion of cartilage breakdown and the activation of the acute phase response2. In 1988, the gene encoding IL1R1 was cloned, but the predicted sequence gave no clues as to the mechanism by which it might signal, as there were no recognizable motifs in the cytosolic domain3. In 1991, this domain was shown to be homologous to the cytosolic domain of a D.melanogaster protein termed Toll4. This was unexpected,
as at that time the only known function of Toll was to promote dorsoventral polarity in the developing D.melanogaster embryo5. Notably, the establishment of dorsoventral polarity in flies had been shown to involve a protein termed Dorsal, which shares a REL homology domain with the other members of the nuclear factor (NFB) family of transcription factors6. At that time, NFB, which was first identified in Bcells as a factor that was activated by the Gram-negative bacterial cell wall constituent LPS7, was emerging as a major component of inflammation and infection. Indeed, remarkable numbers of genes with roles in immunity and inflammation were shown at that time to be regulated by NFB. Moreover, IL1 had been found to activate NFB signalling 8. This suggested that the same highly efficient signalling mechanism might be used both in development in D.melanogaster and in pro-inflammatory signalling in mammals. So, it was established that the highly similar proteins Toll and IL1R1 have important NFdependent roles in two different contexts; this was particularly evident after Toll and IL1R1 were shown to share common amino acids that are essential for NF signalling 9. In 1994, the next important step in the development of the TLR field involved the characterization of a plant protein that confers resistance to tobacco mosaic virus the N protein10. The amino-terminal domain of the N protein was found to be similar to the cytoplasmic domains of Toll and IL1R1, which indicated that this conserved domain is involved in host defence in two disparate kingdoms the plant and the animal kingdoms. This conserved domain was named the TollIL1resistence (TIR) domain. Importantly, at around the same time (in 1993) Michael Levine and colleagues11 reported that Dif, another member of the NFB family in D.melanogaster, translocated from the cytoplasm to the nucleus following bacterial infection or injury in the larval fat body. This study also demonstrated that Dif binds to a Blike sequence in the promoter of the gene that encodes the antimicrobial peptide cecropin. Furthermore, it was shown that Dif was activated by the constitutively
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Timeline | The history of Toll-like receptors
CD14 and LPS-binding protein are identified as components of the LPS receptor complex (19931996). Pathogen-specific immune signalling found to involve induction of antimicrobial peptides by members of the NF-B family in Drosophila melanogaster The first human homologue of Toll receptor is cloned (hToll; later renamed TLR4) A role for MYD88 in IL1 receptor signalling is identified LPS signalling is found to require MYD88 The requirement of MD2 for TLR4 responsiveness to LPS is identified (20002002). Ligands for TLR2heterodimeric complexes are identified
IL1R1 is cloned
1988
1989
1990
1991
1993
1994
1996
1997
1998
1999
2000
Charles Janeway proposes the concept of pattern-recognition receptors
Sequence similarity between Toll and IL1R1 identified
Plant protein N is shown to be involved in disease resistance and to have a TIR domain that is similar to Toll and IL1R1
The Toll pathway is shown to regulate the antifungal response in D. melanogaster
Four further human TLRs are identified TLR4 is identified as the signalling receptor for LPS
Viral antagonists of TLRs are identified TLR9 is characterized as the receptor for CpG-DNA
IL-1R1, interleukin-1 receptor type1; LPS, lipopolysaccharide; MAL, MYD88-adaptor-like protein; MD2, myeloid differentiation factor 2; MYD88, myeloid differentiation primary-response protein 88; SARM1, sterile-- and armadillo-motif-containing protein 1; ssRNA, single-stranded RNA; TIR, TollIL1resistence; TLR, Toll-like receptor; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adaptor protein inducing IFN.
active mutant of Toll, termed Toll10b (REF.11). These studies built on earlier work by Hans Boman12, who was the first to describe the antimicrobial peptides cecropin and attacin in the moth Hyalophora cecropia12. The apparent association of a Toll mutant with the Dif-dependent induction of antimicrobial peptides, together with the earlier described link between Toll and Dorsal, led Jules Hoffmanns laboratory to postulate that Toll might regulate not only developmental processes but also immune gene expression. Definitive proof for this hypothesis was provided in 1996by Bruno Lemaitre13, a member of the Hoffman laboratory, in the context of the Tollmediated induction of the antifungal peptide Drosomycin13. Indeed, Lemaitre showed that, after microbial infection, Drosomycin expression was upregulated following activation of the Toll pathway 14 work for which the Nobel Prize was awarded. It was subsequently shown that Dif, and not Dorsal, was the main regulator of antimicrobial peptide expression in adult flies15. Mammalian proteins that were more similar to Toll than to IL1R1 had been spotted in the PubMed database as early as 1994 (REF.16). These proteins were predicted to have TIR domains, as well as leucine-rich repeats that are similar to those of Toll, and to differ from IL1R1 in terms of their lack of immunoglobulin domains. In 1997, one of these mammalian Toll homologues, which was termed hToll at the time, was cloned and studied by Ruslan Medzhitov and Janeway17. They showed that transfection of human monocytes with a CD4hToll chimeric protein (predicted to be constitutively active in the absence of ligand) led to the
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activation of NFB and to the expression of NFBdependent genes, including the gene encoding CD80 (REF.17). CD80 is a protein that provides costimulation via CD28 to Tcells, and this highly important finding provided one of the first observed links between innate and adaptive immunity, as innate hToll signalling in antigen-presenting cells is associated with CD80 expression and Tcell activation. This landmark discovery of the function of hToll fulfilled the criterion that had been postulated by Janeway for the identification of PRRs1 that they would provide an important link between innate and adaptive immunity. In 1998, five mammalian Toll homologues were described and named Toll-like receptors (TLRs) these included hToll which was renamed as TLR4 (REF.18). At the time, no function was ascribed to mammalian TLRs but, as Toll was involved in innate immunity in D.melanogaster, there was a strong suspicion that TLRs would be involved in innate immunity TLRs were the ideal PRR candidates1. Moreover, LPS was an obvious candidate PAMP that might be sensed by a TLR, given its ability to activate NFB. So, almost 10years after the publication of his monograph, Janeways contribution to the field of TLRs was shown to be crucial, as it had inspired several researchers (including Medzhitov, who was working with Janeway) to engage in the search forPRRs.
The LPS receptor uncovered LPS had been studied in great detail as an important component of endotoxin the substance that had been identified as the ill-defined causative agent of Gram-negative bacteria-induced sepsis. However, it was a
challenging reagent to work with, as there is diversity in the polysaccharides present on any given individual strain of bacteria. Few receptors in the history of immunology had received more attention than the LPS receptor, but attempts to determine the identity of this receptor were hampered by the difficulties in working with a ligand that was so structurally heterogeneous and impossible to purify to homogeneity. The importance of lipid A the lipid moiety of LPS for the inflammatory activity of LPS was first described in 1954 (REF.19). However, the correct structure of lipid A was not properly recognized until 1983 (REF.20). Synthetic forms of lipid A, which were devoid of impurities and much easier to work with than natural LPS, became available in 1985 and provided an important way to determine whether previously reported effects of endotoxin were mediated by LPS or simply by biological contaminants. Between 1960 and 1965, a spontaneous mutation occurred in the C3H/HeJ mouse colony at The Jackson Laboratory that rendered the colony resistant to LPS toxicity. The genetic basis of that resistance was determined to be under the control of a single autosomal gene named Lpsd. In 1978, Lpsd was mapped to mouse chromosome fourby backcross-linkage analysis between the C3H/HeJ and the C57BL/6J mouse strains. In the early 1980s, a binding protein for LPS was discovered in serum and was termed lipopolysaccharide-binding protein (LBP)21. Later, in 1986, LPS was shown to activate NF-B7; subsequently, Jiahuai Han etal.22 reported that LPS also activated p38 mitogen-activated protein kinase (MAPK)22.
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for TLR4, but in 2001 it was demonstrated that a soluble (extracellular) form of MD2 functions as a TLR4 coreceptor, which suggested that the MD2LPS complex was the essential ligand for TLR4 (REF.36). Indeed, cocrystallization structures of the TLR4 MD2 complex with a rough mutant form of LPS bound in the lipid A binding pocket were reported in 2009 (REF.37) and showed TLR4MD2 dimers to be the basic signalling units for the LPS receptor. Umeharu Ohto etal.38 more recently published findings using cocrystallization structures of the mouse and human forms of the TLR4MD2 complex bound to a synthetic lipid A precursor known as lipid IVa38. This study demonstrated that an inducible conformational change in MD2, which is brought about by the binding of the ligand to MD2, results in the formation of an m-shaped complex; the authors showed that this is the principal event that is required for LPS activity 38. A clear molecular understanding of the mechanism of recognition of LPS, the causative agent of Gram-negative bacteriainduced septic shock, had finally been achieved and the role of TLR4 as a PRR had been established.
