Journal of Immunological Methods 281 (2003) 65 78 www.elsevier.

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Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation
Michael R. Betts *, Jason M. Brenchley, David A. Price, Stephen C. De Rosa, Daniel C. Douek, Mario Roederer, Richard A. Koup
Laboratory of Immunology, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Bethesda, MD 20892, USA Received 22 January 2003; received in revised form 25 June 2003; accepted 7 July 2003

Abstract Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNg. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8 + T cells expressing cognate T cell receptors (TCR). D 2003 Elsevier B.V. All rights reserved.
Keywords: Degranulation; T lymphocyte; Intracellular cytokine; CD107a; CD107b

Abbreviations: APC, allophycocyanin; CFSE, carboxyfluorescein diacetate succinimidyl ester; CMTMR, chloromethyl-benzoyl-aminotetramethyl-rhodamine; CMV, cytomegalovirus; CTL, cytotoxic T lymphocyte; ELISpot, enzyme-linked immunospot; FCS, fetal calf serum; FITC, fluorescein isothiocynate; ICS, intracellular cytokine staining; HIV, human immunodeficiency virus; HLA, human leukocyte antigen; IFNg, interferon-gamma; LAMP, lysosomal associated membrane protein; MHC, major histocompatibility complex; MIP1h, macrophage inflammatory protein-1-beta; NK, natural killer cell; PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; PerCP, peridinin chlorophyll protein; PHA, phytohemagglutinin; qPCR, quantitative polymerase chain reaction; SEB, staphylococcus enterotoxin-B; TNFa, tumor necrosis factor alpha. * Corresponding author. Tel.: +1-301-594-8612; fax: +1-301-480-2779. E-mail address: mbetts@mail.nih.gov (M.R. Betts). 0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0022-1759(03)00265-5

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1. Introduction After MHC-mediated recognition of cognate peptide, CD8+ cytotoxic T lymphocytes exhibit two general effector functions: production of soluble factors, including cytokines and chemokines, and target cell killing. Recent technological advances in multiparameter flow cytometry have enabled the enumeration of antigen-specific CD8+ T cells (MHC-class I tetramer staining) and a direct assessment of their ability to produce cytokine (intracellular cytokine staining (ICS)) (Altman et al., 1996; Kern et al., 1998; Appay et al., 2000; Betts et al., 2000). These techniques have advantages over other methods, such as ELISpot analysis, in that they allow for precise phenotypic characterization of the T cell populations of interest. CD8+ T cell-mediated target cell killing, however, has historically been assessed by the standard chromium (51Cr) release assay (Brunner et al., 1968), or, more recently, by methods that monitor the release of fluorescent dyes from target cells (Sheehy et al., 2001; Liu et al., 2002). These techniques are cumbersome, semi-quantitative, and potentially insensitive. Importantly, none of these methods directly examine the CD8+ T cells that mediate killing; rather, they examine the death of target cells, essentially the aftermath of CD8+ T cell effector function. Cytotoxic CD8+ T lymphocytes (CTL) mediate the killing of target cells via two major pathways, a granule-dependent (perforin/granzyme) and independent (ligand ligand induced cell death, e.g. fas-fasL) mechanism (reviewed in Trapani and Smyth, 2002). The granule-dependent pathway does not require de novo synthesis of proteins by the effector CD8+ T cell, which instead utilize pre-formed lytic granules located within the cytoplasm (Trapani and Smyth, 2002). The lytic granules are membrane-bound secretory lysosomes that contain a dense core composed of various proteins, including perforin and granzymes (Peters et al., 1991). The core is surrounded by a lipid bilayer containing lysosomal associated membrane glycoproteins (LAMPs), including CD107a (LAMP-1), CD107b (LAMP-2), and CD63 (LAMP-3) (Peters et al., 1991). These proteins are not normally found on the surface of T cells, although they have been observed on the cell surface of PHA-activated lymphocytes (CD107a and b), and ionomycin treated CD4+ and CD8+ CTL clones (CD63) (Kannan et al., 1996; Bossi

