Genetic and Small Molecule Disruption of the

AID/RAD51 Axis Similarly Protects

Nonobese Diabetic Mice from Type 1
Diabetes through Expansion of Regulatory B
This information is current as Lymphocytes
of May 2, 2017.
Jeremy J. Ratiu, Jeremy J. Racine, Muneer G. Hasham,
Qiming Wang, Jane A. Branca, Harold D. Chapman, Jing
Zhu, Nina Donghia, Vivek Philip, William H. Schott, Clive
Wasserfall, Mark A. Atkinson, Kevin D. Mills, Caroline M.
Leeth and David V. Serreze
D
J Immunol published online 1 May 2017 o
http://www.jimmunol.org/content/early/2017/04/29/jimmun w
nl
ol.1700024 oa
de
d
fr
Supplementary o
http://www.jimmunol.org/content/suppl/2017/04/29/jimmunol.170002 m
Material 4.DCSupplemental htt
p:/
Subscription Information about subscribing to The Journal of Immunology is online at: /w
http://jimmunol.org/subscription w
w.
Permissions Submit copyright permission requests at: ji
m
http://www.aai.org/About/Publications/JI/copyright.html m
Email Alerts un
Receive free email-alerts when new articles cite this article. Sign up at: ol.
http://jimmunol.org/alerts or
g/
by
gu
est
on

The Journal of Immunology is published twice each month by The American Association of Immunologi
1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright 2017 by The American Association of
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Published May 1, 2017, doi:10.4049/jimmunol.1700024
The Journal of Immunology

Genetic and Small Molecule Disruption of the

AID/RAD51 Axis Similarly Protects Nonobese
Diabetic Mice from Type 1 Diabetes through
Expansion of Regulatory B Lymphocytes
Jeremy J. Ratiu,*,1 Jeremy J. Racine,*,1 Muneer G. Hasham,*,1
Qiming Wang,*, Jane A. Branca,* Harold D. Chapman,* Jing Zhu,
Nina Donghia,* Vivek Philip,* William H. Schott,* Clive
x x {
Wasserfall, Mark A. Atkinson, Kevin D. Mills, Caroline M.

Leeth, and David V. Serreze*
B lymphocytes play a key role in type 1 diabetes (T1D) development by serving as a subset of APCs
preferentially supporting the expansion of autoreactive pathogenic T cells. As a result of their
pathogenic importance, B lymphocytetargeted therapies have received considerable interest as
potential T1D interventions. Unfortunately, the B lymphocytedirected T1D interventions tested to
date failed to halt b cell demise. IgG autoantibodies marking humans at future risk for T1D indicate
that B lymphocytes producing them have undergone the affinity-maturation processes of class switch
recombination and, possibly, somatic hyper- mutation. This study found that CRISPR/Cas9-mediated
ablation of the activation-induced cytidine deaminase gene required for class switch
recombination/somatic hypermutation induction inhibits T1D development in the NOD mouse model. The
activation- induced cytidine deaminase protein induces genome-wide DNA breaks that, if not repaired
through RAD51-mediated homologous recombination, result in B lymphocyte death. Treatment with the
RAD51 inhibitor 4,49-diisothiocyanatostilbene-2, 29-disulfonic acid also strongly inhibited T1D
development in NOD mice. The genetic and small moleculetargeting approaches expanded CD73 + B
lymphocytes that exert regulatory activity suppressing diabetogenic T cell responses. Hence, an
initial CRISPR/Cas9- mediated genetic modification approach has identified the AID/RAD51 axis as a
target for a potentially clinically translatable pharmacological approach that can block T1D
development by converting B lymphocytes to a disease-inhibitory CD73 + regula- tory state. The
Journal of Immunology, 2017, 198: 000000.
subsequent processing and presentation to

A
lthough the autoimmune destruction of diabetogenic T cells (10, 12). Similar populations
insulin-producing pancreatic b cells of pathogenic B lymphocytes also likely contribute
underlying the development of type 1 to T1D development in humans, given the presence
diabetes (T1D) is ultimately mediated by of circulating b cell Ag-specific autoantibodies
the combined that are critical biomarkers for identifying
activity of CD4+ and CD8+ T cells, it is clear in individuals at high risk for future disease
the NOD mouse model, and likely in humans, that B (13).
Most autoantibodies in humans with, or at risk
lymphocytes play an additional for, T1D are of an IgG isotype, indicating that
key pathogenic role (19). Studies in NOD mice the B lymphocytes producing them have undergone
indicate that B lymphocytes contribute to T1D affinity maturation (13). Affinity maturation is
by being the subset of APCs that most efficiently the process occurring within germinal centers
supports the expansion of pathogenic T cell (GCs) by which B lymphocytes undergo Ig
responses (1012). This is due to the presence of diversification and clonal selection. Ig
B lymphocytes expressing plasma membranebound Ig
molecules capable of efficiently capturing and
internalizing b cell autoantigens for

*The Jackson Laboratory, Bar Harbor, ME 04609; Graduate Program

in Genetics, Sackler School of Graduate Biomedical Sciences,
Tufts University, Boston, MA 02111; Department of Animal and
Poultry Sciences, Virginia Polytechnic and State University,
Blacksburg, VA 24061; xDepartment of Pathology, University of
Florida, Gainesville, FL 32610; and {Cyteir Therapeutics,
Cambridge, MA 02138
1 or Dr.
J.J. Ratiu, J.J. Racine, and M.G.H. are cofirst authors.
ORCIDs: 0000-0001-5514-3074 (J.J. Racine); 0000-0002-4498-0210
(M.G.H.); 0000-0002-
8014-2855 (J.Z.); 0000-0002-5909-7623 (C.M.L.); 0000-0001-7614-
5925 (D.V.S.).
Received for publication January 5, 2017. Accepted for
publication April 4, 2017.
This work was supported by National Cancer Institute Grant
P30CA034196. M.A.A. is supported by National Institutes of Health
Grant P01-AI42288. D.V.S. is supported by National Institutes of
Health Grants DK-46266, DK-95735, and OD-020351. C.M.L. is
supported by National Institutes of Health Grant DK101735. K.D.M.
is supported by National Institutes of Health Grant CA138646 and
The Jackson Laboratory Principal Investigator Grant TJL DIF FY13
KDM. J.J. Racine is supported by National Institutes
of Health Fellowship
1F32DK111078.
J.J. Ratiu and J.J. Racine designed and conducted experiments,
interpreted data, and contributed to writing of the manuscript;
M.G.H. designed and conducted experi- ments and helped to
interpret data; Q.W., J.A.B., J.Z., N.D., and C.W. conducted
cytometry strategies; M.A.A. helped to interpret data; K.D.M. and
C.M.L. contrib- uted to study conception and helped to interpret
data; and D.V.S. contributed to study conception and supervised
experiments and writing of the manuscript.
Address correspondence and reprint requests to Dr. David V. Serreze
Caroline
M. Leeth, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME
04609 (D.V.S.) or Virginia Polytechnic and State University,
Department of Animal and Poultry Sciences, 175 West Campus Drive,
MC 0306, Litton Reaves Hall, Room 3280, Blacksburg VA 24061
(C.M.L.). E-mail addresses: dave.serreze@jax.org (D.V.S.) or
cmcphee@vt.edu (C.M.L.)
The online version of this article contains supplemental
material.
Abbreviations used in this article: Aicda, activation-induced
cytidine deaminase gene; AID, activation-induced cytidine
deaminase protein; APCP, a,b-methyleneadenosine 59-diphosphate;
A2aR, adenosine A2a receptor; B6, C57BL/6J; Breg, regulatory B
lymphocyte; CSR, class switch recombination; DIDS, 4,49-
diisothiocyanatostilbene-2-29disulfonic acid; DSB, dsDNA break;
GC, germinal center; HR, homologous recombination; IAA,
scid b
insulin autoantibody; NOD-scid, Emv30 /Dvs; PLN, pancreatic
NOD.Cg-Prkdc lymph
experiments; H.D.C. conducted experiments and helped to
interpret data; V.P. con- ducted statistical analyses;
W.H.S. conducted experiments and helped to design flow
node; qPCR, quantitative PCR; sgRNA, single-guide RNA; SHM, somatic
hypermutation; Copyright 2017 by The American Association of Immunologists, Inc.
Tfh, follicular helper T; T1, transitional-1; T1D, type 1 diabetes; T2, 0022-1767/17/$30.00
transitional-2; UMI, unique molecular identifier; WT, wild-type.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700024

D
o
w
nl
oa
de
d
fr
o
m
htt
p:/
/w
w
w.
ji
m
m
un
ol.
or
g/
by
gu
est
on
2 TARGETING AID-ACTIVE B CELLS FOR TYPE I DIABETES PREVENTION