Mammalian TLR ligands Once TLR4 was shown to be involved in LPS recognition, several other microbial products were tested as possible ligands for the other TLRs. In the meantime, it was shown that there are tenTLR genes in humans and 12 in mice39. From 1999 to the present day, the work of Akira and colleagues, who generated multiple TLRand adaptor molecule-knockout mice, has been invaluable for resolving which specific ligands are recognized by eachTLR. TLR2 was shown to sense bacterial lipopeptides: it heterodimerizes either with TLR1 to recognize triacylated lipopeptides or with TLR6 to recognize diacylated lipopeptides, and the structures of both dimers were eventually solved in2007 (REFS4044) (FIG.1). TLR2 has also been shown to recognize a wide variety of other non-lipopeptidic PAMPs from various pathogens39. In 2001, TLR5 was shown to sense bacterial flagellin a protein component of flagella and further work showed that TLR5 could regulate both innate and adaptive responses to bacteria in the intestine4547. A role for TLR10 has not yet been shown, although the sequence of this receptor is known to be most similar to TLR1, and, therefore, TLR10 might heterodimerize with TLR2. Mouse TLR11 has been shown to detect a component of uropathogenic
(20022009). Endogenous ligands for TLRs are identified TRIF is discovered
TLR7 and TLR8 are reported to recognize viral ssRNA
The first function for mammalian SARM1 (a regulator of TRIF) is reported
2001
2002
2003
2004
2006
2007
The first TLR that recognizes viral components is identified (TLR3) Flagellin is identified as a ligand for TLR5 MAL (also known as TIRAP) is discovered
TRAM is discovered
(20072009). Structures of several TLRligand complexes (including TLR4, TLR2TLR1, TLR2TLR6 and TLR3) are solved
It was known that p38 MAPK could also be activated by IL1, as had been shown to be the case for NF-B, which suggested that the receptor for LPS resembled IL1R1 in terms of their downstream-activated signalling pathways. Many individuals attempted to identify a signalling receptor for LPS. Scavenger receptors, such as scavenger receptor A (SRA), were shown to bind to the lipid A moiety of LPS, but were subsequently shown to be involved in the degradation of lipid A rather than in LPS-induced signalling 23. In 1990, an 1825kDa LPS-binding protein was identified that was almost certainly the LPS coreceptor myeloid differentiation factor 2 (MD2), but at that time there was little further investigation into this observation24. Shortly after this, seminal papers showed that LBP works together with CD14 to initiate LPS signalling, and that CD14 increases the sensitization of cells to LPS by a factor of 1,00010,000 (REFS25,26). A popular concept at the time was that CD14 which is a glycosylphosphatidylinositol (GPI)-anchored protein initiates signalling via its GPI anchor, perhaps by directly activating a protein kinase. Later reports provided strong evidence that, contrary to what had been initially thought, CD14 was not a signalling receptor, as both a soluble and a transmembrane form of CD14 were able to mediate LPS responses27,28. These observations initiated a thorough search for a CD14associated signal transducer. Despite considerable effort, the immuno logical community only began to make real progress in the hunt for the LPS receptor following the identification in 1997 of TLR4
(REF.17) (discussed above) and the subse-
quent finding about a year later by Ruey-Bing Yang and colleagues29 that expression of a TLR conferred cellular responsiveness to preparations of endotoxin29. The study by Yang and colleagues was extremely important for the immunological community because it demonstrated that TLRs were in fact true PRRs. However, this work proved to be partly flawed, as the LPS receptor was misidentified as TLR2. Expression of TLR2 did in fact confer LPS responsiveness to transfected HEK293 cells, but only because the LPS preparations that had been used were contaminated with small but active amounts of bacterial lipoprotein30, which is a potent TLR2 agonist. Indeed, despite the strength of this early paper by Yang and colleagues, Craig Gerard31 warned in an accompanying news piece that, on the basis of the chromosomal location of the known TLRs18, the Lpsd gene was likely to encodeTLR4 (REF.31). The breakthrough came later, in 1998, when Bruce Beutlers32 group positionally cloned the Lpsd gene work for which the Nobel Prize was awarded. This study definitively identified Lpsd as Tlr4 (REF.32). Shortly after that, Danielle Malos33 group also reported that Lpsd was in fact Tlr4 (REF.33). Furthermore, a study published later, in 1999,by ShizuoAkira and colleagues34 showed that TLR4knockout mice failed to respond to LPS, which confirmed that TLR4 is the signalling receptor forLPS34. However, TLR4 was soon found to lack LPS-binding activity and, therefore, it was unclear how it would function as an LPS receptor until MD2 was shown in 1999 to confer LPS responsiveness to TLR4 (REF.35). Initially, MD2 was thought to be a chaperone
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Triacylated lipopeptides or diacylated lipopeptides TLR2TLR1 or TLR2TLR6
Flagellin
Uropathogenic bacteria
LPS
TLR5
TLR11
TLR4 Cytoplasm
TIR domain
MAL MYD88
Endocytosed TLR4 Endosome dsRNA IRAK4 IRAK1 IRAK2 TRAF6 RIP1 TRAM TRIF ssRNA TLR3 CpGDNA rRNA
TLR13 TAB2 TAB3 TLR7 TLR8 TRAF3 TLR9
TAK1
MKK3 or MKK6 p38 CREB
MKK4 or MKK7 JNK AP1
IKK IKK TBK1 IKK NF-B IRF3
TRAF6
IRAK4 IRAK1 IRAK2 TRAF3 IKK IRF7
CREB
AP1
NF-B
Pro-inammatory cytokines
IRF3
IRF7
Type I IFNs (IFN and IFN)
Nucleus
Figure 1 | Mammalian TLR signalling pathways. A detailed knowledge of how mammalian Toll-like receptors (TLRs) signal has developed over the past 15years. TLR5, TLR11, TLR4, and the heterodimers of TLR2TLR1 or TLR2TLR6 bind to their respective ligands at the cell surface, whereas TLR3, TLR7TLR8, TLR9 and TLR13 localize to the endosomes, where they sense microbial and host-derived nucleic acids. TLR4 localizes at both the plasma membrane and the endosomes. TLR signalling is initiated by ligandinduced dimerization of receptors. Following this, the TollIL1resistence (TIR) domains of TLRs engage TIR domain-containing adaptor proteins (either myeloid differentiation primary-response protein 88 (MYD88) and MYD88adaptor-like protein (MAL), or TIR domain-containing adaptor protein inducing IFN (TRIF) and TRIF-related adaptor molecule (TRAM)). TLR4 moves from the plasma membrane to the endosomes in order to switch signalling from MYD88 to TRIF. Engagement of the signalling adaptor molecules stimulates downstream signalling pathways that involve
interactions between IL1Rassociated kinases (IRAKs) and the adaptor molecules TNF receptor-associated factors (TRAFs), and that lead to the Naturekinases Reviews(MAPKs) | Immunology activation of the mitogen-activated protein JUN N-terminal kinase (JNK) and p38, and to the activation of transcription factors. Two important families of transcription factors that are activated downstream of TLR signalling are nuclear factorB (NFB) and the interferon-regulatory factors (IRFs), but other transcription factors, such as cyclic AMP-responsive element-binding protein (CREB) and activator protein 1 (AP1), are also important. A major consequence of TLR signalling is the induction of pro-inflammatory cytokines, and in the case of the endosomal TLRs, the induction of typeI interferon (IFN). dsRNA, doublestranded RNA; IKK, inhibitor of NF-B kinase; LPS, lipopolysaccharide; MKK, MAP kinase kinase; RIP1, receptor-interacting protein 1; rRNA, ribosomal RNA; ssRNA, single-stranded RNA; TAB, TAK1binding protein; TAK, TGF-activated kinase; TBK1, TANK-binding kinase 1.
bacteria48 and, in cooperation with mouse TLR12, to bind to the Toxoplasma gondii profilin protein4951. Moreover, mouse TLR13 has been very recently shown to recognize bacterial ribosomal RNA5254.
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Before the discovery of TLR3 in 2001, it was thought that TLRs were not involved in antiviral responses; however, the previous discovery, in 2000, that viral proteins antagonized TLR4 signalling (REF.55), together
with the observation that the fusion protein from respiratory syncytial virus mediated responses via TLR4 (REF.56), had already prompted the idea that TLR ligands might be derived from viruses as well as from
PERSPECTIVES
bacteria. Indeed, in 2001, TLR3 was shown to recognize double-stranded RNA, which is a major component of many viruses, and to mediate activation of the NFB and typeI IFN signalling pathways57 (FIG.1). Structural studies indicated that a common mechanism might underly the activation of all TLRs, as the receptorligand structures for TLR2 and TLR3 (REF.58) complexes both adopted an m-shaped TLR dimer, as was reported for TLR4 (REF.59). In 2012, the crystal structure of zebrafish Tlr5 in complex with a fragment of flagellin was resolved, and, although the receptorligand interaction modes were distinct from those of other non-protein TLR ligands, flagellin binding also caused the formation of an mshaped receptor dimer 60. TLR9 was the next TLR to be implicated in antiviral responses. In 2000, TLR9 had been identified as the receptor for CpG-rich hypomethylated DNA motifs61, which are frequent in bacteria but rare in vertebrates. It was later found that TLR9 also responds to herpesvirus DNA6264. In 2002, TLR7 was characterized as another antiviral TLR, as it was shown to sense the synthetic chemical imiquimod, which was known to stimulate antiviral responses65. Subsequently, in 2004, both TLR7 and the related receptor TLR8 were shown to sense single-stranded viral RNA6668. In recent years, TLRs have also been shown to recognize endogenous ligands. From 2002 to 2005, host nucleic acids were found to function as ligands for TLR9, TLR7 and TLR8 in certain contexts6972. All of these findings established that TLRs are a family of receptors that can initiate innate immunity and inflammation in response to danger signals in the form of infection or tissuedamage.
Elucidation of TLR signalling The intricate signalling pathways that are downstream of mammalian TLRs have been resolved over the past 15years (FIG.1). In 1997, myeloid differentiation primaryresponse protein 88 (MYD88) a protein that was initially recognized to be involved in myeloid cell differentiation was shown to bind to IL1R1 to drive signalling through the NF-B pathway73,74. MYD88 was found to have a TIR domain, which suggested that TIR-homotypic interactions with TIR domain-containing receptors were involved in MYD88 signalling. MYD88 was also shown to have a death domain that interacts with the death domain of a protein kinase termed IL1Rassociated kinase 1 (IRAK1). Following on from this finding, other IRAKs