and Griffiths, 1999). Although CD107a and b expression on the cell surface of peripheral blood cells has been shown to enhance lymphocyte vascular adhesion (Kannan et al., 1996), the function of these proteins, if any, on the surface of activated T cells remains to be determined. The presence of CD107a and b in the cytotoxic granular membrane has been proposed to protect against the leakage of contents from the granule itself by coating the interior of the membrane (Peters et al., 1991). CD107a, CD107b, and CD63 are constitutively expressed on the surface of activated platelets, and are found ubiquitously in lysosomal and endosomal membranes of numerous other cell types (reviewed in Fukuda, 1991). Degranulation of activated CD8+ T cells occurs rapidly after TCR stimulation, as a result of the polarized mobilization of microtubules that transport the lytic granules towards the immunological synapse formed between the CTL and the target (reviewed in Barry and Bleackley, 2002). Once the granules reach the plasma membrane of the CTL, the membranes fuse (Peters et al., 1991), releasing the granule contents into the immunological synapse, ultimately resulting in the death of the target cell. Degranulation is a requisite process of perforin-granzyme mediated killing, and is a critical step required for immediate lytic function mediated by responding antigen-specific CD8+ T cells. To date, there are no flow cytometric assays to monitor this process directly. Monitoring the presence or absence of perforin and granzymes A and B gives no indication as to the ability of T cells to degranulate. Furthermore, identifying a cell as lacking perforin does not mean it had perforin prior to stimulation. Finally, CD8+ T cells specific for certain viral antigens have low baseline levels of perforin (Appay et al., 2000, 2002; Sandberg et al., 2001), causing difficulty in measuring a further loss after antigen stimulation. We developed a novel assay that directly measures degranulation in primary responding antigen-specific CD8+ T cells by multi-parameter flow cytometry. This assay measures the cumulative exposure of granular membrane proteins (CD107a and b) on the cell surface of responding antigen-specific T cells, providing a positive marker of degranulation. Significant expression of cell surface CD107a and b can be observed as early as 30 min following stimulation of primary CD8+ T cells, and reaches maximum by 4 h. As expected, inhibition of degranulation dramatically reduces the

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acquisition of cell surface CD107a and b. This assay can be combined with existing methods that assess cytokine production in responding antigen-specific CD8+ T cells directly ex vivo, thus providing simultaneous assessment of two critical CD8+ T cell effector functions.

2. Materials and methods 2.1. Patient samples Peripheral blood mononuclear cells (PBMC) were obtained from 11 anonymous healthy donors at the National Institutes of Health Department of Transfusion Medicine. PBMC from two HIV-1 infected patients were obtained from both the Amelia Court HIV Clinic at the University of Texas Southwestern Medical Center and the National Institutes of Health OP-8 and OP-11 clinics. These donors signed informed consent required by the institutions Review Boards. 2.2. Antibodies and reagents The following anti-human monoclonal antibody reagents were obtained from BD Pharmingen, San Diego, CA (purified antibodies to some cell-surface markers were conjugated to various fluorochomes in our laboratory (Roederer), as indicated by an asterisk following the fluorochrome used): anti-CD28, antiCD49d, anti-IFN-g [fluorescein isothicyanate (FITC), allophycocyanin (APC)], anti-CD3 [phycoerythrin (PE), peridinin chlorophyll protein (PerCP)], antiCD4 (FITC), anti-CD8 (PerCP), anti-CD16 (FITC), anti-CD20 (FITC), anti-CD107a (FITC, PE*, APC*), anti-CD107b (FITC, PE*, APC*), anti-CD63 (PE), and anti-perforin (PE). Anti-granzyme B conjugated to PE was obtained from Caltag, Burlingame, CA. CMV-A2 tetramers were obtained from Beckman Coulter Immunomics, San Diego, CA. 2.3. Cell stimulation Fresh PBMC were isolated using Hypaque-Ficoll (Pharmacia, Uppsala, Sweden) density centrifugation. In some instances PBMC were frozen (90% fetal calf serum (FCS)/10% DMSO) at 140 jC until use. 106 PBMC were incubated with 1 Ag/ml each of anti-