diversification occurs through somatic K.D.M. by Dr. T. Honjo (Graduate School of Medicine,
hypermutation (SHM) and class switch recombination Kyoto University). CRISPR-Cas92/2 technology was used to
(CSR), whereas clonal selection re- sults from directly generate NOD.Aicda mice by cytoplasmic
competitive interaction with follicular helper T microinjection of NOD/ShiLtDvs zygotes with 100
(Tfh) cells (14). Selective pressures within GCs ng/ml Cas9 mRNA and 50 ng/ml the following single-guide
RNAs (sgRNAs), with the upper case letters being the
result in the preferential expansion of B complement to the targeted genomic sequence: 59-
lymphocytes with greater affinity for their gaaattaatacgactcactataggAGTCACGCTGGAGAC-
cognate Ag. In autoimmune diseases, such as T1D, CGATAgttttagagctagaaatagc-39 or 59-
aberrant selection processes lead to expansion of gaaattaatacgactcactataggACTTCTT-
TTGCTTCATCAGAgttttagagctagaaatagc-39 targeting exon 1 or
self-reactive B lymphocytes, which may become 2, respec- tively, of Aicda (Supplemental Fig. 1A). Exon 1
autoantibody-secreting cells or retain their and 2 sgRNAs were microinjected into 47 and 39 zygotes,
surface Ig to serve as potentially more effective respectively. These microinjected zygotes were then
transplanted into three and two recipient females, re-
APCs (15). Although previous findings suggest that spectively. Tail DNA from surviving progeny was sequenced
affinity maturation is important to T1D path- and identified 100 and 14.3% targeting efficiency for
ogenesis (16), the significance of CSR/SHM exon 1 (14/14) and exon 2 (2/14). Mosaic founder mice
identified as carrying a mutation in the targeted region
processes to disease progression has yet to be of Aicda were backcrossed to NOD/ShiLtDvs mice. The
elucidated. Furthermore, it remains un- clear resulting N1 progeny were screened for germline
whether B lymphocytes must undergo CSR/SHM to transmitted mutations by PCR ampli- fication of exon 1
become effective autoreactive APCs supporting T1D with the primers 59-TCACACAACAGCACTGAAGC-39 and 59-
ACCCAAAAGACCTGAGCAGA-39 or exon 2 with the primers: 59-
pathogenesis. CGCTCAGCTACCTTGCCTAT-39 and 59-CGAAGTCCAGTGAGCAGGA-
Because of their role in supporting pathogenic T 39. PCR products were purified and analyzed by sequencing
cell responses, there has been considerable on an ABI 3730 DNA analyzer (Applied Biosystems) using
interest in determining whether B the forward or reverse primer. Mutant sequences were
lymphocytetargeted approaches could provide an
effective T1D intervention. A clinical trial found separated from wild-type (WT) using the Poly Peak Parser
that transient treatment with the B lymphocyte package (25) for R. A 2-bp deletion along with a 309-bp
depleting CD20-specific rituximab Ab allowed for insertion was selected for a line targeting exon 1
early (1 y), but not long-term (2 y), preservation (referred to as Line 1 ,em1Cml.
in the text; formal D
of C-peptide pro- o
duction in recent-onset T1D patients (17). The name: NOD/ShiLtDvs- /Dvs), and a 13-bp deletion
lack of long-term Aicda was se- w
protection may be attributable, at least in part, previ- ously (24). B6.Cg-Aicda,tm1Hon./HonRbrc mice were nl
to the rebound of B lymphocytes following kindly provided to oa
transient rituximab treatment. However, in NOD de
mice, pancreatic isletinfiltrating B lymphocytes
lose cell surface expression of CD20 and, thus, are d
rendered resistant to de- pletion by a rituximab- fr
like murine anti-CD20 Ab (18). These results o
indicate a need to identify alternative strategies m
that may provide a more effective B lymphocyte
directed T1D-intervention approach. In the current htt
study, we evaluated the contribution of CSR/ SHM p:/
to T1D development and whether specifically /w
targeting B lymphocytes undergoing these processes w
could provide an effec- tive therapeutic
intervention. As a first step, we used CRISPR-Cas9 w.
technology to directly ablate, in NOD mice, the ji
activation-induced cytidine deaminase gene (Aicda) m
necessary for initiating CSR/ SHM processes (19 m
21). Aicda ablation significantly inhibited
T1D development. un
The Aicda-encoded activation-induced cytidine ol.
deaminase protein (AID) initiates SHM and CSR by or
inducing point mutations and dsDNA breaks (DSBs) g/
in Ig gene sequences. Additionally, AID generates by
DSBs elsewhere throughout the genome (22, 23) that gu
are normally repaired by RAD51 complexmediated
homo- logous recombination (HR). In the absence of est
HR, B lymphocytes in which SHM/CSR has been on
initiated undergo cell death (23). The small
molecule 4,49-diisothiocyanatostilbene-2, 29-
disulfonic acid (DIDS) inhibits RAD51-mediated HR
(23). DIDS has been used as a model agent in
previous studies, indicating that RAD51 blockade
could be considered an intervention for
eliminating AID+ B cell lymphomas (23). In this
study, we used DIDS treat- ment to determine
whether targeting the AID/RAD51 axis could provide
a B lymphocytedirected intervention for T1D.
Interest- ingly, genetic and DIDS-mediated
disruption of the AID/RAD51 axis increased the
+
numbers of CD73 B lymphocytes that exerted
regulatory processes actively suppressing T1D.
Together, these studies indicate that therapies
capable of expanding regulatory B lymphocytes,
potentially including those targeting affinity-
maturation processes, may ultimately represent
clinically translatable T1D- intervention
strategies.
Materials and Methods
Mice
NOD and C57BL/6J (B6) mice are maintained at The
Jackson Labora- tory under specific pathogenfree
conditions. Lymphocyte-deficient NOD.Cg-
PrkdcscidEmv30b/Dvs (NOD-scid) mice were described
The Journal of
lected for a line targeting exon 2 (referred to as Line
Immunology
26 in the text; formal
name: NOD/ShiLtDvs-Aicda,em2Cml./Dvs). N1 mutants were
intercrossed to fix the mutations to homozygosity,
after which lines were maintained by brothersister
matings. Mutant mice were genotyped by amplification-
length polymorphisms using the same primers used for
sequencing (Supplemental Fig. 1B). Mice were matched
by age and sex for experimentation, but no specific
randomization method was performed to form
experimental groups. The Jackson Laboratorys
Institutional Animal Care and Use Committee approved
all protocols involving mice.
RT-PCR and gene-expression analysis
B lymphocytes were purified from 8-wk-old female NOD or
B6 mice. Total RNA was extracted using an RNeasy Micro
Kit (QIAGEN). Primers for Aicda RT-PCR (26) are 59-
CAGGGACGGCATGAGACCT-39 and 59-TC-
AGCCTTGCGGTCTTCACA-39, and primers for Gapdh are 59-
GAGAA- ACCTGCCAAGTATGATGAC-39 and 59-
TGATGGTATTCAAGAGAGTAGG-
GAG-39 (27). RNA was used to synthesize cDNA with a
MessageSensor RT Kit (Thermo Fisher) and Random
Decamers (Invitrogen). Power SYBR Green (Applied
Biosystems) was used to determine the expres- sion of
Aicda and Gapdh. Quantitative PCR (qPCR) was run using
an Applied Biosystems ViiA7, and data were analyzed
using A&B RUO software.
Monitoring of T1D development
Development of T1D was assessed by weekly monitoring of
glycosuria with Ames Diastix (Bayer), with disease
3
onset defined by two consecutive readings $ 0.25%
(corresponds to blood glucose $ 300 mg/dl).
Histological assessment of insulitis
Quantitative mean insulitis scores were determined using
the previously described calculation method (28).
Briefly, Bouins fixed pancreata were sectioned at three
nonoverlapping levels. Slides were stained with al-
dehyde fuchsin and H&E. Islets were scored by a blinded
observer as follows: 0, no visible lesions; 1, peri-
insular noninvasive leukocytic ag- gregates; 2, ,25%
islet destruction; 3, 2575% islet destruction; and 4,
75100% islet destruction. The final score was
determined by dividing the cumulative score for each
pancreas by the number of total islets ($20 per
mouse) examined. If no b cellcontaining islets were
found across three sections, the analyzed mouse received
an insulitis score of 4. The final insulitis score
incorporates the proportion of islets in analyzed mice
that had undergone each level of destruction.
Islet-associated leukocyte isolation
Infiltrating islet-associated leukocytes were isolated
for flow cytometry, as previously described (18).
In vitro CSR assay
Splenic B lymphocytes were purified using anti-CD43
MicroBeads (Miltenyi Biotec), according to the
manufacturers protocol. Purified B lymphocytes were
cultured at a concentration of 1 3 106 cells per
milliliter in RPMI 1640 (Life Technologies)
supplemented with 2 mM L-glutamine (Life
Technologies), 71.5 mM 2-ME (BP176-100; Fisher X-VIVO 20 medium supplemented with 10 mM HEPES
Scientific), 100 U/ml penicillin (Sigma-Aldrich), 100 (Lonza), 1 mM sodium pyruvate (Life Technologies),
mg/ml streptomycin (Sigma-Aldrich), and 10% (v/v)
heat-inactivated FBS (Atlanta Biologicals) under stimu- 71.5 mM 2-ME (Fisher Scientific), 100 U/ml penicillin,
lation with recombinant murine IL-4 (25 ng/ml; 100 mg/ml strep-
PeproTech) and anti- CD40 (1 mg/ml; BD Biosciences) or
LPS (25 mg/ml; Sigma-Aldrich) at 37C (5% CO 2) for 48
h. At 48 h, cultures were restimulated as de- scribed