were discovered and IRAK4 was shown to be the most important receptor-proximal kinase (reviewed in REF.75). IRAK1 was shown to interact with TNF receptorassociated factor 6 (TRAF6), which is a member of the TRAF family of proteins that are known to activate the NFB pathway. Signalling to NFB, p38 MAPK and JUN Nterminal kinase (JNK) was shown to occur downstream of TRAF6, and the kinase cascades, adaptor proteins and ubiquitylation reactions that are involved in these signalling pathways have now been well characterized. The same signals were shown to be induced downstream of TLR2, TLR5, TLR7 and TLR9 activation (FIG.1), which is consistent with the idea that these TLRs are all MYD88dependentTLRs76. At the same time, it was realized that certain TLRs signal from endosomes. Initially, it was observed that CpG-DNA localizes to lysosomal compartments, where it recruits MYD88 (REF.77), and, subsequently, biochemical evidence confirmed that TLR3, TLR7 and TLR9 all localize to endosomes78. CpG-DNA was shown to induce the trafficking of TLR9 from the endoplasmic reticulum to the endolysosome for signalling 79. Following this, interferon-regulatory factor7 (IRF7) was identified as an important MYD88dependent transcriptional regulator that was downstream of endosomal TLR7 and TLR9 and that has a role in the induction of typeI IFN gene expression80,81 (FIG.1). The next TIR adaptor protein to be discovered was MYD88adaptor-like protein (MAL; also known as TIRAP)82,83. In 2001, important studies using MYD88deficient mice showed that the induction of the typeI IFN response via IRF3, as well as the delayed NFB activation and dendritic cell (DC) activation that are downstream of TLR4, were MYD88independent responses8486, suggesting that MYD88independent signalling can be activated downstream of TLRs. However, MAL was not shown to fulfil this MYD88independent role, but was instead shown to be a bridging adaptor that links MYD88 to TLR4 and, to a lesser extent, to TLR2 (REFS8790). By contrast, TIR domain-containing adaptor protein inducing IFN (TRIF; also known as TICAM1), a third TIR adaptor that was characterized between 2002 and 2003, was shown to be involved in the MYD88independent TLR4 pathway, as well as in the MYD88independent TLR3 signalling pathway 9194 (FIG.1). Following this, a fourth TIR adaptor, TRIF-related adaptor molecule (TRAM; also known as TICAM2), was shown to link TRIF to TLR4
(REFS9597). Therefore, TLR4 was shown to
have the most complex signalling arrangement of all the TLRs, activating either the MALMYD88 pathway to induce NFB signalling or the TRAMTRIF pathway to induce IRF3 signalling (FIG.1). The TRAM TRIF pathway is activated downstream of endosomal TLR4, TLR7 and TLR9, which illustrates the importance of subcellular localization for differential signalling by TLRs (FIG.1). In 2006, a fifth TIR adaptor, sterile-- and armadillo-motif-containing protein 1 (SARM1), was shown to inhibit TRIF98. In addition, as SARM1 is the most evolutionarily conserved of the TIR adaptor molecules, it is thought that there are additional functions for SARM1 that remain to be discovered. Interestingly, SARM1 was recently shown to control axonal degeneration in both mice and flies99. Finally, a protein termed Bcell adaptor for PI3K (BCAP; also known as PIK3AP1), which is produced by Bcells, has a domain that is related to the TIR domain and has been proposed as a sixth TIR adaptor molecule. BCAP has been shown to modulate Bcell activation by TLRs100,101. In addition to BCAP, a large number of negative regulators of TLRs have been identified over the past decade; this, as well as the fact that overactivation of TLRdependent innate immune responses can kill the host, shows the importance of TLR modulation.
TLRs in disease As soon as it was realized that TLR4 was involved in LPS sensing and, therefore, that it could have a role in sepsis, it was predicted that targeting of TLRs might be important for the treatment of several diseases. In addition to interfering with TLR responses to treat pathogen infections, an obvious clinical application of the knowledge gained from TLR studies was to use TLR ligands as vaccine adjuvants102. Indeed, in 2005, it was demonstrated that TLR activation is an important aspect of adjuvancy in vaccines, as antigens alone fail to induce an antibody response (unless they are haptenated or aggregated)103. This understanding followed earlier studies that provided the first indications that TLR signalling is an important link between the innate and adaptive immune responses. Another earlier demonstration of the link between TLRs and adaptive immunity came from the finding that the vaccine adjuvant monophosphoryl lipid A, which is a less toxic version of LPS, promotes antibody responses via TLR4 activation104. Moreover, in 2000, TLRs were shown to be expressed
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Box 1 | Prizes won for discoveries in the field of TLRs
Key scientists in the field of Toll-like receptors (TLRs) have won important prizes for their work. This success attests to the importance of the discovery of TLRs for science and medicine. In 2009, Hofmann and Medzhitov shared the Rosenstiel award for distinguished work in basic medical science. Moreover, in 2010, Hoffmann and Akira shared the Keio medical science prize for the discovery of the insect innate immune system and the Toll receptor, and for the elucidation of the molecular mechanisms of innate immune response to microorganisms, respectively. In 2011, 1 year later, Medzhitov, Hoffmann and Beutler were awarded the Shaw Prize in life science and medicine for their discovery of the molecular mechanism of the initiation of innate immunity, which is the first line of defence against pathogens. In the same year, Hoffmann and Akira shared the Canada Gairdner international award, and Hoffmann, Beutler and Steinman were awarded the Nobel Prize for Medicine or Physiology.
on the major antigen-presenting cells, DCs105; and the BCG (bacillus Calmette Gurin) vaccine against tuberculosis was shown to cause DC maturation via TLR2 and TLR4 signalling 106. Bacterial lipo peptides were also shown to stimulate DC maturation via TLR2 1year later (REF.107). Furthermore, Medzhitov and colleagues17 had demonstrated that, in human monocytes, overexpression of what became known as TLR4 could induce expression of the costimulatory molecule CD80 (REF.17) and mechanistically this was shown to involve TRIF108. Finally, imiquimod, which was already being used to treat genital warts caused by the papilloma virus, was identified as a TLR7 ligand in 2002 (REF.65). These and other findings showed the importance of the activation of TLRs in promoting adaptive immunity against pathogens and, consequently, in sustaining host defence. TLR inhibition has also been attempted in the clinic, the goal of which is to limit excessive inflammation that is presumably driven by the overactivation of a particular TLR. Therapies involving the synthetic small-molecule inhibitor of TLR4 eritoran (also known as E5564) were trialed in patients with sepsis as early as in 2007. However, it ultimately had only marginal effects109,110, possibly because it was administered at a late time point in the disease course. In 2010, oligonucleotide-based inhibitors of TLR7 and/or TLR9 were shown to have therapeutic potential in animal models of systemic lupus erythematosus, which is known to involve aberrant immune responses to host nucleic acids111. Finally, in 2012, an inhibitory antibody to TLR2 was shown to efficiently limit ischemiareperfusion injury in the hearts of pigs112 and in the kidneys of mice113. This indicates that endogenous danger signals that are generated by ischemia-induced tissue damage are sensed by TLR2, which, in turn, promotes inflammation and tissue necrosis.
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An important insight into the role of TLRs in human disease has come from the analysis of human genetic variation (reviewed in REF.114). Although single nucleotide polymorphisms in the genes encoding TLRs have been shown to confer a greater risk of developing infectious diseases, the overall effects are modest. By contrast, loss-of-function mutations in TLR-related genes have been strongly associated with susceptibility to infection; in 2007 TLR3 mutations were linked with increased risk of herpesvirus infection115, whereas, at approximately the same time, mutations in the genes encoding MYD88 (REF.116) and IRAK4 (REF.117) were found to correlate with increased susceptibility to pyogenic infections. Moreover, MYD88 mutations are frequently found in certain lymphomas, as was first reported in 2011 (REF.118). Notably, MYD88 deficiency can be lethal in childhood but it seems that if patients survive until adulthood, adaptive immunity is sufficient to protect them in later life116. The fact that individuals with deletions in the important signalling molecule MYD88 are only susceptible to a restricted range of pathogens challenges our view of the importance of TLRs and IL1 in human host defence; indeed, it might indicate that there is redundancy among TLRs and other PRRs, such as the NOD-like receptors (NLRs) and RIGIlike receptors (RLRs). It is also possible that TLRs have a more important role in repairing damaged tissue than in antimicrobial defence; this TLR function might be involved in inflammatory conditions, as in the case of ischemiareperfusion injury (discussed above). Therefore, the true therapeutic potential of TLRs has not yet been realized.
Concluding comments It was an exciting event for many immunologists when the 2011 Nobel Prize for Medicine or Physiology was partly awarded for the discovery of TLRs. However, the
choice of researchers to whom the prize was awarded proved to be somewhat controversial perhaps no surprise given the number of investigators who were involved in the many important discoveries in the field. It is worth noting that other prestigious prizes have also been awarded for the discovery of TLRs: Medzhitov and Hoffmann shared the 2009 Rosenstiel prize; Medzhitov, Hoffmann and Beutler shared the 2011 Shaw Prize; and Hoffmann and Akira shared both the 2011 Canadian Gairdner and the 2010 Keio prizes (BOX1). Overall, the field has benefited from many successful collaborations between laboratories all over the world, including the United States, Europe and Japan. We can all be grateful for the discoveries that have been made in the TLR field and that have led to a renaissance of interest in innate immunity, and we anticipate many more discoveries to come in a field that is in many ways still in its infancy. These discoveries will hopefully have major clinical implications for many diseases.
Luke A.J.ONeill and Andrew G.Bowie are at the School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. Douglas Golenbock is at the Division of Infectious Diseases & Immunology, University of Massachusetts Medical School, Plantation Street, Worcester, MA 01605, USA. Correspondence to L.A.J.ON.and D.G. e-mails: laoneill@tcd.ie; douglas.golenbock@umassmed.edu doi:10.1038/nri3446 Published online 17 May 2013
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Acknowledgements
The authors would like to thank Science Foundation Ireland, the European Research Council and the EU FP7 programme for financial support.
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VIEWPOINT
The plasticity and stability of regulatory Tcells