CD28 and anti-CD49d and 2 Ag/ml of appropriate peptide (when used) in a 1-ml volume. In some experiments, staphylococcus enterotoxin B (SEB, 1 Ag/ml, Sigma, St. Louis, MO) or anti-CD3 (clone HIT3a, BD Pharmingen, 5 Ag/ml) was used to activate the cells. Conjugated antibodies to the granular membrane proteins CD107a and CD107b were added to the cells prior to stimulation, unless otherwise noted. In every experiment a negative control (anti-CD28/CD49d) was included to control for spontaneous production of cytokine and/or expression of CD107a/b. The cultures were incubated for 1 h at 37 jC in a 5% CO2 incubator, followed by an additional 4 5 h in the presence of the secretion inhibitor monensin (BD Pharmingen) or Brefeldin A (Sigma). In some experiments, colchicine (Sigma) was added to inhibit granule release. 2.4. Immunofluorescent staining/analysis Immediately following stimulation, PBMC were washed once, and surface stained with directly conjugated antibodies. The cells were washed and then fix/permeabilized using 750 Al of fixation/permeablization solution, consisting of FacsLyse (Becton Dickinson Immunocytometry Systems, San Jose, CA) diluted to a 2 concentration in dH2O and 0.05% Tween-20 (Sigma). After permeabilization, the cells were washed twice, and stained with directly conjugated antibodies specific for intracellular markers. The cells were washed a final time and resuspended in 1% paraformaldehyde (Electron Microscopy Systems, Fort Washington, PA) in PBS. Six-parameter flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems). List mode data files were analyzed using FlowJo software (Tree Star, San Carlos, CA). In all cases at least 100,000 live events were collected for analysis. 2.5. Direct ex vivo cytotoxicity assay Autologous B cells were isolated from PBMC by positive selection using magnetic Microbeads coated with anti-CD19 monoclonal antibody according to the manufacturers instructions (MACS, Miltenyi Biotec, Germany). Purified B cells were then labeled with either carboxyfluorescein diacetate succinimidyl ester

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(CFSE) or chloromethyl-benzoyl-amino-tetramethylrhodamine (CMTMR) (Molecular Probes, Eugene, OR). For CFSE staining, 5 106 cells/ml were incubated with 0.25 AM CFSE in PBS at 37 jC for 7 min, then washed three times in RPMI-1640 (BioWhittaker, Walkersville, MD)/20% FCS. For CMTMR staining, 2 106 cells/ml were incubated in pre-warmed RPMI/ 10% FCS (R10) supplemented with 5 AM CMTMR at 37 jC for 30 min, washed twice in pre-warmed R10, and then incubated in R10 alone for 1 h prior to a final wash. Labeled cells were protected from light during all subsequent procedures. Cells labeled with CFSE were either pulsed for 90 min with 200 nM CMV pp65 peptide 495 503 (NLVPMVATV) and then washed three times in R10, or mock-pulsed in parallel; all cells labeled with CMTMR were mock-pulsed. Cytotoxicity assays used 200,000 each of CFSE and CMTMRlabeled B cells in FACS tubes; PBMC were added in R10 to give a range of effector to target (E/T) ratios. Assays were incubated at 37 jC with 5% CO2 with the tubes placed at an angle. The elimination of peptidepulsed CFSE-labeled cells relative to the unpulsed CMTMR-labeled internal negative control served as a measure of specific cytotoxicity. Parallel assays with mock-pulsed CFSE-labeled cells were set up for each E/T ratio to control for non-specific toxic effects of CFSE itself.

3. Results 3.1. CD107a and b are expressed on the surface of CD8+ T cells that degranulate in response to SEB In order to identify degranulation by activated CD8+ T cells, we examined the expression of

CD107a and b on the cell surface of CD8+ T cells following activation with SEB. PBMC from a normal donor were stimulated with SEB and incubated for 6 h in the presence of the secretion inhibitor Brefeldin A. After stimulation, the cells were stained with antibodies to T cell markers (CD3 and CD8) and a mixture of FITC-labeled antibodies to CD107a and b. A total of 8.4% of CD8+ T cells expressed surface CD107 after stimulation (Fig. 1A, left panel), indicating that degranulation had likely occurred within the responding CD8+ T cells. While this initial result appeared promising, the frequency of the responding population was not comparable to that observed by intracellular cytokine staining for IFNg (approximately 12%, data not shown). We reasoned that it was likely that cell surface expression of CD107 on T cells may be transient. Previous studies have shown that CD107a and b are targeted primarily to lysosomal/ endosomal membranes, and that any CD107a and b externalized to the cell surface is rapidly retrieved via the endocytic pathway (Fukuda, 1991). This suggested that as the cells degranulate, they would become positive for cell surface CD107 for a brief period of time before those proteins were internalized. Therefore, we included the antibodies to CD107a and b for the duration of the stimulation, rather than just staining post-stimulation. Any transient surface expression of CD107 would lead to antibody binding and either surface retention or uptake of the CD107/ antibody complex. In either case, even transient surface expression of CD107 would lead to fluorescent labeling of that cell. This modification enhanced the detection of responding cells, such that 12% of the CD8+ T cells were observed to express surface CD107a and b in response to SEB (Fig. 1A, center panel). This protocol did not result in an increase of