above. 6At 72 h, cultures were diluted with media back to
1 3 10 cells per milliliter. Class switching to IgG1
isotype was quantified by flow cytometric analysis
after 96 total hours in culture.
Flow cytometry
Leukocytes among the indicated samples were phenotyped
by flow cytometry using LSRII SORP (BD Biosciences) or
Attune Cytometer (Thermo Fisher Scientific)
instrumentation, and data were2/2
analyzed using FlowJo
software (TreeStar). For Aicda experiments, single-cell
sus- pensions of splenocytes were lysed with Geys
buffer to remove RBCs, as previously described (29). For
all experiments, propidium iodide or DAPI was used for
live/dead discrimination. All analyses were done on
gated singlets (live) cells; gating strategies are shown
in Supplemental Fig. 2A, 2B. For DIDS-based+ experiments,
prior to singlet/live gating, TER-119 cells were
excluded (Supplemental Fig. 2C) via gating strategies in
lieu of lysis procedures. The following fluorochrome-
conjugated Abs (clones) were used for these studies:
CD95 (J02), GL-7 (GL7), CD43 (S7), CD69 (H1.2F3), CD21
(7G6), CD45.1 (A20), CD62L (MEL-14), CD8a (536.7),
CD19 (1D3), B220 (RA3-6B2), CD4 (GK1.5), CD23 (B3B4),
TER-119
(TER119), and streptavidin (all from BD Biosciences); D
CXCR5 (L138- D7), CD21 (7G6), IgD (11-26c.2a), CD80 o
(16-10A1), GL-7 (GL7), w
CD73 (TY/11.8), CD39 (Duha59), CD45.1 (A20), PD-1 (RMP1-
30), PD- L2 (TY25), CD4 (GK1.5), CD23 (B3B4), B220 (RA3-
nl
6B2), and TCRb (H57-597) (all from BioLegend); CD19 oa
(1D3), IgM (II/41), CD45.1 (A20), de
and CD44 (IM7) (all from eBioscience); CD8a (53-6.7), d
CD19 (1D3), and CD16/CD32 (2.4G2) (all from Tonbo fr
Biosciences); and IgG1 (1070-09) (SouthernBiotech). o
Splenic B lymphocyte subsets were characterized by flow m
cytometry, as previously described (30). htt
Serum Ig isotype ELISA p:/
/w
Quantification of serum Ig isotypes was performed as
described (31). Coating Abs were as follows: IgG1, IgG2b, w
IgG3, and IgM (SouthernBiotech). De- tection Ab was goat w.
anti-mouse k coupled to alkaline phosphatase (South-
ernBiotech). Ab standards included IgG1 and IgG2b ji
(SouthernBiotech) and IgG3 and IgM (Sigma-Aldrich). Plates m
were analyzed using an Infinite M200 Pro running Magellan
7.0 software (Tecan). m
Adoptive transfers un
ol.
Splenic B lymphocytes and + T cells were purified by+ or
negative depletion of CD11b , CD11c+, TER-119+, and CD3
populations and, in some ex- periments,
+ +
CD73+ (for
+
B g/
lymphocyte
+
purification) or CD11b , CD11c , TER-119 , and by
B220 cells (for T cell purification), using biotin-
conjugated Abs and streptavidin MicroBeads (Miltenyi gu
Biotec). NOD-scid recipients were injected i.v. with the
indicated B lymphocytes and/or T cells and were est
monitored for T1D development. The following specific on
biotinylated Abs were used: CD11b (M1/70), CD11c (HL3),
CD3 (145-2C11), TER-119
(TER119), and B220 (RA3-6B2) (all from BD Biosciences). CD73
(TY/11.8) was obtained from BioLegend.
+
CD73 B lymphocytemediated T cell suppression assay
B lymphocytes from 68-wk-old NOD.Aicda2/2 or NOD mice
were pu- rified from pooled spleens and pancreatic lymph
nodes (PLNs) by negative depletion of CD11b+, CD11c+,
CD3+, and TER-119++ cells using Biotin Binder Dynabeads
(Invitrogen). CD73 and CD732 B lymphocytes from the
purified pools were then sorted directly into Heat-
Inactivated HyClone FBS (GE Healthcare Life Sciences)
using a FACSAria II SORP sorter (BD
2
Biosciences) equipped
with an 85-mm nozzle. CD4+ CD73 T cells were purified
from spleens of + 68-wk-old NOD mice by negative
depletion + of CD11b , CD11c +, TER-119+ , B220+, CD8+,
and CD73 cells using
streptavidin MicroBeads (Miltenyi Biotec) and the above-
2
described Abs. Purified CD4 + CD73 T cells were labeled
with 5 mM Cell Proliferation Dye eFluor 670
(eBioscience). Sorted B lymphocytes and labeled T cells
were washed with serum-free X-VIVO 20 medium (Lonza)
three times to remove residual serum and were cocultured
under stimulation with soluble anti-CD40 (1 mg/ml),
plate-bound anti-CD3 (5 mg/ml), and soluble anti- CD28
(2 mg/ml; all from BD Biosciences) in the presence of 10
mM AMP and 0 or 100 mM a,b-methyleneadenosine 59-
diphosphate (APCP; all from Sigma-Aldrich) in serum-free
tomycin (both from Sigma-Aldrich), and nonessential
amino acids (Lonza) at 37C for 4 d. T cell
proliferation was assessed by flow cytometry, and data
were analyzed using FlowJo software. Percentage
suppression was calculated relative to the mean
proliferation index of T cells in the pres- ence of
2
CD73 B lymphocytes with 0 mM AMP and 0 mM APCP.
ELISA measurement of IL-10 production
CD73+ or CD732 B lymphocytes were sorted as described
above and cultured at 5.0 3 105 cells per milliliter in
X-VIVO 20 medium with 0 or 10 mg/ml LPS (Sigma-
Aldrich) for 3 d. IL-10 concentration in culture
supernatants was determined using a Mouse IL-10 ELISA
MAX Kit (BioLegend).
DIDS treatments
Beginning at 6, 8, or 10 wk of age, female NOD mice
were injected i.p. weekly with 0, 10, or 50 mg/kg DIDS
(CAS 67483-13-0; Santa Cruz Biotechnology) for the
indicated periods of time. Vehicle was 0.1 M po-
tassium bicarbonate.
Insulin autoantibody status
Serum insulin autoantibodies (IAAs) were detected as
described as part of the International Workshop on
Lessons From Animal Models for Human Type 1 Diabetes
(32).
Ig repertoire library preparation
Library preparation and data analysis for Ig repertoire
sequencing were performed as previously described (33),
with minor alterations. Briefly, female NOD/ShiLtDvs mice
received weekly DIDS (50 mg/kg) or vehicle treatment from
8 to 16 wk of age, after which PLN B lymphocytes
(CD45.1+ CD19+ B220+) were sort purified using a FACSAria
II. Simi- larly, purified PLN-resident B2/2 lymphocytes
from 8-wk-old unmanipulated NOD and NOD.Aicda mice served
as controls. Purified cells were washed with PBS,
resuspended in Buffer RLT, and snap-frozen. Total
RNA was purified using an RNeasy Micro Kit (QIAGEN).
RNA integ- rity number determined, using the Bioanalyzer
RNA 6000 Pico Kit (Agilent), for all samples was 10.
Total RNA was reverse transcribed using SMARTScribe
Reverse Transcriptase (Takara) with IgH isotype-specific
primers (1 mM each) IgHG1/2: 59-CAGGGATCCAKAGTTC-39,
IgHG3: 59-CAGGGCTCCATAGTTC-39, IgHM: 59-
GATGACTTCAGTGTTGT-39,
and IgHD: 59-AGTGGCTGACTTCCAA-39; and 1 mM template-
switch adapter (59-
AAGCAGUGGTAUCAACGCAGAGUNNNNUNNNNUNN-
NNUCTTrGrGrGrG-39, U, deoxyuridine) to introduce a unique
molecular identifier (UMI) sequence to the 39-end of each
cDNA molecule. Reverse- transcription reactions were set-
up as follows: 5 ml of RNA and 2 ml of IgH isotype-
specific primers (10 mM each) were heated to 72C for 3
min and then incubated at 42C for 3 min to anneal
primers. Then the following components were added to each
reaction for a final concentration of 13 first-strand
buffer, 2 mM DTT, 1 mM 59-template switch adapter, and
1 mM each dNTP (Invitrogen), 40 U RNaseOUT (Invitrogen),
and 200 U SMARTScribe Reverse Transcriptase. cDNA was
then treated with 5 U uracil-DNA glycosylase (New England
Biolabs) to degrade residual tem- plate switch adapter
and then purified using a NucleoSpin Gel and PCR
Purification Kit (Takara). First PCR was performed using
cDNA equivalent to 2.5 ng of RNA and Platinum SuperFi DNA
Polymerase (Invitrogen) with the following primers:
M1SS: 59-AAGCAGTGGTATCAACGCA-39, mIGG12_r2: 59-
ATTGGGCAGCCCTGATTAGTGGATAGACHGATG-39, mIGG3_r2: 59-
ATTGGGCAGCCCTGATTAAGGGATAGACAGATG-39, mIGM_r2: 59-
ATTGGGCAGCCCTGATTGGGGGAAGACATTTGG-39, and mIGD_r2:
59-ATTGGGCAGCCCTGATTCTCTGAGAGGAGGAAC-39. First PCR pro-
ducts were purified using a NucleoSpin Gel and PCR
Purification Kit and then amplified, using Platinum
SuperFi DNA Polymerase to introduce sample- specific dual-
end barcodes, with the primers M1S: 59-(N)46(XXXXXX)CA-
GTGGTATCAACGCAGAG-39 and Z: 59-(N)46(XXXXXX)ATTGGGCAG-
CCCTGATT-39, where XXXXXX represents a sample-specific 6-nt
barcode.
Second PCR products were purified using a NucleoSpin Gel
and PCR Puri- fication Kit. For each sample, 300 ng of
purified second PCR product was used for individual
sequencing library preparation using a NEBNext Ultra II DNA
Library Prep Kit with NEBNext Singleplex Oligos for
Illumina (New England Biolabs). Concentrations for each
library were determined using a Qubit dsDNA BR Assay Kit
(Life Technologies); an equal quantity of each library was
pooled and run on a 1.5% agarose gel, and a 600800-bp
band was ex- cised and extracted using a NucleoSpin Gel and
PCR Purification Kit. Pooled, gel-extracted libraries were
then quality controlled using a Bioanalyzer High
Sensitivity Kit (Agilent) and quantified by qPCR using a
Library Quantifica- tion Kit/Illumina GA/ABI Prism with the
Illumina Internal Control (both from Kapa Biosystems).
Ig repertoire sequencing and data analysis We then compared the female rate of T1D
development
2/2
for each
Sequencing libraries were spiked with a 30% PhiX Control NOD.Aicda line with that of WT NOD controls.
v3 and loaded at Both NOD.
10 pM concentration for asymmetric 400 + 225bp paired- Aicda
2/2
lines exhibited similar significantly