Shimon Sakaguchi, Dario A.A.Vignali, AlexanderY.Rudensky and Rachel E.Niec, and Herman Waldmann
Abstract | Regulatory T (T Reg) cells are crucial for the prevention of fatal autoimmunity in mice and humans. Forkhead box P3 (FOXP3)+ TReg cells are produced in the thymus and are also generated from conventional CD4+ Tcells in peripheral sites. It has been suggested that FOXP3+ TReg cells might become unstable under certain inflammatory conditions and might adopt a phenotype that is more characteristic of effector CD4+ Tcells. These suggestions have caused considerable debate in the field and have important implications for the therapeutic use of TReg cells. In this article, Nature Reviews Immunology asks several experts for their views on the plasticity and stability of TReg cells.
What defines the TReg cell phenotype and is it stable?
Shimon Sakaguchi. The cardinal pheno-
typical features of thymus-derived CD4+ regulatory T (TReg) cells include their constitutive expression of the transcription factor forkhead box P3 (FOXP3), their cell surface expression of CD25 (the interleukin2 receptor -chain (IL2R); a component of the high affinity IL2R) and their cell surface and cytoplasmic expression of the coinhibitory receptor cytotoxic T lymphocyte antigen 4 (CTLA4)1. Each molecule has its own indispensable function in TReg cells: CD25 is required for TReg cell survival (and presumably for IL2 absorption as part of TReg cell-mediated suppression); CTLA4 is involved in the suppressive function of TReg cells by downregulating CD80 and CD86 expression on antigen-presenting cells; and FOXP3 is essential for TReg cell development and suppressive activity, partly through the maintenance of high levels of CD25 and CTLA4 expression. Regarding phenotypical markers in mice, FOXP3 is the most specific marker for delineating TReg cells from other Tcells, whereas CD25 and CTLA4 are less specific, because they can also be expressed by conventional Tcells. These molecules are stably expressed in physiological states, but can be temporally downregulated under certain conditions. For example, when FOXP3+ TReg cells are transferred to a lymphopenic environment, a fraction of FOXP3+ T cells lose the expression of
FOXP3 and CD25 (REF.2). However, such Tcells appear to be capable of reexpressing FOXP3 and of reacquiring TReg cell suppressive activity upon T cell receptor (TCR) stimulation, presumably because they retain a TReg cell-specific epigenetic status that facilitates the expression of TReg cell-associated molecules, including FOXP3, upon antigenic stimulation3 (see below). In addition, IL2 that is produced by conventional Tcells can inhibit the downregulation of FOXP3 expression in these cells; cotransfer of conventional Tcells with FOXP3+ Tcells into lymphopenic hosts prevents the conversion of FOXP3+ Tcells into FOXP3 Tcells because the conventional Tcells provide IL2 (REF.2). Thus, the phenotype of thymusderived TReg (tTReg) cells is essentially stable even if TReg cells may occasionally and temporally downregulate the expression of FOXP3 and other regulatory-associated molecules.
Dario A. A. Vignali. There are three key
characteristics of the TReg cell phenotype. First, a potent suppressive capacity is clearly the main phenotypical characteristic of TReg cells. The diversity of suppressive mechanisms at their disposal is impressive and is an important feature that endows them with the capacity to influence a very broad range of cell populations and to mediate suppression in a variety of anatomical locations and disease scenarios4. I would speculate that there are important suppressive mechanisms that remain to be identified. Second, it is clear that FOXP3 is an
essential transcription factor required to manifest the TReg cell phenotype5,6. However, it is also clear that FOXP3 does not function alone and that the expression of additional transcription factors is required to define the TReg cell phenotype and to establish their characteristic transcriptional programme7. It is also becoming clear that unique epigenetic changes that are partly induced by TCR signalling are typical of the TReg cell lineage8. Collectively, this epigenetic landscape and transcriptional profile both give rise to a unique TReg cell signature that is characterized by the expression of molecules that can be used to facilitate their identification5,6,9,10. Third, TReg cell development and survival are very dependent on a number of key factors and signals, including IL2, transforming growth factor (TGF) and costimulatory molecules (such as CD28). It is possible that additional key factors remain to be identified. There are some features of the TReg cell phenotype that have been proposed that I feel may be misleading and, thus, are worth discussing here. First, TReg cell suppression is not predominantly contact dependent as has been suggested. While TReg cells can certainly suppress other cells via contactdependent mechanisms, cytokines clearly have a key role4. This misunderstanding may be partly explained by our observation that it is the potentiation or boosting of TReg cell function that has a crucial contactdependent requirement, rather than the suppression itself 11. Second, the notion that TReg cells suppress in a non-antigen-specific manner is misleading. It is certainly true that TReg cells can suppress Tcells that recognize a different antigenic epitope, but TReg cells still recognize antigen and require TCR signalling for optimal activation and function. Third, the notion that TReg cells have a low proliferative capacity is inaccurate and is merely an invitro characteristic owing to the lack of IL2 in the culture medium. In fact, TReg cells proliferate robustly in inflammatory environments (such as in tumours and in the islets of NOD mice), often to a greater extent than other Tcell subsets. The question of TReg cell stability is more complex. There are two FOXP3+ TReg cell populations: tTReg cells and peripherally derived induced TReg (pTReg) cells12. Subtle differences in these two populations, such as the methylation status of conserved noncoding sequence 2 (CNS2; also known as the TReg cell-specific demethylated region (TSDR)) in the FOXP3 locus, may underlie much of the contentious debate over the
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The contributors*
Alexander Y. Rudensky is Chairman of the Immunology Program at the Memorial Sloan-Kettering Cancer Center (MSKCC), New York, New York, USA, and Investigator with the Howard Hughes Medical Institute, USA. His research is focused on investigating the development of T cells, as well as their function and role in the regulation of immune responses to infection and inflammation and in the prevention of autoimmunity. His research is also focused on developing novel methods of tumor immunotherapy and intervention in a wide range of autoimmune and inflammatory diseases, as well as in chronic infections. As a member of the Rudensky laboratory, Rachel E. Niec has focused her research on regulatory T cell lineage stability and peripherally generated TReg cells. Rachel is a member of the Tri-institutional M.D./Ph.D. programme. Shimon Sakaguchi obtained his M.D. and Ph.D. in Japan, and trained as a pathologist and immunologist in Japan and the United States. He is currently the Vice Director for human immunology and is Professor at the World Premier International Immunology Frontier Research Center, Osaka University, Japan. His main research interest is in immunological tolerance and immune regulation. Dario A. A. Vignali received his Ph.D. in 1988 from the London School of Hygiene & Tropical Medicine, University of London, UK. He then held postdoctoral positions at the German Cancer Research Center, Heidelberg, Germany, and then at Harvard University, Cambridge, Massachusetts, USA. He is currently Vice Chair and Member (Full Professor equivalent) of the Department of Immunology, St. Jude Childrens Research Hospital, Memphis, Tennessee, USA. His research focuses on the molecular and cellular aspects of regulatory T cell function, immune regulation by inhibitory receptors and inhibitory cytokines (interleukin35) in tumour immunity, mucosal immunity and type 1 diabetes. He also studies proximal events in T cell receptorCD3 signalling. Herman Waldmann is Emeritus Professor of Pathology at the University of Oxford, UK, after he recently retired as Head of the Sir William Dunn School of Pathology, University of Oxford. His main research interests have been to therapeutically harness the bodys own tolerance processes to enhance the acceptance of transplants and the reversal of autoimmune diseases. His laboratory was responsible for the discovery and academic development of the CAMPATH1 antibodies that culminated in the licensing of alemtuzumab (Lemtrada). His current research focuses on the mechanisms by which regulatory T cells mediate dominant (infectious) tolerance.
*Listed in alphabetical order
features of TReg cells and, therefore, its expression does not unequivocally mark the cells to be TReg cells. Indeed, FOXP3 can be expressed by non-regulatory Tcells; cells can initiate and then discontinue FOXP3 expression or they can express FOXP3 in a non-permissive chromatin landscape. Multiple studies have demonstrated transient activation-induced upregulation of FOXP3 expression in both mouse and human Tcells19,20. These observations of transient FOXP3 expression do not necessarily indicate TReg cell lineage instability, as it is possible that these cells might never have fulfilled the strict phenotypical and molecular criteria necessary to be defined as bonafide TReg cells. Indeed, the examples of transient expression of FOXP3 refer to cells that never fully engaged in the positive autofeedback loop at the Foxp3 locus (discussed below) and thus have not committed to the TReg cell lineage. The conditions of cellular activation might induce a state of functional adolescence an intermediate maturation stage in which newly generated TReg cells might engage in some eclectic behaviour before establishing the full TReg cell character and commitment.
Herman Waldmann. The errant description