Fig. 1. Characterization of CD107a and b staining. (A) PBMC from a normal donor were stimulated with SEB and incubated with or without antibodies to CD107a and b FITC as shown in the presence of Brefeldin A for 6 h, then stained with CD3, CD8, and CD107a and b antibodies (where appropriate). Events shown are gated for CD3 and CD8. (B) A comparison of the differential effects of Brefeldin A (dashed line) and monensin (solid line) on the fluorescence of FITC CD107a and b in SEB stimulated CD8+ T cells. Cells were stimulated in the presence of FITC anti-CD107a and b for 6 h in the presence of either inhibitor, then stained with CD3 and CD8 antibodies. Events shown are gated for CD3 and CD8. Values shown represent the mean fluorescence of the indicated population (C) Comparison of different CD107 antibody conjugates. Cells from one donor were SEB-stimulated, pre-stained with anti-CD107a and b FITC, PE, or APC, and incubated in the presence of monensin for 6 h, followed by staining of CD3 and CD8 molecules. Events shown are gated for CD3 and CD8. (D) Coordinate staining of CD107a and b on SEB stimulated cells. Cells from the same donor used in (C) were surface stained with CD107a APC and CD107b PE, and then stimulated as described in (C) and stained with CD3 FITC and CD8 PerCP. Events shown are gated for CD3 and CD8. (E) Effect of colchicine on degranulation in SEB stimulated PBMC. Cells were stimulated, stained, and analyzed as described in (C) in the presence or absence of varying concentrations of colchicine, as depicted on the figure.

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background staining for CD107a and b (Fig. 1A, right panel). Our initial experiments utilized antibodies to CD107a and b conjugated to FITC, the fluorescence of which is sensitive to acidic pH (Roederer et al., 1987). Because CD107 is internalized from the cell surface following degranulation, likely into an acidic endosomal or lysosomal compartment, the fluores-

cence of FITC would be quenched. Therefore we examined the differential effects of Brefeldin A and monensin on the mean fluorescence of the responding cells (Fig. 1B). While both Brefeldin A and monensin serve as inhibitors of secretion, monensin also neutralizes the pH within endosomes and lysosomes (Mollenhauer et al., 1990). The mean fluorescence of CD107a and b-FITC in activated cells incubated

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with monensin (mean fluorescence = 78.3) was substantially higher than cells incubated with Brefeldin A (mean fluorescence = 41.8)(Fig. 1B). This result suggests that a substantial proportion of the FITC-labeled CD107a and b antibodies were internalized into an endosomal or lysosomal compartment, further supporting the need to label these proteins during stimulation and use monensin to achieve optimal detection of responding CD8+ T cells. In order to further optimize the staining of CD107, we conjugated the CD107a and b antibodies to PE and APC. The PE and APC conjugates were substantially brighter than the FITC conjugate, without greatly affecting the fluorescence of the non-responding population (Fig. 1C). One observation of note is that the PE-CD107 antibody conjugate appears to have a higher background in non-stimulated cells than the FITC and APC conjugates (Fig. 1C, upper row), perhaps related to the hydrophobicity of PE. We therefore utilized either the FITC or APC CD107 conjugates in our remaining experiments. In general, background expression of CD107a and b in unstimulated CD8+ T cells varied between 0.05% and 0.5%. The background expression of CD107a and b can be a result of several factors, including the presence of dead/apoptotic cells, platelets, granulocytes, monocytes, or B cells within the cultures. In particular, removal of monocytes and B cells through the use of a dump channel can dramatically reduce the background CD107 labeling observed (data not shown). We next examined the coordinate expression of CD107a and b on the cell surface of SEB-activated CD8+ T cells. Both CD107a and b are coordinately expressed on the majority of the responding cells, although the CD107a signal appears to be stronger in most cells than CD107b with these reagents (Fig. 1D). CD107a and b are likely differentially regulated within all cell types, as they are encoded on different chromosomes, and expressed at different copy numbers within the cell (Fukuda, 1991). It remains to be determined if these two proteins are differentially expressed in cytotoxic granules within individual CD8+ T cells or subpopulations of CD8+ T cells. Therefore, to ensure greatest sensitivity, we chose to use a mixture of the two antibodies in future experiments. In order to ensure that CD107a and b expression on the cell surface occurred as a result of degranulation, we examined the effect of the microtubule inhibitor