end sequencing performed using a MiSeq sequencer running
a MiSeq Reagent Kit v3 (all from Illumina). Sequencing reduced rates of T1D (Fig. 1D). Heterozygous
data were processed using MIGEC (34), as follows. First, +/2
raw reads were demultiplexed based on each samples two NOD.Aicda mice developed T1D at an intermediate
6-nt barcodes and then split into molecular identifier rate (data not shown). Interestingly, Lines 1 and
2/2
groups (MIGs) consisting of reads sharing the same UMI. 26 NOD.Aicda mice remaining free of overt T1D at
MIGs consisting of fewer than five reads were discarded. the end of the incidence study were still
Then, using only the first reads, consensus se- quences
were determined independently for 59- and 39-end reads characterized by significant levels of insulitis
within each MIG. 59- and 39-end read consensus sequences (Fig. 1E). Next, we compared the kinetics of
were then merged using the MiTools Merge Utility 2/2
(https://github.com/milaboratory/mitools), with the insulitis progression in female NOD and NOD.Aicda
minimum sequence similarity set to 70% for reads with mice at 7, 11, and 18 wk of age. Insulitis
overlapping parts, to generate full-length IgH sequences 2/2
progression was not delayed in Line 1 NOD. Aicda
for each MIG. Using MiXCR (35), full-length Ig sequences mice (Fig. 1F, 1G), indicating that this could not
were then mapped to the mouse reference germline
sequences, and the resulting alignments were used to explain their resistance to overt T1D. Together,
assemble clonotypes. When assembling 24clonotypes, a these data indicate that,
specific mutation proba- bility of 10 was used for despite the continued presence of islet-
frequency-based correction of PCR or se- quencing errors. 2/2
Assembled clonotypes were then exported for diversity infiltrating leukocytes in NOD.Aicda mice, the
analyses using VDJtools (36). Clonotypic diversity diabetogenic nature of these cells has been
estimates were calculated based on CDR3 nucleotide altered by genetic ablation of AID.
sequence and V- and J-segment usage.
2/2
Statistical analysis NOD.Aicda total B lymphocytes are numerically increased,
D
and GC and memory-like subsets are proportionally expanded o
All graphs and statistics were generated using Prism 6
(GraphPad) or Excel (Microsoft). Specific statistical To initially investigate the basis for the T1D w
analyses are listed within the respective figure legends. 2/2
The MannWhitney, Wilcoxon, and t test analyses were all resistance of NOD. Aicda mice, we compared them nl
two-tailed. with WT controls for the pres- ence of various oa
immature and mature B lymphocyte populations within
Results spleens and PLNs. Gating strategies for subsequently de
de- scribed flow cytometric studies are depicted in d
NOD B lymphocytes display increased Aicda expression, and Supplemental Fig. 2. Line 1 mice were used for all fr
its ablation significantly retards T1D development subsequent studies because of better breeding o
proclivity and ease of genotyping compared with the
B lymphocytes in NOD mice can undergo CSR/SHM, equally T1D-resistant Line 26 mice. m
enhancing their capacity to process and present Selection processes to prune autoreactive B htt
autoantigenic epitopes to diabetogenic T cells lymphocytes operate at the immature transitional-1 p:/
(16). However, it is unknown whether CSR and SHM (T1) stage in the spleen (38). The frequency of
are naturally important contributors to the 2/2 /w
diabetogenic activity of B lymphocytes in NOD splenic T1 B lymphocytes is increased in NOD.Aicda w
mice, and by extension, in disease-susceptible mice, whereas subsequent developmental stage
transitional-2 (T2) cells are decreased (Fig. 2A). w.
humans. An initial finding supporting this
possibility was that basal expression levels of A shift in the ratio of T1/T2 cells might reflect ji
Aicda necessary to induce CSR/SHM was higher in increased tolerogenic culling of autoreactive B m
2/2
NOD B lymphocytes than in those from lymphocytes in NOD.Aicda mice (39). Alternatively, m
nonautoimmune-prone B6 mice (Fig. 1A). These data this shift may be the result of the decreased
2/2 un
support the presence of a significantly higher susceptibility of Aicda B lymphocytes to
baseline level of autoantigen-activated Aicda- apoptosis at the T1 stage (40). Despite the dif- ol.
expressing B lymphocytes in NOD mice than in B6 fering distribution of T1 and T2 subsets, total or
controls and are consistent with previous obser- splenic and PLN B lymphocyte yields were higher g/
2/2
vations that GCs are expanded in autoimmune-prone in NOD.Aicda mice than in WT controls (Fig. 2B). by
strains (37). We next tested whether Aicda Consistent with reports of AID ablation in other
expression by NOD B lymphocytes was critical to gu
strains (41), Fas+ GL7+ GC B lymphocytes were est
their diabetogenic activity. CRISPR-Cas9 technology expanded in the spleens and PLNs of NOD.Aicda
2/2
was used to directly target exon 1 or 2 on
(containing a potential alternative start site) mice (Fig. 2C, 2D). This correlates with the large
2/2
of Aicda in NOD zygotes (Supplemental Fig. 1AD). GC sizes observed in spleens of Aicda mice (data
This allowed for the subsequent generation of pure not shown). Also, the proportions of splenic CD80 +
NOD background stocks carrying the exon 1 (Line and PD-L2+ memory-like B lymphocytes (42) were
1)targeted or exon 2 (Line 26)targeted Aicda increased in NOD. Aicda
2/2
mice (Fig. 2E).
allele in a homozygous state (Supplemental Fig. Furthermore, the IgM-dominated serum isotype
1AD). We then assessed the ability of puri- fied 2/2
profile of NOD.Aicda mice parallels that of other
B lymphocytes from the Line 1 or 26 stocks to 2/2
undergo CSR to the IgG1 isotype following anti- Aicda strains (Fig. 2F), as well as those seen in
CD40 and IL-4 stimulation in vitro. Similar to AID-deficient patients (43). Finally, the
the case reported for a B6 background stock (22), B percentages of B lymphocytes among islet-
+
2/2
lymphocytes from Lines 1 and 26 NOD.Aicda mice infiltrating leukocytes (CD45.1 ) were similar in
2/2
displayed significantly decreased CSR (Fig. 1B, NOD and NOD.Aicda mice (Fig. 2G). The proportions
1C). Analyses were also carried out to confirm 2/2
2/2
of islet-infiltrating B lymphocytes in NOD.Aicda
that the reduced ability of NOD.Aicda B mice expressing the CD69 activation and CD80 or
lymphocytes to undergo CSR was not the result of PD-L2 memory-like markers (42) were similar and
off-target mutations caused by CRISPR-Cas9. To do greater, respectively, than those in NOD controls
2/2
this, each NOD. Aicda line was crossed to the (Fig. 2H). Together, these data indicate that
established B6.Cg-Aicda
,tm1Hon. genetically ablat- ing AID in NOD mice
increases the numbers of peripheral B
/HonRbrc stock, and purified B lymphocytes from the lymphocytes displaying a more predominant GC and
resulting F1 hybrids were tested for CSR capacity. memory- like phenotype.
The observation of similarly reduced CSR in F1
offspring for each line demonstrates that this AID ablation in NOD mice expands T cell populations but
phenotype is the result of our novel direct-in-NOD
disruption of Aicda (Supplemental Fig. 1E, 1F). does not directly affect their diabetogenic activity
T cells are the ultimate mediators of b cell
destruction in T1D. Therefore, we tested whether T
2/2
cells in T1D-resistant NOD. Aicda mice were
quantitatively and/or qualitatively distinct from splenic CD4+ and CD8+ T cells were increased in
those in NOD controls. Surprisingly, yields of 2/2
NOD.Aicda mice (Fig. 3A).
 D
o
w
nl
oa
de
d
fr
o
m
htt
p:/
/w
w
w.
ji
m
m
FIGURE 1. NOD B lymphocytes spontaneously express high levels of Aicda, and ablation of this gene inhibits T1D
development. (A) B lymphocytes purified from individual spleens of 8-wk-old NOD (n = 3) and B6 (n = 3) female mice un
were tested for Aicda expression via qPCR. Data are representative of three independent experiments. (B) ol.
Representative flow cytometry plots showing class switching to IgG1 of purified B lymphocytes from NOD and NOD. or
Aicda2/2 Lines 1 and 26 mice stimulated in culture with anti-CD40 (1 mg/ml) and murine IL-4 (25 ng/ml) for 96 h; g/
quantitative data pooled from three experiments (NOD = 7, Line 1 = 5, Line 26 = 5) are summarized in ( C). (D) Female
2/2 2/2
T1D incidence in NOD controls compared with NOD.Aicda Line 1 (p , 0.0001) and NOD.Aicda Line 26 (p = 0.001) mice.
by
(E) Insulitis scores on a scale of 0 (no visible lesion) to 4 (75100% islet destruction) for female NOD.Aicda2/2 Lines gu
1 and 26 mice remaining free of overt T1D at the end of the disease incidence study. (F) Insulitis for female NOD and est
2/2
NOD.Aicda Line 1 mice at 7, 11, and 18 wk of age. (G) Representative islet for each insulitis scoring level. Arrows on
point to lymphocytic infiltration of islets. Scale bar, 200 mm. All bar graphs and scatter plots show mean 6 SEM. The
Student t test was used to determine the p value shown for gene expression, and the Mann Whitney test was used for class
switching. The p values for T1D incidence were determined using MantelCox log-rank tests.