issue of stability 5,6. Most researchers agree that under normal circumstances tTReg cells are very stable and long-lived13. However, it has been argued that under inflammatory or pathogenic conditions some TReg cells may become unstable, losing FOXP3 expression and developing features that are more characteristic of effector Tcell populations (termed exTReg cells)14. However, the prevalence of this instability remains contentious, as does the origin of this unstable population. There are three possibilities. First, it is possible that a genuine tTReg cell population becomes unstable. Second, it is possible that this perceived instability only affects pTReg cells, which cannot yet be unambiguously distinguished from tTReg cells. Third, it has been suggested that transient FOXP3 expression in a small Tcell population may inadvertently define these cells as exTReg cells but these cells may never have been destined to become a stable bonafide TReg cell population3. Although more analysis is required, the bottom line is that FOXP3+ TReg cells are predominantly stable. However, it is possible that a small percentage may become unstable under unique and/or extreme circumstances.
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Alexander Y. Rudensky and Rachel E. Niec.
TReg cells are defined by having a suppressive phenotype endowed by high and sustained expression of the transcription factor FOXP3, as well as by other TReg cell signature genes. The TReg cell lineage is highly stable owing to the multiple mechanisms that maintain high levels of FOXP3 expression after the establishment of the TReg cell transcriptional programme13 (see below). FOXP3 is considered to be the lineage specification factor for this Tcell subset. Indeed, forced FOXP3 expression in CD4+ Tcells results in a regulatory phenotype and function, whereas experimental deletion of the Foxp3 gene in differentiated TReg cells results in the loss of their suppressive capacity 1517. However, FOXP3 cooperates with other nuclear factors, the activity of which must precede or coincide with the expression of FOXP3 to establish the molecular signature and function of TReg cells. FOXP3 binds to a set of pre-existing regulatory elements that become accessible in pre cursor cells in response to TCR signals of a particular strength, as well as in response to other cues8,18. Thus, although it is required, FOXP3 expression alone does not impart the characteristic suppressive and homeostatic
of suppressor Tcells in the 1970s has been followed by a general acceptance in the early 1990s that some CD4+ Tcells can and do regulate immune responses. Among these suppressive cells is a population of Tcells that express the transcription factor FOXP3. Even this population of cells has been shown to exhibit significant heterogeneity, some of which is related to a physiological need for these regulators to express certain features of the different Tcell subsets that they control21, while a further source of heterogeneity is the socalled stability and plasticity of their functional status. The currently used terms stability and plasticity do not distinguish between epigenetic stability within individual TReg cells and competition with cellular contaminants, uncommitted cells or cells that have reverted from more committed cells in the particular sample population under investigation. In addition, although we are really concerned with functional stability, what is often measured is the stability of FOXP3 expression, as though that alone were the basis for suppressive function. However, it is becoming clear that the post-translational state of FOXP3 may be important to its function and that proinflammatory cytokines can affect this22. Not surprisingly, these types of uncertainties all contribute to the current confusion regarding the stability and plasticity of TRegcells.
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Some of the controversies about TReg cell stability may not be as irreconcilable as they might appear at first, but could well reflect the limitations of the read-out systems that are used; indeed, in these systems the context (for example, inflammatory rather than quiescent environments, or lymphopenic rather than replete immune systems) determines the extent of the epigenetic changes that are associated with increasing commitment to the TReg cell phenotype3,13,2325. Moreover, as most studies investigating the stability of TReg cells have used bulk populations rather than single cells26, the seemingly robust interpretations of such studies may not completely reflect the characteristics of individual cells at different stages of develop ment. The most stable TReg cell, even when it is fully differentiated, may still have the capacity for destabilization if given the appropriate stimulus. How might TReg cell stability or plasticity be regulated? To what extent does the context of antigen exposure determine the stability of TReg cells?
D.A.A.V. I prefer to view stability and
plasticity as different terms, with stability determined by whether a FOXP3+ TReg cell becomes a FOXP3 exTReg cell, and plasticity determined by whether a TReg cell changes its migratory or functional capabilities but still maintains its fundamental FOXP3+ TReg cell identity. Although FOXP3+ TReg cells are predominantly stable, I feel it is very probable that there are several external signals and factors (such as Toll-like receptor (TLR)induced IL6) that could induce TReg cell instability 27 and, thus, that there are several counteractive mechanisms that are required to maintain TReg cell stability, particularly in inflammatory environments. Determining the factors and mechanisms on both sides of this equation will be important. These factors and mechanisms could act by impinging on the expression and function of FOXP3 (or other transcription factors that are required for the TReg cell phenotype), by modulating the expression of effector Tcell programmes that need to be repressed in TReg cells, or by modulating the expression of survival and/or quiescence factors that need to be enhanced in TRegcells. TReg cell plasticity is probably regulated by a complex network of cooperative and counteractive transcription factors. While much still remains to be clarified, it does appear that certain lineage-specific transcription factors can be expressed by
TRegcells, endowing them with an enhanced capacity to regulate effector T helper (TH) cell populations that use the same transcription factors (such as interferon-regulatory factor 4 (IRF4), signal transducer and activator of transcription 3 (STAT3) or Tbet)2830. The extent to which this is a selective process as opposed to a regulated process is unclear. There is also the important issue of functional plasticity and whether all TReg cell-associated suppressive mechanisms can be used by every TReg cell, or whether there are subpopulations that have different subspecialities that allow them to adapt to different environments. It seems probable that TReg cells have mechanisms that allow them to adapt to highly varied targets and microenvironments, either on a single-cell basis or as a population. The context of antigen exposure or, more specifically, the extent of TCR and/or costimulatory signalling is likely to affect TReg cell stability. Indeed, TReg cells in inflammatory sites are probably exposed to three sets of signals that will determine their stability: one set of signals from the TCR and costimulatory molecules, another set of signals that drive instability and a final set of signals (both cell-intrinsic and cell-extrinsic) that actively maintain stability. Indeed, the latter may constitute the primary basis for the TReg cell stability observed. However, the molecular details of these signals and how they might interact remain unknown.
A.Y.R. and R.E.N. Once the TReg cell tran-
those that have been generated invitro in the presence of TGF or those that have been recently generated invivo and that fail to demethylate the CpG island, lose FOXP3 expression despite the fact that it was initially expressed at high levels3,19,33. These cells may represent uncommitted precursor cells that have yet to choose a lineage. Furthermore, other transcription factors have been shown to bind to CNS2 and other regulatory elements at the Foxp3 locus and may contribute to stable FOXP3 expression34; these transcription factors include nuclear factor of activated T cells (NFAT), cyclic AMP-responsive element-binding protein (CREB)ATF, nuclear factor-B (NFB) and ETS1 (which are downstream of TCR activation), as well as STAT5 and SMADs (which are activated through IL2 and TGF receptor signalling). Although the extent to which these factors and persistent IL2R signalling stabilize FOXP3 expression is not yet clear, FOXP3 expression alone may be insufficient to maintain lineage commitment. Thus, the stability of the TReg cell lineage is probably acquired in a two-step manner: through the establishment of an epigenetic state, including the demethylation of CNS2, that is favourable for continued FOXP3
Glossary
Asymmetric Tcell division
A process by which two daughter cells can inherit different amounts of immune receptors and signalling components from a parent cell during Tcell division. It has been suggested that this process occurs because of the polarity of the dividing cell that is associated with immunological synapse formation and that it could specify different fates to the progeny of an individual Tcell.
scriptional programme is established, there are multiple mechanisms in place to endow TReg cells with remarkable stability; that is, the capacity to heritably maintain their transcriptional and functional features. FOXP3 expression maintains the TReg cell suppressive phenotype; when FOXP3 is experimentally ablated in mature TReg cells, the resulting cells are capable of producing effector cytokines and causing inflammation17. FOXP3dependent lineage commitment at least partly relies on the demethylation of a CpG island that is located in a regulatory element in CNS2 of the FOXP3 locus31,32. Deletion of CNS2 does not affect the initial acquisition of high levels of FOXP3 expression, but it results in cell division-dependent loss of FOXP3 expression. FOXP3, with the assistance of the transcription factor runtrelated transcription factor1 (RUNX1), binds to CNS2 in the demethylated state to maintain FOXP3 expression32. Accordingly, some FOXP3expressing cells, for example,
Conserved non-coding sequence 2