colchicine on the expression of CD107a and b. Colchicine has a potent effect on the expression of cell surface CD107a and b following activation of CD8+ T cells with SEB (Fig. 1E). This result, along with the observation that CD107a and b are expressed on the cell surface in the presence of the secretion inhibitors Brefeldin A and monensin, indicates that CD107a and b are indeed expressed on the cell surface as a result of degranulation. 3.2. Cell surface expression of CD107a and b is associated with the loss of intracellular perforin Perforin is released from cytotoxic granules during the degranulation process; therefore, acquisition of cell surface CD107a and b on CD8+ T cells after stimulation should be associated with a loss of intracellular perforin. To address this, we stimulated PBMC with anti-CD3, and performed a time course to compare the levels of intracellular perforin with cell surface CD107a and b. As shown in Fig. 2, CD107a and b was rapidly expressed after stimulation, concomitant with a loss of perforin expression. After a 5h stimulation, nearly every perforin expressing cell had degranulated, indicating that acquisition of cell surface CD107a and b occurs as a result of degranulation, rather than de novo production. 3.3. Comparison of CD107a and b expression with CD63 expression following degranulation CD63 (LAMP-3) can also be found within the membrane of cytotoxic granules (Peters et al., 1991), and has been previously used as a positive marker associated with the deposition of fas ligand on the cell surface during degranulation in ionomycin-stimulated CD4+ and CD8+ T cell clones (Bossi and Griffiths, 1999). We examined the expression of CD63 in comparison with CD107a and b in CD8+ T cells that degranulate in response to SEB (Fig. 3). While a portion of the responding CD8+ T cells expressed CD63, CD107a, and CD107b, the majority of responding cells were CD63 low or negative. Furthermore, the CD63 background was substantially higher than that observed for CD107a and b. These results show that CD107 expression on ex vivo activated CD8+ T cells provides a more accurate assessment of degranulation than does CD63 expression.

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Fig. 2. Acquisition of cell surface CD107 is correlated with a loss of intracellular perforin. PBMC were stimulated with anti-CD3 (5 Ag/ml), and incubated for up to 5 h in the presence of anti-CD107a and b FITC and monensin. Baseline perforin expression was approximately 20% (data not shown). At the time-points designated on the figure, aliquots were removed, washed, and permeabilized, followed by staining for perforin, CD3, and CD8. Events shown are gated for live CD3+ CD8+ T cells.

3.4. Cytotoxic-granule containing lymphocytes express higher levels of CD107a and b than non-granule containing lymphocytes While all lymphocytes express CD107a and b on endosomal and lysosomal membranes, not all lymphocytes have cytotoxic granules that contain perforin. Such granules are expressed by CD8+ T cells, NK cells,

and a subset of CD4+ T cells. We therefore examined the relationship between intracellular expression of CD107 and perforin expression. Nearly all lymphocytes express detectable CD107a and b when staining for these molecules following permeabilization (data not shown). Cells that contain perforin, however, express even higher levels of CD107a and b. For example, NK cells, which typically express high levels

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Fig. 3. Comparison of CD63 expression with CD107a and b expression. PBMC were stimulated with SEB in the presence of antibodies to CD63 PE and CD107a and b FITC and monensin, incubated for 6 h, and then stained with CD3 and CD8 antibodies. Events shown are gated CD3+ CD8+ T cells.