Corresponding with increased GC B lymphocytes, ficiently induced T1D development in NOD-scid

fully activated CXCR5+ PD-1+ Tfh cells were also recipients. This indicated that T1D resistance in
2/2
expanded in spleens and PLNs of NOD.Aicda mice 2/2
NOD.Aicda mice does not result from a T cell
(Fig. 3B, 3C). Although numerically ex- panded, we intrinsic effect. Therefore, we assessed how AID-
sought to determine whether the intrinsic deficient B lymphocytes might influence such
2/2
diabetogenic activity of T cells in NOD.Aicda mice pathogenic effectors. To initially test this,
2/2
was decreased. Purified NOD or NOD.Aicda T cells equal numbers of purified NOD splenic T cells
2/2
did not differ in their ability to transfer T1D and purified NOD or NOD.Aicda splenic B
to lymphocyte-deficient NOD-scid recipients (Fig. lymphocytes were transferred into NOD-scid
3D). Together, these data indicate that genetic recipients. NOD T cells induced T1D far less
ablation of AID in NOD mice leads to an expansion efficiently in NOD-scid recipients when
of total peripheral and T fh subset T cells, and 2/2
cotransferred with Aicda B lymphocytes than when
the observed disease protection is not due to a
decrease in their diabetogenic potential. cotransferred with WT B lymphocytes (Fig. 4A).
2/2
These results indicated that Aicda B lymphocytes
NOD T cells adoptively transfer T1D less efficiently in the may actively suppress diabetogenic T cell
presence of Aicda-deficient B lymphocytes characterized by an responses.
expanded CD73+ subset CD39 and CD73 are ectoenzymes that catalyze the
conversion of extracellular ATP to AMP and then to
As described above, when transferred in adenosine, respectively. There is evidence that
alterations to the CD39/CD73 purinergic metabolic
the absence of B lymphocytes, purified T pathway play a major role in several autoimmune
2/2
cells from NOD.Aicda donors ef- dis- eases (44). Adenosine signaling through the
adenosine A2a receptor
 D
o
w
nl
oa
de
d
fr
o
FIGURE 2.
2/2
NOD.Aicda B lymphocytes are numerically increased and display a more predominant GC phenotype than do m
those from NOD controls. htt
p:/
(A) Flow cytometric analyses comparing splenic T1 (B220 + CD19+ CD212 CD232), T2 (B220+ CD19+ CD21hi CD23+), marginal
zone (MZ; B220+ CD19+ CD21hi CD232), and follicular (FO; B220+ CD19+ CD21+ CD23+) B lymphocyte subsets in 8-wk-old female /w
NOD and NOD.Aicda
2/2
mice (n = 8 per group; one representative of three experiments). (B) Enumeration of total B w
lymphocytes (CD45.1+ B220+ CD19+) in spleen and PLNs of 710-wk-old NOD.Aicda2/2 mice (n = 21) compared with NOD controls w.
(n = 19) pooled from four individual experiments. (C) Representative flow cytometry contour plots showing single-cell ji
live B lymphocytegated events for analysis of splenic and PLN GC (Fas + GL7+) B lymphocytes in 7-wk-old female NOD and m
2/2
NOD.Aicda mice; quantitative data (n = 7 per group) from one experiment are summarized in (D). (E) Percentage of
CD80 or PD-L2+ cells among splenic or PLN B lymphocytes in 10-wk-old female NOD (n = 8) and NOD.Aicda2/2 (n = 10) mice
+ m
from one experiment. (F) Quantification, by ELISA, of serum Ig isotypes in 67-wk-old male NOD mice (n = 7) and un
2/2
NOD.Aicda mice (n = 10) pooled from two experiments. (G) Analysis of islet-infiltrating B220+ cells among CD45.1+ ol.
leukocytes in 1012-wk-old female NOD mice (n = 18) and NOD.Aicda2/2 mice (n = 16) pooled from five experiments. (H) or
Proportions of islet-infiltrating B lymphocytes with a CD69 +, CD80+, or PD-L2+ phenotype in 1012-wk-old female NOD g/
2/2
mice (n = 18 for CD69 and CD80, n = 13 for PD-L2) or NOD.Aicda mice (n = 16 for CD69 and CD80, n = 14 for PD-L2) pooled
from five experiments. All bar graphs show mean 6 SEM. All p values were calculated using MannWhitney analyses. by
gu
est
(A2aR) can inhibit TCR signaling and prevent retain increased CD73 expression (Fig. 5A). Thus, we
cellular activation (45). In a murine colitis on
model, CD73 expression reportedly marks a subset of assessed whether the expanded CD73+ B lymphocytes in
B lymphocytes with the capacity to suppress T cell
re- sponses through adenosine production (46).
Thus, we compared the extent to which B
lymphocytes from Aicda-intact and -deficient NOD
mice express CD39 and/or CD73. Levels of CD73+ B
lympho- cytes were greater within spleens, PLNs, and
2/2
islets of NOD.Aicda mice compared with NOD
controls (Fig. 4B, 4C). We observed higher
+
proportions of CD73 B lymphocytes that either did
or did not coexpress CD39 within spleens, PLNs,
2/2
and islets of NOD. Aicda mice compared with NOD
controls but found no dif- ferences in the CD39+
2
CD73 fraction (Fig. 4DF). Together, these data
indicate that the ability of NOD T cells to
induce T1D development is reduced in the presence
2/2
of NOD.Aicda B lymphocytes, which contain an
expanded CD73+ subset that was previously reported
to exert regulatory activity (46).
2/2
CD73+ B lymphocytes in NOD.Aicda mice exert regulatory
activity suppressing diabetogenic T cell responses
As noted above, CD73 expression can mark a
2/2
population of suppressive B lymphocytes. Aicda B
lymphocytes engrafted into NOD-scid recipients
 2/2
NOD.Aicda mice exert T1D suppressive effects in lymphocytes from AID-intact and -deficient NOD
adoptive- transfer experiments. Purified NOD T mice suppressed T cell prolifera- tion equally and
cells transferred T1D to NOD-scid recipients with to a significantly greater extent than did the
2
significantly greater efficiency when admixed CD73 subset in the presence of AMP; addition of
2/2
with Aicda B lymphocytes that had been depleted APCP di- minished this effect (Fig. 5C,
Supplemental Fig. 3A).
of the CD73+ subset (Fig. 5B). We then carried It has been reported previously that B
out in vitro studies to determine whether Aicda- lymphocytes with an ability to suppress T1D
deficient NOD B lymphocytes exert suppressive development in NOD mice primarily do so through
effects on such pathogenic effectors through IL-10 secretion (47). Similarly, the expanded
regula- tory activity mediated by the expanded 2/2
2
CD73+ subset. Sorted CD73+ and CD73 B lymphocytes CD73+ B lymphocyte population in NOD.Aicda mice,
2/2 which exhibit the capacity to suppress diabetogenic
from NOD or NOD.Aicda mice were cultured with T cell responses, secrete signifi- cantly greater
2
anti-CD40, anti-CD3/CD28stimulated CD73 CD4+ WT levels of IL-10 upon LPS stimulation than do their
T cells, and AMP, with or without the CD73 2
CD73 counterparts (Fig. 5D). Together, these
inhibitor APCP. On a per-cell basis, CD73+ B results indicate that
 upon stimulation. These collective results also
indicate that genetic ablation of AID inhibits T1D
development in NOD mice by quan- titatively
increasing, rather than qualitatively changing,
immuno- regulatory CD73+ B lymphocytes.
DIDS treatment decreases the Ig repertoire diversity of NOD
B lymphocytes
The results of our genetic studies show that AID-
dependent processes play an important role in B
lymphocyte contributions to T1D devel- opment in NOD
mice. By inhibiting RAD51-mediated HR repair of
AID-initiated off-target DSBs, treatment with the
small molecule DIDS induces the death of B
lymphocytes in which SHM and CSR processes have
been initiated (23). Therefore, we hypothesized
that targeting the AID/RAD51 pathway by DIDS
treatment might pro- vide an effective B
lymphocytedirected T1D intervention. To ini-
tially test this possibility, we stimulated purified
NOD B lymphocytes with anti-CD40 and IL-4 in the
FIGURE 3. Numbers of total peripheral and GC presence of vehicle or 150 mM DIDS in vitro.
phenotype CD4+ T cells are increased in NOD.Aicda2/2 Similar to the case for those from B6 control
mice. (A) Yields of splenic or PLN-resident CD4 mice, after 4 d of stimulation, fewer NOD B
lymphocytes were recovered from DIDS-containing
(CD45.1+ TCRb+ CD4+) and CD8 (CD45.1+ TCRb+ cultures (Fig. 6A). In addition, fewer anti-
CD8+) T cells in 710-wk-old female NOD (n = 19) and CD40/IL-4stimulated B lymphocytes were recovered D
NOD.Aicda2/2 from cultures containing (E)-5-acetamido-2-(4-(3- o
(n = 21) mice pooled from four experiments. (B) isopropylthioureido)- 2-
Representative flow cytometric contour plots showing sulfonatostyryl)benzenesulfonate, another small w
single-cell live CD4 T cellgated events for analysis molecule RAD51 inhibitor that is 1500-fold more nl
of splenic and PLN full T fh cells (CXCR5+ PD-1+) in 7-wk- potent than DIDS (Fig. 6A, Supplemental Fig. 4). oa
old female mice, with quantitative data (n = 7 per group)
summarized in (C), one representative of three de
experiments. (D) T1D development
6
in NOD- d
scid recipients injected with purified T cells from 7 We next determined whether DIDS treatment affected fr
2.5 3 10 8-wk-old the Ig rep-
female NOD.Aicda2/2 or NOD mice. All bar graphs show
o
mean 6 SEM. ertoire of NOD mice in a similar manner to genetic m
ablation of AID. We reasoned that the inability of htt
All p values in bar graphs were calculated using Mann NOD.Aicda
2/2
B lymphocytes to initiate CSR/SHM
Whitney analysis. T1D incidence p value was calculated processes would result in a decreased Ig gene coding p:/
using MantelCox analysis. repertoire diversity relative to WT controls. By /w
extension, we also hypothesized that DIDS-mediated w
disruption of the AID/RAD51 axis would recapitulate w.
2/2 such decreases in Ig repertoire diversity. Therefore,
diminished T1D development in NOD.Aicda mice is using established protocols (33), we used high- ji
due, at least in part, to an expansion of CD73+ B throughput sequencing to characterize full-length IgH m
lymphocytes with the capacity to suppress mRNAs from purified PLN-resident B lymphocytes of m
pathogenic T cell responses. CD73+ B lymphocytes individual 8-wk-old NOD and NOD.
suppress T cell responses through the generation of un
adenosine by this ectoenzyme and potentially by ol.
their ability to secrete IL-10 or
g/
by
gu
est
on