(CNS2). The element engaged in the positive auto-feedback loop that confers heritable maintenance of forkhead box P3 (FOXP3) expression once demethylated. Also known as the TSDR (TReg cell-specific demethylated region).
CpG island
A sequence of 0.52 kilobases that is rich in CpG dinucleotides. CpG islands are mostly located upstream of housekeeping genes and also upstream of some tissue-specific genes. They are constitutively non-methylated in all animal cell types.
Infectious tolerance
The ability of a tolerized population of Tcells to induce tolerance in a new, naive population of Tcells. Tolerance might be to the same antigens or to new antigens that are encountered in the same context (linked suppression). Newly tolerized Tcells can, in turn, induce tolerance in other Tcells.
NOD mice
Non-obese diabetic (NOD) mice spontaneously develop a form of autoimmunity that closely resembles human type1 diabetes.
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expression and through the subsequent stabilization of this state by FOXP3 autoregulation at the demethylatedCNS2. Cells that only transiently express FOXP3 for the aforementioned reasons or that fail to establish an epigenetic state that is favourable for the binding of FOXP3 at target sites do not represent fully differentiated and functionally competent TReg cells; rather, they may represent an uncommitted intermediate cell state or a cell that has aborted TReg cell differentiation. The multistep process of TReg cell commitment affords uncommitted precursor cells the option of differentiating into alternative cell lineages as instructed by environmental cues. Once the stabilizing FOXP3 positive feedback regulatory pathway has been established, circumstances invivo that disrupt this regulatory programme of lineage stability are probably rare but may exist in extreme inflammatory conditions or upon IL2 deprivation13,14,35.
S.S. FOXP3+ TReg cells are functionally and
phenotypically stable after many cell divisions in inflammatory sites13. This indicates that stability is maintained by particular types of constant cell-extrinsic stimuli (such as antigen, costimulation and cytokines) and/or by a cell-intrinsic mechanism that induces TReg cells to express TReg functionassociated molecules. Regarding the cell-intrinsic mechanism, there is accumulating evidence to suggest that the epigenetic status of the TReg cell is crucial for the maintenance of its function and phenotype8,36,37. Among several epi genetic mechanisms, DNA hypomethylation of specific genes is the most important for sustaining TReg cells as a cell lineage because it is highly inheritable, relatively stable and linked to gene expression. A recent genomewide study has shown that TReg cells retain a TReg-specific DNA hypomethylation pattern, especially in the genes encoding TReg function-associated or TReg-specific molecules such as CTLA4 and the zinc finger proteins Helios and Eos8. The induction of this TReg-type epigenetic pattern is FOXP3 independent, and both TReg cell epigenetics and FOXP3 expression depend on TCR stimulation. Developing TReg cells that have these epigenetic changes but lack expression of FOXP3 express low levels of CD25 and CTLA4; however, FOXP3 appears to sustain the high and constitutive expression of these molecules in TReg cells. Thus, FOXP3expressing TReg cells with these TReg-type epigenetic changes are functionally and phenotypically stable. However, a small fraction of FOXP3+ Tcells without
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stable epigenetic modifications would be unstable and would lose FOXP3 expression8. By contrast, those Tcells with these epigenetic changes but without expression of FOXP3 are primed to express FOXP3 following TCR stimulation and, once FOXP3 is expressed, would be driven to a stable TReg cell lineage3. As seen in the process of TH cell differentiation from naive Tcells after antigen exposure, TReg cells show adaptive properties by sensing environmental cues28,29. For example, like TH1 cells, FOXP3+ TReg cells that are recruited to a site of TH1type inflammation express Tbet and CXC-chemokine receptor3 (CXCR3), and they apparently produce interferon- (IFN)28; similarly, TReg cells expressing STAT3, retinoic acid receptorrelated orphan receptor-t (RORt) and CC-chemokine receptor 6 (CCR6) as well as producing IL17 accumulate at sites of TH17 cell-mediated inflammation29. Although the production of pro-inflammatory cytokines by TReg cells may appear to contradict their immunosuppressive function, this does not necessarily mean that TReg cells are plastic and have pro-inflammatory functions for the reasons discussedbelow. First and most importantly, TReg cells do not exert suppressive activity until they receive strong antigenic stimulation, as illustrated by studies carried out invitro38,39. Upon TCR stimulation, FOXP3 and other transcription factors, as well as corepressor and coactivator molecules, assemble to repress or to activate a particular gene (for example, repression of Ifng in TReg cells) depending on the combination of factors assembled. In addition, the demonstration of cytokine production at the level of mRNA in TReg cells that have been stimulated with the mitogens PMA (phorbol 12myristate 13acetate) and ionomycin, as has been shown in many reports, does not necessarily mean that TReg cells actually secrete these cytokines in significant amounts. Thus, it is necessary to determine whether TCR and/or CD3 stimulation can induce the secretion of these proinflammatory cytokines by TReg cells and not that they merely induce the expression of the cytokine mRNA.
H.W. Antigen, in an appropriate context,
is clearly a driver for the induction of TRegcells40. Historically, there are many examples of regulation being destabilized when antigen is made unavailable41. However, if one wishes to understand how the context of antigen experience determines long-term stability, then more information is required.
First, information about markers of differentiation is necessary. Our best indicators of FOXP3+ Tcells with a tractable level of commitment to regulation are currently FOXP3 expression together with demethylation of some key genes Foxp3 at intron1 (the position of CNS2), tumour necrosis factor receptor superfamily member 18 (Tnfrsf18; the gene that encodes GITR) at exon 5, Ctla4 at exon 2 and Ikaros family zinc finger protein4 (Ikzf4; the gene that encodes Eos) at intron1 (REF.8). Given that TReg cells lack many immune effector functions, the methylation status of the genes that mediate these functions may provide additional guidance. In due course we should expect other technologies, such as modern proteomic approaches, to offer further markers to guide us in determining the long-term stability of TRegcells. Second, understanding the heterogeneity of TReg cells is important. In terms of the preexisting heterogeneity in the TReg cell population, it would be helpful to establish epigenetic and functional parameters of TReg cell clones that have been isolated from both mice and humans, and to correlate these parameters with the functional status of the cell. A recent report clearly demonstrated that single-cell isolates of human natural TReg cells exhibit significant heterogeneity, which is not visible in bulk cultures26. Following various forms of inflammatory stimuli it would be helpful to understand what features of the TReg cell, when called to action, enable it to selectively repress the initiating inflammation, but that may be inappropriate in other inflammatory settings (for example, comparing TH1-, TH2- and TH17based inflammatory events). Third, it is important to understand the decision-making processes that are involved in the expansion of the TReg cell population. What has been missing in the analyses of TReg cell plasticity, especially in the case of pTReg cells, has been the assessment of outcomes in sequential divisions of daughter cells that are derived from individual TReg cells. Recent work with other lymphocyte populations indicates that there are a range of factors that can determine whether divisions progress symmetrically or asymmetrically (asymmetric Tcell division)42; asymmetric division provides the potential for the diversification of cell fates and for daughter TReg cells to be diluted in an expanding population. Modern flow cytometry imaging approaches should allow a better understanding of how daughter cells progress under diverse stimulatory conditions and should provide guidelines for how to steer division to give rise to more homogeneous or heterogeneous progeny, as might be desired.
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Is there a difference in the stability of mouse and human TReg cells? FOXP3+ Tcells in mice and humans are heterogeneous in their function, phenotype and epigenetic status. When considering the argument for TReg cell plasticity, this heterogeneity needs to be taken into consideration because plasticity could be a property of only a particular subpopulation of TReg cells rather than being a general property of all FOXP3+ TReg cells. This is especially the case in humans20. FOXP3+ T cells in humans can be divided into three populations: FOXP3lowCD45RAhiCD25low TReg cells (designated as resting or naive TReg cells); FOXP3hiCD45RAlowCD25hi TReg cells (known as effector or activated TReg cells); and FOXP3lowCD45RAlowCD25low non-TReg cells, which are not suppressive and are capable of producing pro-inflammatory cytokines43. The TReg-type epigenetic status, including DNA hypomethylation of the CNS2 region in FOXP3, is also different among these populations of cells: naive and effector TReg cells are more demethylated than non-TReg cells. Upon TCR stimulation, naive TReg cells upregulate FOXP3 expression, proliferate and differentiate into effector TReg cells, which are highly proliferative and terminally differentiated, and the majority of effector TReg cells die by apoptosis after exhibiting strong suppressive activity. Therefore, there is a possibility that the production of pro-inflammatory cytokines (which does not necessarily mean cytokine secretion) by FOXP3+ Tcells can be attributed to the presence of non-TRegcells in the population sample. It needs to be determined in humans whether the observations that have been ascribed to TReg cell plasticity could be, at least partly, due to the heterogeneity of FOXP3+ T cells, and how each population of FOXP3+ T cells differentiate to or from other FOXP3+ or FOXP3 Tcells. The heterogeneity of FOXP3+ T cells is less clear in mice than in humans. The use of cell-fate reporter mice has revealed the presence of phenotypically unstable plastic FOXP3+ T cells14. FOXP3+ T cells with a full TReg-type epigenetic pattern are highly stable in terms of function and phenotype, whereas FOXP3+ T cells that have not yet established this epigenetic pattern are unstable (some of them differentiate into full TReg cells with a complete TReg-type epigenetic pattern, whereas others lose FOXP3 expression and revert to naive Tcells)8.
S.S. Recent studies have shown that
In addition, using cell-fate reporter mice, one can detect a small population of cells that has once expressed FOXP3 and has then lost its expression3. These exTReg cells appear to retain the TReg-type epigenetic pattern and can easily be reconverted to FOXP3+ T cells. In addition, as discussed above, the conversion of FOXP3+ Tcells into FOXP3 Tcells can be prevented by IL2 secretion by other Tcells or by the converted Tcells themselves2. It is currently unclear whether such exFOXP3expressing T cells constitute a significant fraction of Tcells in the physiologicalstate. Taken together, these findings in humans and mice indicate that, although some FOXP3+ T cells might lose FOXP3 expression, this might not be a general property of FOXP3+ TReg cells; indeed, these exTReg cells may reexpress FOXP3 if they retain the TReg-type epigenetic status. These findings also suggest that the conversion of FOXP3 TReg cells to FOXP3+ TReg cells might not be frequent in the physiological state because conventional Tcells secrete IL2, which prevents this process and sustains the expression of FOXP3 by TReg cells.