of perforin, have higher CD107a and b expression than B cells or CD4+ T cells. Interestingly, although only a subset of CD8+ T cells is perforin+, the CD8+ T cell population as a whole expresses more CD107a and b than B cells or CD4+ T cells. This suggests that CD8+ T cells have a higher granular content, similar to NK cells. To support this conclusion, we also compared granzyme B, another granular component expressed by a higher percentage of CD8+ T cells than perforin, with CD107a and b content. CD8+ T cells and NK cells that express granzyme B also have high levels of CD107a and b (data not shown). 3.5. CD107a and b are expressed on the cell surface of activated peptide-specific CD8+ T cells The standard for examining antigen-specific CD8+ T cell effector responses by flow cytometry is measurement of intracellular cytokine production, typically IFNg, after stimulation with cognate peptide. We therefore compared production of IFNg in response to peptide stimulation with acquisition of cell surface CD107a and b (Fig. 4A D). PBMC isolated from four different patients were stimulated with peptides derived from either CMV or HIV, stained for CD107a and b, then incubated for 5 h in the presence of monensin. After stimulation, the cells were permeabilized and stained for intracellular IFNg. As can be seen in Fig. 4, nearly all of the CD8+ T cells that produce IFNg in response to specific peptide also express

CD107a and b. This indicates that acquisition of cell surface CD107a and b occurs in an antigen specific manner, and that nearly all CD8+ T cells which produce cytokine in response to cognate antigen degranulate. As shown in Fig. 4C, a mixture of peptides can also be used to stimulate CD8+ T cells to degranulate, suggesting that measurement of cell surface CD107a and b could be used in peptide response mapping procedures. Interestingly, in some patients a population of CD107a+ and b+ cells that did not produce IFNg could be observed (Fig. 4B and C). This suggests that measuring CD8+ T cell responses by IFNc production alone may underestimate the total response. 3.6. Kinetics of CD107a and b expression as compared to IFNg production Having established that cell surface expression of CD107a and b occurs in an antigen specific manner, we examined in more detail the rate at which cells became surface positive for CD107a and b after stimulation. Intracellular expression of IFNg has previously been shown to plateau between 5 and 6 h, thus we previously had stimulated the cells for this length of time to compare CD107 and IFNg expression. It is known, however, that degranulation occurs very rapidly following triggering of the T cell receptor complex. We therefore expected to detect acquisition of cell surface CD107a and b rapidly follo-

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Fig. 4. CD107a and b is expressed by ex vivo activated antigen-specific CD8+ T cells. PBMC from four different donors were stimulated with specific peptides in the presence of anti-CD28/49d, anti-CD107a and b FITC or APC, and monensin for 6 h. Peptides used include HLA-A2 restricted CMV pp65 NLVPMVATV (A, B), overlapping HIV-Gag derived 15-mers (15-mer peptides overlapping by 11 amino acids, C), and HLA-B57 restricted HIV Gag KAFSPEVIPMF (D). All peptides were used at a final concentration of 2 Ag/ml. Events shown are gated CD3+ CD8+ T cells.

wing stimulation. Expression of cell surface CD107 can be detected as early as 30 min to 1 h following stimulation with anti-CD3 (Fig. 2) or specific peptide (Fig. 5), and peaks between 4 and 5 h. IFNg, which requires de novo synthesis following activation, also peaked at 4 5 h post stimulation, although no IFNg production was detected at 1 h post-stimulation. Thus, optimal expression of both cell surface CD107a and b and IFNg occurs between 4 and 6 h post-stimulation. 3.7. CD107a and b can be used to measure degranulation in activated MHC-class I tetramer+cells The functionality of tetramer-binding CD8+ T cells can be examined by staining for IFNg production after the stimulation of tetramer stained cells with cognate peptide. However, since cytokine production alone does not provide a full functional assessment of the tetramer binding cells, we examined the ability of tetramer stained cells to degranulate, an example of which is shown in Fig. 6. Approximately 0.8% of

CD8+ T cells in this individual are capable of binding to the CMV-A2 MHC-class I tetramer (Fig. 6, left panel). After stimulation with peptide, only 25% of the tetramer + cells produced IFNg (Fig. 6, center panel), while a substantially higher proportion (>50%) of the same tetramer + cells degranulated following stimulation (Fig. 6, right panel). Similar results were observed in four additional individuals (data not shown). These data demonstrate that examination of cytokine production alone may not provide sufficient information regarding the full functionality of tetramer + cells. 3.8. CD107a and b expression directly correlates with cytotoxic activity While measurement of the ability of CD8+ T cells to degranulate provides an indication of cytotoxic potential, it still does not prove that degranulating cells are capable of killing targets. To address this question more directly, we examined the cytotoxic activity of a CMV-specific CD8+ T cell population known to degranulate in response to an HLA-A2 restricted CMV-derived peptide (NLVPMVATV,