FIGURE 4. NOD T cells adoptively transfer T1D less efficiently in the presence of Aicda-deficient B lymphocytes
characterized by an expansion of the CD73 + subset. (A) T1D development in NOD-scid recipients injected with 2.5 3 106
purified T cells from 78-wk-old female NOD donors admixed with
2/2
2.5 3 106 B lymphocytes from 78-wk-old NOD mice (n = 10) or NOD.Aicda mice (n = 9). (B) Representative contour plots
for B220 versus CD73 staining pattern of splenic B220 CD19 B lymphocytes from female NOD and NOD.Aicda2/2 mice. (C)
+ +

Quantification of the percentage of CD73+ cells among spleen, PLN, and islet B lymphocytes in 712-wk-old NOD mice
(spleen/PLN: n = 15, islets: n = 18) and NOD.Aicda2/2 mice (spleen/PLN: n = 17, islets: n = 16). Spleen and PLN data are
pooled from three experiments; islet data are pooled from five experiments. Quantification of the percentage of CD39+ CD73+,
2 2
CD39+ CD73 , and CD39 CD73+ cells among splenic (D), PLN (E), or islet (F) B220+ CD19+ B lymphocytes in 712-wk-old female
NOD mice (spleen/PLN: n = 15, islets: n = 7) and NOD.Aicda2/2 mice (spleen/PLN: n = 17, islets: n = 8) pooled from two
individual experiments. T1D incidence p value was calculated using MantelCox analysis. All bar graph p values were
calculated using MannWhitney analysis, and all bar graphs show mean 6 SEM.
 NOD B lymphocytes are as susceptible to DIDS as
previously reported for those from the B6 strain
(23), and treatment with this small molecule
decreases IgH repertoire diversity in a manner
similar to Aicda ablation.
Molecular inhibition of the AID/RAD51 axis inhibits T1D
development
We next tested whether in vivo treatment with DIDS
exerted T1D protective effects in NOD mice.
Starting at 6, 8, or 10 wk of age, female NOD mice
received weekly i.p. injections of vehicle or DIDS
at 10 mg/kg (low-dose) or 50 mg/kg (high-dose; 6
and 8 wk start only). Regardless of start time or
dose, DIDS treatment exerted strong T1D protective
effects to 24 wk of age, at which time the disease
incidence in controls reached 90% (Fig. 7AC).
The presence of IAAs is an important criterion that
is used to identify humans at high future risk for
T1D for inclusion in possible disease-intervention
trials (13). Thus, just prior to treatment
initiation, serum was collected from mice depicted in
Fig. 7A and retrospectively typed for IAAs.
Importantly, DIDS treatment prevented progression to
overt T1D when initiated in already IAA+ NOD mice
2
(IAA+: vehicle 2/3, DIDS 0/9; IAA : vehicle 6/7, DIDS
0/11) (Fig. 7D). Additionally, NOD mice treated with D
DIDS from 8 wk of age had decreased levels o
w
nl
oa
de
d
fr
FIGURE 5. Expanded AID-deficient CD73 + B lymphocytes o
exert regulatory-like activity suppressing diabetogenic T m
cell responses. (A) + Splenic htt
engraftment levels B lymphocytes from NOD (n = 9) of insulitis compared with controls (Fig. 7E). p:/
of CD73 or NOD.
2/2 6 These collective data /w
Aicda (n = 8) donors admixed with NOD T cells of indicate that DIDS treatment inhibits progression to w
(2.5 3 10 each overt T1D, even w.
cell type) 4 wk posttransfer into NOD-scid recipients.
Results from one when initiated in NOD mice that had already developed ji
experiment. (B) T1D development in NOD-scid recipients high ongoing levels of b cell autoimmunity marked by m
injected with the presence of IAAs. m
2.5 3 106 purified T cells from 78-wk-old female NOD un
mice admixed with 2.5 3 106 total or CD73-depleted B DIDS treatment alters B lymphocyte profiles in a manner ol.
lymphocytes purified from 78-wk- old female or
NOD.Aicda2/2 donors. (C) A total of 1.0 3 105 CD73- similar to that elicited by Aicda ablation
+ g/
depleted purified CD4 T cells from 710-wk-old male To initially investigate how the reagent may elicit
NOD mice were labeled with Cell Proliferation Dye by
T1D protection,
eFluor 670 and cocultured for 4 d with 1.0 3 105 gu
CD73+ or B lymphocytes from pooled spleens of 710- NOD mice treated with DIDS from 8 to 16 wk of age est
2
CD73 wk-old male 2/2 were evaluated on
NOD (n = 6 biological replicates) or (n = 10 for phenotypic changes in B lymphocyte populations.
NOD.Aicda biological As observed
replicates) mice under stimulation conditions originating from single cDNA molecules to form
consisting of soluble anti- consensus reads. Rarefaction and extrapolated Chao
CD40 (1 mg/ml), plate-bound anti-CD3 (5 mg/ml), and diversity estimate
soluble anti- CD28 (2 mg/ml), with 0 (baseline) or 10 (48) analyses revealed decreased IgH diversity among B
mM AMP in the presence or absence of 100 mM APCP. lymphocytes from NOD.Aicda
2/2
and DIDS-treated NOD
Quantification of percentage suppression from baseline. mice compared with untreated and vehicle-treated
(D) A total of 5 3 104 sort-purified NOD (n = 5 NOD controls, respectively (Fig. 6B, 6C). To
biological replicates) or NOD.Aicda2/2 (n = 10 biological ensure that sampling bias resulting from var-
replicates) CD73+ or CD732 B lymphocytes were cultured iances in sequencing depth was not responsible for
for 3 d with 0 or 10 mg/ml LPS, and culture supernatant the observed differences in diversity, each sample
IL-10 levels were measured by ELISA. CD73- mediated in was down-sampled to 500 consensus reads. After 500
vitro suppression and IL-10 production data shown are iterations of resampling, Aicda
2/2
and DIDS-
combined from three and two individual experiments,
respectively. All bar graphs show mean 6 SEM. T1D treated B lymphocytes had decreased diversity, as mea-
incidence study p value was cal- culated using Mantel sured by the observed diversity and lower bound total
Cox analysis. The Wilcoxon test was performed for the diversity (Efron Thisted) estimate (49), compared
suppression assay. MannWhitney analysis was performed with their respective controls (Fig. 6D, 6E).
for IL-10 production. Together, the in vitro and in vivo data indicate
that
2/2
Aicda mice, as well as from NOD mice treated
with vehicle or 50 mg/kg DIDS from 8 to 16 wk of
age. To minimize overestimations of diversity, the
protocol uses template switch reverse transcription
to incorporate a 12-bp UMI at the 39-end of each
cDNA molecule. This allows for correction of
sequencing and PCR errors by grouping multiple reads
following genetic ablation of AID, NOD mice
treated with 50 mg/kg DIDS from 8 to 16 wk of age
were characterized by increased numbers of total
splenic and PLN B lymphocytes (Fig. 8A). Ad-
ditionally, similar to the case elicited by
genetic ablation of AID, DIDS treatment led to
the expansion of splenic and PLN GC B recirculating mature (Hardy frac- tion F) B
lymphocyte compartments (Fig. 8B). B lymphocytes
were also proportionally increased among islet-
infiltrating lymphocytes (Fig. 8C). Further
2/2
phenocopying NOD.Aicda mice, the propor- tions of
splenic, PLN, and islet-resident CD73+ B
lymphocytes with potential immunosuppressive
capacity were increased by DIDS treatment (Fig.
8D). Furthermore, the DIDS-elicited increase in
islet CD73+ B lymphocytes occurred in the CD73+
2
CD39+ and CD73+ CD39 subpopulations (Fig. 8E).
Finally, islet-infiltrating B lymphocytes had
similar levels of CD69 but increased CD80 and
PD-L2 expression (Fig. 8F). This was also similar
to the ex- pansion of B lymphocytes with a memory-
like phenotype within the islets of NOD.Aicda
2/2
mice. Despite this observed expansion of islet-
infiltrating memory-like B lymphocytes in DIDS-
treated mice, flow cytometric analyses of bone
marrowresident B lymphocytes revealed that,
relative to vehicle-treated controls, DIDS
treatment led to a proportional decrease in
lymphocytes (Fig. 8G, Supplemental Fig. 2F).
DIDS treatment effects on B lymphocytes indirectly suppress
diabetogenic T cell responses
DIDS treatment and genetic ablation of Aicda elicit
similar phe- notypic alterations in NOD B
lymphocytes. Although not elicited by Aicda
ablation, we considered it important to determine
whether DIDS treatment directly affects the
diabetogenic capacity of NOD T cells. Total CD4+ and
CD8+ T cells were numerically increased in the PLNs,
but not spleens, of DIDS-treated mice (Fig.
9A). As observed upon genetic ablation of AID, the
proportions
FIGURE 6. DIDS diminishes in vitro expansion and Ig usage diversity of NOD B lymphocytes. (A) Cellular yields of D
purified B lymphocytes from B6 or NOD (n = 3 biological replicates per group) mice cultured for 96 h (1 3 106 cells per o
milliliter) with anti-CD40 (1 mg/ml) and murine IL-4 (25 ng/ml) in the presence of vehicle, 150 mM DIDS, or 100 nM w
(E)-5-acetamido-2-(4-(3-isopropylthioureido)-2-sulfonatostyryl)benzenesulfonate. Data are representative of one of
three experiments. (B) A total of 136,000400,000 purified PLN B lymphocytes from the indicated NOD experimental nl
groups was sequenced for IgH gene usage diversity (n = 3 mice per group) in one experiment. Sequences with early stop oa
codons or frame-shift mutations were filtered to display only functional clones. Data are presented in a de
rarefaction plot showing clonal diversity as a function of unique cDNA molecules sequenced. Solid and dashed lines are
interpolated and extrapolated regions, respectively, with points marking the exact sample size and observed diversity. d
The shaded area represents the 95% confidence interval. (C) Chaos diversity estimate index. Observed diversity (D) fr
and EfronThisted estimate (E) after 500 iterations of down- sampling to 500 reads. Scatter plots show mean 6 SEM. o
The p values were calculated using the Student t test. m
htt
of Tfh cells were increased in the spleens and PLNs p:/
in the absence of B lymphocytes. Purified splenic
of DIDS-treated NOD mice (Fig. 9B). The proportion T cells from 56- wk-old female NOD mice were /w
of total T cells was decreased among islet- transferred into NOD-scid recip- ients. Starting 3 w
infiltrating leukocytes (Fig. 9C) after DIDS d posttransfer, the NOD-scid recipients began
treatment. Additionally, there was a decrease in the w.
proportion of the CD8+ subset among islet- weekly 50 mg/kg DIDS or vehicle treatment and were ji
monitored for T1D development. After engraftment
infiltrating T cells (Fig. 9D). Islet CD4+ T cells with NOD T cells, NOD- scid recipients treated with m
displayed increased CD69 expression after m
DIDS treatment (Fig. 9E). DIDS treatment did not DIDS or vehicle developed T1D at an equivalent
significantly alter CD69 expres- sion of islet CD8+ rate (Fig. 9H). These results indicate that DIDS un
T cells, but it marginally increased IL-7Ra levels treat- ment does not directly affect the ol.
(Fig. 9E). Finally, compared with vehicle-treated diabetogenic activity of NOD T cells. Thus, the or
controls, in DIDS- treated mice, islet- diminished ability of T cells from DIDS-treated
NOD mice to adoptively transfer T1D cannot be g/
infiltrating CD4+ T cells had a decreased naive and attributed to a direct reduction in their by
increased central memory phenotype, whereas CD8 + T pathogenicity; rather, it is likely due to DIDS
cells had a decreased effector and an increased gu
limiting the ability of B lymphocytes to support
central memory pheno- type (Fig. 9F). Together, the ex- pansion of pathogenic effectors. est
these data indicate that DIDS treatment reduces on
effector CD8+ T cells and drives the accumulation +
CD73 B lymphocytes expanded by DIDS treatment suppress
of central memory CD4+ and CD8+ T cells within the
islets of NOD mice. diabetogenic T cell responses
We next tested whether DIDS treatment had lasting We tested whether coinfusion of B lymphocytes from
effects on T cell diabetogenic activity. Female NOD control or DIDS-treated NOD mice differentially
mice, treated once weekly with vehicle or 50 mg/kg affected the ability of di- abetogenic T cells to
DIDS from 8 to 16 wk of age, then became donors of transfer disease to NOD-scid recipients. NOD female
purified splenic T cells that were transferred into mice were injected once weekly, from 8 to 16 wk of
NOD-scid recipients that were subsequently monitored age, with vehicle or 50 mg/kg DIDS. Purified
for T1D development. T cells from DIDS-treated mice splenic T cells from the vehicle-treated mice were
transferred T1D to NOD-scid recipients with sig- then cotransferred into NOD-scid re- cipients with
nificantly less efficiency compared with those from equal numbers of purified total B lymphocytes from
control donors (Fig. 9G). We envisioned two possible vehicle or DIDS-treated donors. As previously
(nonexclusive) explanations for this result: B noted, similar to the case of NOD.Aicda
2/2
mice,
lymphocytes in the DIDS-treated donors expand autore- +
active T cell populations less efficiently than in DIDS treatment expands CD73 B lymphocyte
vehicle-treated mice and DIDS can act directly to populations. Therefore, we also transferred T
suppress diabetogenic T cell activity. cells from vehicle-treated controls with CD73-
depleted B lymphocytes from DIDS-treated mice. T1D
To distinguish between the above possibilities, development was significantly de- creased in NOD-
we tested the direct effect of DIDS on the scid recipients of pathogenic T cells coinfused
diabetogenic function of NOD T cells
The Journal of 11
Immunology
Discussion
We have demonstrated that disruption of the
AID/RAD51 axis, through genetic or pharmacological
means, strongly inhibits T1D development in NOD
mice, and this protection is due, at least in
part, to the expansion of specific CD73 + B
lymphocyte populations with capacities to regulate
pathogenic T cell responses. Addi- tionally, we
provide the first evidence, to our knowledge,
that, although CSR and SHM processes are important
B lymphocyte intrinsic processes in T1D
pathogenesis, their absence does not diminish
autoreactive T cell development. Although purified
2/2
T cells from NOD.Aicda mice are effective at
adoptively transferring T1D to NOD-scid recipients,
inhibiting CSR and SHM processes leads to an
expansion of Bregs controlling these effectors. These
findings reveal an unexpected link between blocking
AID/RAD51- dependent affinity-maturation processes
and Breg development. Thus, disruption of the
AID/RAD51 axis may represent a previ- ously
unrealized means of in vivo Breg expansion.

FIGURE 7. DIDS treatment inhibits T1D development.

Starting at 6 (A), 8 (B), or 10 (C) wk of age, female
NOD mice were treated with ve- hicle or DIDS (10 or 50
mg/kg) on a weekly basis and monitored for T1D
development. The p values were calculated using Mantel
Cox analysis.
(D) Serum was harvested from the cohort of mice in (A)
at the initiation of treatment and retrospectively typed
for IAAs. Graph represents the per- centage of IAA + or
IAA2 mice in each treatment group that did or did not
progress to T1D (IAA+ DIDS: n = 9; IAA+ Vehicle: n = 3;
2 2
IAA DIDS: n = 11; IAA Vehicle: n = 7). The p values
were calculated using the Fisher exact test. (E)
Insulitis scores for NOD female mice treated weekly
starting at 8 wk of age with vehicle (n = 10) or DIDS at
a dose of 10 mg/kg (n = 8) or 50 mg/kg (n = 14). Data
show mean 6 SEM. The p values were calculated using
MannWhitney analysis.

with total B lymphocytes from DIDS-treated donors increase in CD73+ B lymphocytes with a capacity to
compared with vehicle-treated donors (Fig. 10A). actively suppress T1D development (Fig. 10A).
However, depletion of the CD73 + subset Next, we tested whether the CD73+ population
significantly enhanced the ability of B expanded by DIDS treatment can directly suppress T
lymphocytes from DIDS-treated donors to support T1D cell responses. On a per- cell basis, CD73 + B
development in NOD-scid recipients coinfused with lymphocytes from vehicle- and DIDS-treated mice
pathogenic T cells (Fig. 10A). Hence, similar to equally suppressed T cell proliferation in response
the case with genetic ablation of AID, DIDS to anti- CD3 and anti-CD28 stimulation to a
treatment of NOD mice induces a quantitative 2
significantly greater extent than did the CD73
10 TARGETING AID-ACTIVE B CELLS FOR TYPE I DIABETES PREVENTION
subset in the presence of AMP (Fig. 10B). This
suppressive effect was diminished upon addition of
APCP (Fig. 10B, Supplemental Fig. 3B). Finally, we
examined IL-10 pro- duction by sorted CD73+ and
2
CD73 B lymphocytes from vehicle-