H.W. Within this framework of uncertainty
regarding TReg cell stability and plasticity, there have also been concerns that human TReg cells are more unstable than mouse TReg cells the cells from which we have gained much of our optimism for therapeutic manipulation. This concern partly derives from our inability to purify human TReg cells owing to poorly defined combinations of surface markers; in mice, the availability of markers that can be expressed under the control of the Foxp3 locus guarantees pure starting populations. Given other confounding factors, such as the uncertainty of what determines a TReg cell that is committed to regulation or the uncertainty of the cellular microenvironment that delivers the final stable functional regulators, it seems premature to make, let alone worry about, such comparisons.
A.Y.R. and R.E.N. The cell-intrinsic mecha-
As in mice, human TReg cells are defined by a suppressive phenotype and by sustained high FOXP3 expression. Human FOXP3expressing T cells can be divided into three categories based on their expression of FOXP3, CD25 and CD45RA. FOXP3lowCD25medCD45RA+ Tcells and FOXP3hiCD25hiCD45RA Tcells appear to represent competent TReg cells with potent suppressive capacities; these cells are characterized by CNS2 demethylation and stable expression of FOXP3 invivo. By contrast, FOXP3lowCD25medCD45RA Tcells are not suppressive, have extensive methylation at the CNS2 and are predictably prone to proliferation-dependent loss of FOXP3 expression43. Thus, as in mice, FOXP3 expression alone is insufficient to identify human TReg cells. Activation of human CD4+ Tcells invitro can result in transient unstable expression of FOXP3 and, while this phenomenon has also been observed in mice, it is more pronounced in humans20. We suggest that in humans, as in mice, the committed, CNS2 demethylated, suppressive TReg cells represent a stable population that serves to mitigate inflammation-induced tissue injury in a variety of settings.
D.A.A.V. This is an important question but
I feel we do not have enough information to provide a definitive answer. Given that TReg cell stability is often determined by loss of expression of FOXP3, the expression of FOXP3 by activated human Tcells serves to further complicate this issue. My general view is that the mechanisms that maintain TReg cell stability and that may drive their instability are likely to be similar in humans and mice. However, there may be important differences in the extent to which pathways that maintain stability are required in different disease settings. This may have significant clinical implications. This important question will be easier to address when we have a full appreciation of all the pathways that maintain and impinge on TReg cell stability. What are the functional implications of TReg cell stability or plasticity? seem preoccupied with understanding the plasticity of TReg cells rather than other CD4+ Tcell subsets? There are very good reasons for this that are related to understanding immunological diseases and their treatment. One reason is that TReg cells with self-reactive TCRs might, if destabilized,
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nisms that confer stability of FOXP3 expression in mouse and human TReg cells are likely to be similar. The characterized regulatory DNA elements at the FOXP3 locus, including CNS2, are well conserved between mice and humans. FOXP3expressing T cells in mice and, more prominently, in humans are heterogeneous in terms of expression levels of FOXP3, CD25, CTLA4 and other factors that are associated with TReg cell activation or function.
H.W. Why does the research community
PERSPECTIVES
cause autoimmune disease23. Other reasons relate to the need to enhance a patients own TReg cells either within the patient themselves or by expanding the cells exvivo and injecting them back into the patient in order to minimize the use of immunosuppressive drugs44. Such strategies would benefit from an understanding of how TReg cells could be operationally stabilized. Finally, there is a growing optimism in cancer immunotherapy that if one could destabilize immune regulation then a more effective mode of tumour immunization would be possible. If we reflect on how immunological homeostasis is maintained, and what might need to be done to reset a dysfunctional immune system, it may well be that therapeutically we only need to modestly tip the balance between regulatory and effector immune function to restore a long-term ceasefire. This might be achieved by encouraging more death of effector Tcells than of TReg cells; by the selective expansion of regulatory populations; or by guiding more wouldbe TReg cells to a committed functional status. Recent studies have described a real potential for epigenetic therapeutics45,46 to enhance commitment to the TReg cell lineage. Once the overall stability threshold of the population of TReg cells has been raised to a certain level, this state might be sustained via intrinsic mechanisms of infectious tolerance44, thereby limiting the duration of treatments and the risks of unwanted side effects. In short, for therapeutic applications we might only need to think about stability and plasticity in quantitative terms rather than as an allornothing dilemma. is to maintain immune self-tolerance and homeostasis. Functional stability is crucial for the ability of FOXP3+ tTReg cells to suppress autoimmune disease because they are more self-reactive than conventional Tcells with respect to their TCR repertoire and are therefore more hazardous when they are converted to FOXP3 non-TReg cells. The functional stability of TReg cells is also necessary to control a variety of inflammatory responses because some of the pro-inflammatory cytokines, such as IL6, may temporarily antagonize the TReg cell suppressive function. Therefore, it is not their plasticity but their capacity to adaptively differentiate (while sustaining FOXP3 expression) that is crucial for TReg cell function. In addition, they can acquire a migratory capacity to specific inflammatory sites, as well as additional
suppressive activity for example, to secrete IL10 or to produce granzyme B and perforin depending on the type, tissue site or chronicity of the inflammatory response47. It remains to be determined whether TReg cell functional instability, for example, due to anomalies in TReg cell epigenetics, contributes to any immunological diseases inhumans. implications of TReg cell stability and plasticity could be substantial. First, TReg cells have been shown to represent a major barrier to effective antitumour immunity as well as to sterilizing immunity to chronic viral infections10,48. Identifying the pathways that maintain TReg cell stability in inflammatory sites could present potentially important new therapeutic targets to undermine intratumoural or antiviral TReg cells and to enhance disease clearance. Second, TReg cells may be unstable or may not function effectively in autoimmune or inflammatory diseases. It is conceivable that the pathways that maintain stability are not optimally used in certain disease settings, which could lead to TReg cell insufficiency. One could potentially enhance pathways that maintain TReg cell stability to limit or to cure disease. Third, clinical trials using invitro generated or expanded TReg cells are in progress. Once the pathways that maintain or undermine TReg cell stability invivo are known, these cells could be manipulated during their invitro preparation and/or following transfer into the patient to optimize their clinical efficacy. Likewise, an in-depth understanding of the pathways that mediate TReg cell plasticity could also be used to limit or to enhance TReg cell activity in different disease settings. requisite for their ability to limit transient, and to prevent lasting, tissue damage in response to various types of inflammation or injury. A loss of TReg cell function in a particular environment can have potentially devastating consequences. Different tissue and inflammatory environments can impose adaptive changes on TReg cells, modifying their functional properties, through the induced expression of transcriptional regulators and of homing and growth factor receptors. Although the stability of FOXP3 expression, which is imprinted as the result of a positive FOXP3 auto-feedback loop, is essential for immune homeostasis, it remains unknown whether particular states of TReg cells that are induced in response to specific tissue or inflammatory cues are reversible (that is, plastic) or lasting. The
A.Y.R. and R.E.N. TReg cell stability is a pre D.A.A.V. The functional and translational
stability of bonafide TReg cells implies that they have robust potential for cellbased therapies. Nevertheless, unstable FOXP3expressing non-TReg cells (as described above) represent a potential complication of these approaches and need to be taken into account. A mechanistic understanding of stable FOXP3 expression will facilitate the use of techniques to manipulate, and assays to validate, stable FOXP3 expression, which should facilitate efficacious use of TReg cells forimmunotherapy.
Shimon Sakaguchi is at the World Premier International Immunology Frontier Research Center, Osaka University, Suita 5650871, Japan and at the Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 6068507, Japan. Dario A. A.Vignali is at the Department of Immunology, St. Jude Childrens Research Hospital, Memphis 381053678, Tennessee, USA. Alexander Y.Rudensky and Rachel E.Niec are at the Immunology Program and Howard Hughes Medical Institute at Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA. Herman Waldmann is at the Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Correspondence to S.S., D.A.A.V., A.Y.R. and H.W. e-mails: rudenska@mskcc.org; shimon@ifrec.osaka-u.ac.jp; vignali.lab@stjude.org; Herman.Waldmann@path.ox.ac.uk doi:10.1038/nri3464 Published online 17 May 2013
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Acknowledgements
S.S. acknowledges the daily discussions he had with his colleagues that helped him to write his comments for this article. D.A.A.V. apologises to those investigators who contributed important observations related to the questions he covered that he could not discuss or quote owing to space limitations. D.A.A.V. is supported by the US National Institutes of Health ( N I H ) ( g ra n t s A I 0 91 9 7 7 , A I 0 3 9 4 8 0 , A I 0 5 21 9 9 , DK089125), American Asthma Foundation (grant100128), National Cancer Institute Comprehensive Cancer Center grant (CA21765) and ALSAC. A.Y.R. is supported by an NIH grant (R37 AI034206) and is an investigator at the Howard Hughes Medical Center. R.E.N. is supported by an NIH Medical Scientist Training Program grant (GM07739) and a National Institute of Neurological Disorders and Stroke grant (1F31NS073203-01). H.W. wishes to acknowledge the help of his colleagues D. Howie, R. Hilbrands and S. Cobbold in writing his comments for this article.
The authors declare competing financial interests. See Web version for details.
FURTHER INFORMATION
Shimon Sakaguchis homepage: www.ifrec.osaka-u.ac.jp Dario A. A. Vignalis homepage: http://www.stjude.org/vignali Alexander Y. Rudenskys homepage: http://www.mskcc.org/research/lab/alexander-rudensky Herman Waldmanns homepage: http://www.path.ox.ac.uk/dirsci/immunology/waldmann
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RESEARCH HIGHLIGHTS