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Fig. 5. Time course of CD107a and b expression as compared to IFNg production. PBMC were stimulated with CMV-A2 NLVPMVATV peptide (2 Ag/ml) for up to 6 h in the presence of anti-CD28/49d, anti-CD107a and b FITC and monensin. At 1, 2, 3, 4 and 6 h post stimulation, aliquots were removed, washed, permeabilized and stained for intracellular IFNg, CD8, and CD3. The frequency of responding CD8+ cells was determined at each time point and the % maximal response was calculated for each time point. (x, CD3+ CD8+ IFNg+ cells; n, CD3+ CD8+ CD107a and b+ cells; D, CD3+ CD8+ CD107a and b+ IFNg+ cells).

CMV-A2). PBMC were isolated from an individual with a high CMV-specific CD8+ T cell frequency (Fig. 7A, left panel). Approximately 10 15% of the circulating CD8+ T cells in this individual are specific for the CMV-A2 peptide as measured by direct tetramer binding, and 80% of the tetramer + cells degranulate in response to the CMV-A2 peptide (Fig. 7A, right panel). We then examined the cytotoxic ability of these cells directly by using a flow cytometry-based killing assay, as described in the Materials and methods. CFSE labeled cells, depicted in green, are selectively

killed by the CD8+ T cells only when CMV-A2 peptide loaded, as demonstrated by a shift into the dead cell population determined by the forward/side scatter profiles (Fig. 7B). The selective loss of peptide loaded CFSE labeled B cells in the presence of CD8+ T cells can also be directly observed by comparing the frequency of CMTMR and CFSE labeled cells within the live cell gate (Fig. 7C). Taken together these results indicate that antigen-specific CD8+ T cells that degranulate, as measured by CD107 expression, mediate cytotoxic activity.

Fig. 6. CD107a and b are expressed by functional MHC-class I tetramer + cells. PBMC were stained with CMV-A2 MHC-class I tetrameric complexes, then activated with CMV-A2 peptide (NLVPMVATV) in the presence of anti-CD28/49d, anti-CD107a and b APC and monensin for 5 h. Following stimulation, the cells were washed, permeabilized, and stained for CD8 and IFNg. All events shown are gated CD8+ cells.

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Fig. 7. Correlation between CD107a and b expression and cytotoxic activity. (A) PBMC were stained with a CMV-A2 MHC-class I tetramer, and anti-CD107a and b APC and treated as follows: Left panel: stained with anti-CD3 and anti-CD8, and analyzed without further incubation. Right panel: stimulated with cognate peptide (NLVPMVATV), incubated for 5 h in the presence of monensin, washed, then stained with antiCD3 and anti-CD8. All events shown are live-gated on CD3 and CD8. (B and C) Multiple parameter overlay plots showing the distribution of CFSE and CMTMR-labeled autologous B cells within the forward/side scatter profile (B, green events = CFSE, red events = CMTMR) or live gate (as depicted in B) only (C). In the example shown, 2 106 PBMC were incubated with 200,000 target cells for 60 h. Left panel: neither population of fluorescent dye-labeled cells was pulsed with cognate peptide. Right panel: the target cells labeled with CFSE only were prepulsed with cognate peptide (NLVPMVATV) at 200 nM.