FIGURE 8. DIDS treatment alters B lymphocyte profiles in

a manner similar to that elicited by Aicda ablation. NOD
female mice were treated with vehicle or 50 mg/kg DIDS
from 8 to 16 wk of age. (A) Yield of total splenic and
PLN CD19+ B220+ B lymphocytes (n = 7 vehicle, n = 8 DIDS;
combined from two experiments). (B) Percentage of splenic
and PLN GC (Fas+ GL7+) B lymphocytes (n = 7 vehicle, n = 8
DIDS; combined from two experiments). (C) Percentage of
B220+ leukocytes among islet CD45.1+ cells (combined from
two experiments). (D) Percentage of CD73+ B cells among
B220+ CD19+ B lymphocytes (n = 7 Vehicle Spleen/PLN, n = 8
DIDS Spleen/PLN, n = 13 Vehicle Islet, n = 14 DIDS Islet;
combined from two experiments). (E) Percentage of CD73+
CD39+, CD732 CD39+, and CD73+

and DIDS-treated mice. Surprisingly, CD73+ B

2
CD39 cells among islet lymphocytes (n = 13 Vehicle, n = 14
lymphocytes from B220+ DIDS; + + D
DIDS-treated mice produced less IL-10 upon LPS combined from two experiments). (F) Percentage of CD69 , o
stimulation than did those from vehicle-treated CD80 , and
w
controls (Fig. 10C). These results indicate that PD-L2+ cells among islet B220+ cells (n = 13 Vehicle, n = 14
pharmacological targeting of RAD51 activity DIDS; combined from two experiments). (G) Proportions of nl
inhibits T1D development in NOD mice in a manner Hardy fraction D (B220+ CD432 IgM2 IgD2), fraction E oa
similar to that elicited by Aicda ablation, (B220+ CD432 IgM+ IgD2), and de
including a quantitative increase in CD73 + 2
fraction F (B220+ CD43 IgM+ IgD+) B lymphocyte subsets in d
regulatory B lymphocytes (Bregs). bone marrow (one experiment). MannWhitney analysis was fr
performed for all bar and scatter plot graphs; data are
mean 6 SEM. o
m
htt
p:/
/w
w
w.
ji
m
m
un
ol.
or
g/
by
gu
est
on
 NOD mice from an effector to a central memory
phenotype (Fig. 9F), paired with increased IL-7Ra
expression (Fig. 9E), further supports the
conclusion that, in this system, diabetogenic T
cells are suppressed through an adenosine-mediated
mecha- nism. Therefore, although not precluding
the possibility of other nonoverlapping adenosine-
independent suppression pathways, the population
of CD73+ B lymphocytes expanded in AID- deficient
or DIDS-treated NOD mice appears to largely
inhibit diabetogenic T cell responses through a
mechanism that is de- pendent on activity of this
ectoenzyme.
It should also be noted that, although CD73-
depleted B lymphocytes from DIDS-treated mice could
support the ability of coinfused pathogenic T
cells to transfer T1D to NOD-scid recipients, they
did so less efficiently than total B lymphocytes
from vehicle- treated control donors. This could
indicate that, in addition to expanding CD73 + Bregs,
DIDS treatment diminishes the ability of other B
lymphocyte populations in NOD mice to support dia-
betogenic T cell activity. This could include DIDS
2
treatment supporting the expansion of CD73 Bregs
capable of suppressing T cells through adenosine-
independent mechanisms. Should this be the
2
case, the mechanism by which such CD73 Bregs
mediate suppression is likely to be IL-10 D
independent, because this population produces o
little of the cytokine in response to stimulation.
It is unknown why CD73+ B lymphocytes from DIDS- w
nl
treated mice produce less LPS-induced IL-10 than
oa
do those from controls.
de
IL-10 secretion has been recognized as a major
means of Breg- mediated immune suppression (51). d
However, there have also been reports of Bregs fr
capable of suppressing systemic lupus o
erythematosus and experimental autoimmune m
encephalomyeli- tis development through IL-10
independent mechanisms (52). IL-10 production by B htt
lymphocytes requires strong stimulation, whereas p:/
adenosine generation by CD73+ B lymphocytes is con- /w
stitutive, providing that its substrate is present
(46). The ability of B lymphocytes from DIDS- w
treated NOD mice to inhibit diabeto- genic T cell w.
responses (Fig. 10A, 10B), despite decreased IL-10 ji
production (Fig. 10C), supports the conclusion
that such disease- protective effects are largely m
the result of CD73-mediated aden- osine m
production. This observation is consistent un
with a report
FIGURE 9. DIDS effects on B lymphocytes indirectly ol.
suppresses di- abetogenic T cell responses. NOD female or
mice were treated weekly with 50 mg/kg DIDS or vehicle
from 8 to 16 wk of age. (A) Percentage of CD4+ and CD8+ T g/
cells among live TER-1192 splenic or PLN-resident leuko- by
cytes (non-RBCs) (n = 7 Vehicle, n = 8 DIDS; combined gu
from two ex- periments). (B) Quantification of est
percentage of full Tfh cells among CD4+ TCRb+ cells in the
spleen and PLN (n = 7 Vehicle, n = 8 DIDS; combined from on
two experiments). (C) Percentage of TCRb+
leukocytes among
CD45.1+ cells within the +islets. (D) or + cel suggesting that Breg-mediated adenosine production
Percentage of CD4 CD8 ls may play a
among islet T cells (n = 13 Vehicle, n = 14 DIDS; more important role in immunosuppression than IL-10
combined from two secretion
2/2
experiments). (E) Percentage and IL- cells among (46). Additionally, it should be noted that IL-10
of CD69+
7Ra+ islet B lymphocytes
CD4+ and CD8+ T cells (n = 13 Vehicle, n = 14 DIDS;
combined from Ablation of AID and DIDS targeting of RAD51
2
two experiments). (F) Percentage of naive (CD44 CD62L+), results in ac- cumulation of CD73+ B lymphocytes
+ 2
effector (CD44 CD62L ), and central memory (CD44+
capable of suppressing T cells, at least in part
through the production of adenosine (Figs. 5C, 10B).
CD62L+) CD4+ and CD8+ T cells in the islets of DIDS- or Previous studies demonstrated that+ adenosine
vehicle-treated mice (n = 13 vehicle, n = 14 DIDS; signaling via A2aR inhibits CD8 T cell
combined from two experiments). (G) Female NOD mice downregulation of IL-7Ra, preventing the
were injected with vehicle or 50 mg/kg DIDS from 8 to 16 differentiation of memory T cells to effectors (50).
wk of age. Splenic T cells were then purified from each +
treatment group and transferred (3 3 106) into NOD-scid Therefore, the shift in islet-infiltrating CD8 T
cell populations in DIDS-treated
recipients (n = 16 per group) that were subsequently
monitored for T1D development. (H) A total of 2.5 3 106
purified total T lymphocytes from 6-wk-old female NOD
mice was transferred into NOD-scid mice that subsequently
began weekly treat- ment with 0 or 50 mg/kg DIDS (n = 10
per group) and were monitored to T1D. Incidence study p
values were calculated using MantelCox analysis. Mann
Whitney analysis was performed for all bar graphs; data
show mean 6 SEM.
have reduced CD73 expression, suggesting that
this cytokine likely directly regulates CD73
expression by B lymphocytes (46). There- fore, DIDS
treatment of mice made genetically deficient in IL-
10 expression or treated with an Ab blocking this
cytokine would not discriminate between the
regulatory contributions of CD73-mediated adenosine
production and direct action by IL-10. The future
2/2
creation of B lymphocytespecific CD73 NOD mice
will help to uncouple the individual regulatory
contributions provided by adenosine generation
and IL-10 secretion.
This study has focused on the role of the
AID/RAD51 axis in GC affinity-maturation
processes contributing to T1D. However, the AID
pathway has been shown to play an important role
in B lymphocytes outside of the splenic or
lymph node GC envi- ronment. For example, AID has
been implicated in the central tolerance of B
lymphocytes in C57BL/6 and BALB/c background
strains (53). Another study demonstrated a role
for AID in central and peripheral tolerance of
human B lymphocytes (54). However, discerning the
contribution of AID to these processes in NOD mice
is complicated by strain-specific defects in B
lymphocyte central tolerance (55). Therefore, the
effect of targeting AID/ RAD51 on B lymphocyte
central tolerance in NOD is unclear and
warrants further investigation. Additionally,
manipulation of
The Journal of 13
Immunology
some B lymphocytes to a T1D-protective CD73+
regulatory phe- notype. Furthermore, because this
immunomodulatory therapy retains efficacy, even
when initiated at a late prodromal autoantibody-
positive stage of T1D development, it also
represents a significant improvement upon previous
B lymphocytetargeted disease- intervention
strategies.
In summary, these studies show that the genetic
or pharmacologic blockade of B lymphocyte
affinity-maturation processes in NOD mice drives
diversion to a CD73+ regulatory phenotype capable
of inhibiting autoimmune T1D development.
Therefore, pharmaco- logical targeting of RAD51 to
block diabetogenic B lymphocyte activity, either
directly or by converting them to an immunoregu-
latory state, might ultimately represent a
clinically translatable disease-intervention
FIGURE 10. approach. Because RAD51 is a multiprotein complex,
B lymphocytes in DIDS-treated mice this area of the AID pathway has other potential
CD73+ are T1D targets that might be exploited. The identification
and use of pharmaco- logical agents that
potentially directly block AID activity as a novel
T1D-intervention approach should also be explored.
These studies