Nature Reviews Immunology | AOP, published online 7 May 2013; doi:10.1038/nri3462

VOLUME 13 | JUNE 2013

The interferon paradox

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Stand by for action!

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TLRs get rhythm

TLR expression by intestinal epithelial cells ... is under circadian control

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Distal signalling in mast cell degranulation

A genetically encoded T cell calcium indicator

Metabolites mangle microbes

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The origins of colonic TReg cells

colonic TReg cells seem to derive predominantly from FOXP3+CD4+ thymocytes

Photod isc/Getty I mages

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Adaptive control of NK cells

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Regenerating muscles the type 2 way

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398 | JUNE 2013 | VOLUME 13

Box 1 | Sensing of mitochondrial stress

IFN-inducible factor Nucleus

Non-canonical NLRP3 inammasome in response to Gram-negative bacteria

2013 Macmillan Publishers Limited. All rights reserved

IFN IFNGR Cytoplasm

Type I IFN IFNAR

Priming via deubiquitylation

NLRP3 inammasome Ub Caspase 1

Pro-IL-1 NLRP3 NLRP3 mRNA miR-223 Degradation NLRP3 pro-IL-1 AIM2

Cryopyrin-associated periodic syndrome

Pro-caspase 1 dsDNA ASC

AIM2 inammasome HIN dsDNA PYD Caspase 1

CARD, caspase activation and recruitment domain.

Nature Reviews | Immunology

P2RX7 Pore formed by bacterial toxins

InsP3R ER Ca2+ Unfolded protein response

Unknown CHOP-independent ER stress response ?

Sources of experimental evidence

Accessory NLR proteins

CARD-containing regulatory proteins

PYD-containing regulatory proteins

Source of experimental evidence

Other regulators (cont.)

2013 Macmillan Publishers Limited. All rights reserved

VOLUME 13 | JUNE 2013 | 409

Competing interests statement

The authors declare no competing financial interests.

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Activated Transcription Suppressing STATs factors cytokines

Canonical Roles in host cytokines protection produced

TH2 cells TH17 cells

GATA3 and MAF RORt and ROR

IL4, IL5 and IL13

IL23 and IL1 IL-6 and IL1 TGF

PU1 and IRF4

IFN and IL27

STAT1 STAT3 STAT5

RORt and AHR

High doses of TGF

TReg cells TFH cells