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4. Discussion We present here a novel assay that measures degranulation in response to antigen-specific stimulation, an essential CD8+ T cell effector function. We demonstrate that the granular membrane proteins CD107a and b are expressed on the cell surface of activated CD8+ T cells due to degranulation. Expression of cell surface CD107a and b on CD8+ T cells often occurs in concert with production of IFNg in response to stimulation, but both functions do not necessarily occur in all antigen-specific CD8+ T cells. Additionally, we show that the same CD8+ T cells which degranulate are capable of cytotoxic activity. Numerous methods exist to examine both the physical presence and functionality of antigen-specific CD8+ T cells. The physical presence of antigenspecific CD8+ T cells can be monitored with MHCclass I tetramers or qPCR clonotype analysis (Altman et al., 1996; Douek et al., 2002). Neither of these techniques, however, provides any indication as to the functionality of the cells detected. Functionality can be assessed using both direct (intracellular cytokine staining and CFSE-based proliferation assays) and indirect (e.g. 51Cr release assays, flow-based killing assays and 3 H proliferation assays) methods (Kern et al., 1998; Sheehy et al., 2001; Brenchley et al., 2002; Liu et al., 2002). The direct methods as a whole, however, provide no information regarding cytotoxic ability, and conversely the indirect methods provide no indication as to the identity of the effector cells. The assay we describe here provides a link between the direct and indirect methods of CD8+ T cell effector analysis by enabling precise phenotypic and functional characterization of responding CD8+ T cells through flow cytometry using a marker that is only expressed during degranulation, the initial event that takes place during target cell lysis. Although the CD107 assay does not directly measure target cell lysis, it does provide an indication of the cytotoxic potential of the responding CD8+ T cells. Current methods to assess CD8+ T cell-mediated target cell killing, including the standard chromium release and flow-based killing assays do not identify or quantify effector cells, instead measuring their downstream effect on target cells. Thus, it has not been possible to characterize directly the full functional capacity and phenotype of the responding

CD8+ T cells. Our CD107 assay can provide an assessment of the capacity, frequency and phenotype of CD8+ T cells that kill in conditions similar to those used in a standard 51Cr release assay. More recently, intracellular cytokine staining has provided a wealth of information regarding the phenotype and functional status of antigen-specific CD8+ T cells. The question, however, has remained whether CD8+ T cells that produce cytokine after stimulation are cytotoxic. By measuring degranulation in the same cells, it is apparent that the majority of the CD8+ T cells that respond to antigen by producing cytokine also degranulate. This suggests, therefore, that cytokine producing CD8+ T cells that degranulate should be capable of killing targets, provided they express the necessary granular components to do so. Interestingly, we observe in some patients that a substantial population of responding CD8+ T cells degranulate but do not produce IFNg. It is well documented that many tetramer + cells do not produce detectable levels of IFNg after direct ex vivo stimulation with cognate peptide (Goepfert et al., 2000; Shankar et al., 2000). Therefore, functional heterogeneity exists within the population of responding CD8+ T cells. Thus, in assessing the frequency of the CD8+ T cell response to any particular antigen, one should also include measurement of degranulation, lest the response frequency be underestimated. Current data suggests that CD8+ T cell populations specific for a single antigen can produce multiple cytokines, but that heterogeneity exists as to the cytokines produced by individual cells within that population (data not shown). Importantly, there does not appear to be a significant population of CD8+ T cells that produce IFNg without degranulating. Although it remains to be determined if the non-IFNg producing CD107+ cells produce other cytokines in response to stimulation, preliminary results indicate that CD107 is expressed on the surface of MIP1h+ and TNFa + CD8+ T cells (data not shown). Thus, CD107a and b expression after stimulation provides a more complete assessment of the total frequency of responding CD8+ T cells than does monitoring production of any one cytokine alone. An important methodological aspect of the CD107 assay is that fixation or permeabilization of the responding cells is not necessary, thus making this a

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suitable procedure for sorting of live cells. Previously, sorting of antigen-specific CD8+ T cells ex vivo has been limited to methods utilizing either tetramer staining or IFNg capture systems. Tetramer-based sorts, while very rapid and specific, are limited by the need for very specific reagents and restricted peptide-MHC combinations. While IFNg capture is not limited by reagent availability, it is inherently more difficult to perform, and is limited to IFNg producing cells. Unlike tetramer-based methods, sorting based on CD107a and b expression is not limited by reagent availability, or to prior knowledge of HLA type or peptide recognition, since mixtures of overlapping peptides can be used. Additionally, sorting based on CD107a and b is not limited only to those cells capable of producing a certain cytokine, as is the IFNg capture assay. In conclusion, we have described a novel method to assess CD8+ T cell effector function based on the ability of these cells to degranulate. This assay is simple, rapid, sensitive, and can be adapted for use in combination with both tetramer and intracellular cytokine assays. Assessment of degranulation alongside cytokine production and phenotypic characterization will greatly enhance our knowledge of the functionality of antigen-specific CD8+ T cells both in disease as well as in vaccine models.

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