suppressive. NOD female mice were treated weekly with 50 pharmaceutical agents targeting the AID/RAD51 axis D
mg/kg DIDS
or vehicle from 8 to 16 wk of age. (A) NOD-scid recipients could convert o
were infused with 2 3 106 purified splenic T cells from w
vehicle-treated donors admixed with an equal number of nl
purified total splenic B lymphocytes from vehicle (Total
VB), CD73-depleted B lymphocytes from DIDS-treated mice oa
(CD732 DB), or total B lymphocytes from DIDS-treated mice de
(Total DB). The NOD-scid recipients were then monitored for d
T1D development over 8 wk. (B) A total of 1.0 3 105 NOD T fr
cells was labeled with Cell Pro- liferation Dye eFluor
670 and cocultured for 4 d with 1.0 3 105 CD73+ or CD732 B o
lymphocytes from pooled spleens of vehicle-treated (n = 6 m
biological replicates) or DIDS-treated (n = 6 biological htt
replicates) mice under stimulation conditions consisting
of soluble anti-CD40 (1 mg/ml), plate-bound anti-CD3 (5 p:/
mg/ml), and soluble anti-CD28 (2 mg/ml) with 0 /w
(baseline) or 10 mM AMP in the presence or absence of w
100 mM APCP. Quantification of percentage of suppression w.
from baseline. (C) A total of 1 3 105 sort-purified
+ 2
CD73 or CD73 B lymphocytes from vehicle- or DIDS-treated ji
NOD mice (n = 6 biological replicates per group) were m
cultured for 3 d with 10 mg/ml LPS, and culture m
supernatant IL-10 levels were measured by ELISA. CD73- un
mediated in vitro suppression and IL-10 production data
shown are each combined from two individual experiments. ol.
All bar graphs show mean 6 SEM. Incidence study p value or
was calculated using MantelCox analysis. The Wilcoxon
test was performed for suppres- sion assay. MannWhitney g/
analysis was performed for IL-10 production. by
gu
AID affects gut-associated B lymphocytes and est
alters intestinal microflora (56). Gut microflora on
alterations can drastically impact T1D development
(57). Thus, examining the impact of disrupting the
AID/RAD51 axis on GALT homeostasis is warranted.
Addi- tionally, disruption of the AID/RAD51 axis
could conceivably result in a decreased ability to
clear infection. Due to institutional policy, we
cannot infect mice with microbes at The Jackson
Labo- ratory. Therefore, the possible effects of
targeting the AID/RAD51 axis on the ability to
clear pathogens will need to be the subject of
other investigators research. Additionally,
agents that might spe- cifically increase CD73+
Bregs without impacting the AID/RAD51 axis should be
investigated.
A previous rituximab-mediated panB
lymphocytetargeting clinical trial did not
permanently halt b cell demise (17). Studies in
NOD mice indicate that this may be due, at least
in part, to downregulation of cell surface CD20
expression by islet-infiltrating B lymphocytes
(18). This provides a likely explanation for why
anti-CD20 immunotherapy only protects NOD mice
from T1D if initiated prior to IAA development.
Other studies have provided evidence that B reg
expansion has a potential to confer strong T1D
inhibitory effects, and pan-B lymphocytedepletion
regimens could deplete these protective populations
(58). Our results provide the first indication, to
our knowledge, that future clinically applicable
 12 TARGETING AID-ACTIVE B CELLS FOR TYPE I DIABETES PREVENTION
also indicate that further research into the role 2. Akashi, T., S. Nagafuchi, K. Anzai, S. Kondo, D. Kitamura, S.
of the purinergic immunoregulatory pathway in T1D Wakana, J. Ono,
pathogenesis is warranted. Furthermore, this M. Kikuchi, Y. Niho, and T. Watanabe. 1997. Direct evidence
for the contri- bution of B cells to the progression of
pathway could also represent a potentially clini- insulitis and the development of diabetes in non-obese
cally relevant immunomodulatory target for the diabetic mice. Int. Immunol. 9: 11591164.
treatment of various other autoimmune diseases, 3. Wong, F. S., I. Visintin, L. Wen, J. Granata, R. Flavell, and
either through increasing adenosine pro- duction or C. A. Janeway. 1998.
through administration of A2aR agonists. Together, The role of lymphocyte subsets in accelerated diabetes in
these initial studies reveal that therapeutic nonobese diabetic-rat insulin promoter-B7-1 (NOD-RIP-B7-1)
targeting of the AID/RAD51 axis in B lymphocytes is mice. J. Exp. Med. 187: 19851993.
a previously unrealized area of research for T1D 4. Noorchashm, H., N. Noorchashm, J. Kern, S. Y. Rostami,
therapy development. C. F. Barker, and
A. Naji. 1997. B-cells are required for the initiation of
Acknowledgments insulitis and sialitis in nonobese diabetic mice. Diabetes 46:
941946.
We thank staff within The Jackson Laboratorys Genome 5. Bouaziz, J. D., K. Yanaba, G. M. Venturi, Y. Wang, R. M.
Technologies group, Genetic Engineering Technologies Tisch, J. C. Poe, and
group, Flow Cytometry service, and Re- search Animal T. F. Tedder. 2007. Therapeutic B cell depletion impairs
Facility for technical support. We also thank Susanne adaptive and autoreactive CD4+ T cell activation in mice. Proc.
Sattler (Imperial College, London, U.K.) for critical
review of the manuscript. Natl. Acad. Sci. USA 104: 2087820883.
6. Fiorina, P., A. Vergani, S. Dada, M. Jurewicz, M. Wong, K. Law,
E. Wu, Z. Tian,
Disclosures R. Abdi, I. Guleria, et al. 2008. Targeting CD22 reprograms
K.D.M. is a founder and shareholder in Cyteir Therapeutics, B-cells and reverses autoimmune diabetes. Diabetes 57: 3013
Inc. K.D.M., M.G.H., and C.M.L. hold United States 3024.
Patent No. US20130184342 A1: Methods and compositions 7. Hu, C. Y., D. Rodriguez-Pinto, W. Du, A. Ahuja, O.
for treatment of cancer and autoimmune dis- ease. The Henegariu, F. S. Wong,
other authors have no financial conflicts of interest. M. J. Shlomchik, and L. Wen. 2007. Treatment with CD20-specific
antibody pre- vents and reverses autoimmune diabetes in mice. J.
Clin. Invest. 117: 38573867.
References 8. Xiu, Y., C. P. Wong, J. D. Bouaziz, Y. Hamaguchi, Y.
1. Serreze, D. V., H. D. Chapman, D. S. Varnum, M. S. Hanson, P. Wang, S. M. Pop,
C. Reifsnyder, R. M. Tisch, and T. F. Tedder. 2008. B lymphocyte depletion by
CD20 monoclonal antibody prevents diabetes in nonobese
S. D. Richard, S. A. Fleming, E. H. Leiter, and L. diabetic mice despite isotype-specific dif- ferences in Fc
D. Shultz. 1996. B lymphocytes are essential for the gamma R effector functions. J. Immunol. 180: 28632875.
initiation of T cell-mediated autoimmune diabetes: analysis
of a new speed congenic stock of NOD.Ig mu null mice. J. 9. Pearson, J. A., F. S. Wong, and L. Wen. 2016. The importance
Exp. Med. 184: 20492053. of the Non Obese Diabetic (NOD) mouse model in autoimmune
diabetes. J. Autoimmun. 66: 7688.
10. Hulbert, C., B. Riseili, M. Rojas, and J. W. Thomas. 29. Julius, M. H., and L. A. Herzenberg. 1974. Isolation of
2001. B cell specificity contributes to the outcome of antigen-binding cells from unprimed mice: demonstration of
antibody-forming cell precursor activity and correlation
diabetes in nonobese diabetic mice. J. Immunol. 167: 55355538. between precursor and secreted antibody avidities. J. Exp. Med.
11. Marino, E., B. Tan, L. Binge, C. R. Mackay, and S. T. 140: 904920.
Grey. 2012. B-cell cross- 30. Silveira, P. A., H. D. Chapman, J. Stolp, E. Johnson,
presentation of autologous antigen precipitates diabetes. S. L. Cox, K. Hunter,
[Published erratum appears in 2013 Diabetes 62: 662.] Diabetes L. S. Wicker, and D. V. Serreze. 2006. Genes within the Idd5
61: 28932905. and Idd9/11 di- abetes susceptibility loci affect the
12. Silveira, P. A., E. Johnson, H. D. Chapman, T. pathogenic activity of B cells in nonobese diabetic mice. J.
Bui, R. M. Tisch, and Immunol. 177: 70337041.
D. V. Serreze. 2002. The preferential ability of B 31. Christianson, G. J., W. Brooks, S. Vekasi,
lymphocytes to act as diabe- togenic APC in NOD mice depends E. A. Manolfi, J. Niles,
on expression of self-antigen-specific im- munoglobulin S. L. Roopenian, J. B. Roths, R. Rothlein, and D. C. Roopenian.
receptors. Eur. J. Immunol. 32: 36573666. 1997. Beta 2-microglobulin-deficient mice are protected from
13. Bonifacio, E., and A. G. Ziegler. 2010. Advances in the hypergammaglobulinemia and have defective antibody responses
prediction and natural because of increased IgG catabolism. J. Immunol. 159: 4781
history of type 1 diabetes. Endocrinol. Metab. Clin. North Am. 39: 4792.
513525. 32. Bonifacio, E., M. Atkinson, G. Eisenbarth, D. Serreze, T.
14. Victora, G. D., and M. C. Nussenzweig. 2012. Germinal W. Kay, E. Lee-Chan,
centers. Annu. Rev. Immunol. 30: 429457. and B. Singh. 2001. International workshop on lessons from
15.Murphy, K., P. Travers, M. Walport, and C. Janeway. 2008. animal models for human type 1 diabetes: identification of
Janeways Immu- insulin but not glutamic acid decar- boxylase or IA-2 as
nobiology. Garland Science, New York. specific autoantigens of humoral autoimmunity in nonobese
16. Wan, X., J. W. Thomas, and E. R. Unanue. 2016. Class- diabetic mice. Diabetes 50: 24512458.
switched anti-insulin antibodies originate from 33. Turchaninova, M. A., A. Davydov, O. V. Britanova,
unconventional antigen presentation in multiple lym- phoid M. Shugay, V. Bikos,
sites. J. Exp. Med. 213: 967978. E. S. Egorov, V. I. Kirgizova, E. M. Merzlyak, D. B.
17. Pescovitz, M. D., C. J. Greenbaum, H. Krause- Staroverov, D. A. Bolotin,
Steinrauf, D. J. Becker, D
S. E. Gitelman, R. Goland, P. A. Gottlieb, J. B. o
Marks, P. F. McGee, w
A. M. Moran, et al; Type 1 Diabetes TrialNet Anti-CD20 Study
Group. 2009. Rituximab, B-lymphocyte depletion, and
nl
preservation of beta-cell function. N. Engl. J. Med. 361: oa
21432152. de
18. Serreze, D. V., H. D. Chapman, M. Niens, R. Dunn, M. R. d
Kehry, J. P. Driver,
M. Haller, C. Wasserfall, and M. A. Atkinson. 2011. Loss of fr
intra-islet CD20 expression may complicate efficacy of B- o
cell-directed type 1 diabetes therapies. Diabetes 60: 2914 m
2921.
19. Chaudhuri, J., M. Tian, C. Khuong, K. Chua, E. Pinaud, htt
and F. W. Alt. 2003. p:/
Transcription-targeted DNA deamination by the AID antibody /w
diversification enzyme. Nature 422: 726730. w
20. McBride, K. M., A. Gazumyan, E. M. Woo, V. M.
Barreto, D. F. Robbiani, w.
B. T. Chait, and M. C. Nussenzweig. 2006. Regulation of ji
hypermutation by activation-induced cytidine deaminase m
phosphorylation. Proc. Natl. Acad. Sci. USA 103: 87988803.
21. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. m
Yamada, Y. Shinkai, and un
T. Honjo. 2000. Class switch recombination and hypermutation ol.
require activation-induced cytidine deaminase (AID), a
potential RNA editing enzyme. Cell 102: 553563. or
22. Hasham, M. G., N. M. Donghia, E. Coffey, J. Maynard,
K. J. Snow, J. Ames, g/
R. Y. Wilpan, Y. He, B. L. King, and K. D. Mills. 2010. by
Widespread genomic breaks generated by activation-induced
cytidine deaminase are prevented by homologous recombination. gu
Nat. Immunol. 11: 820826. est
23. Lamont, K. R., M. G. Hasham, N. M. Donghia, J.
Branca, M. Chavaree, on
B. Chase, A. Breggia, J. Hedlund, I. Emery, F. Cavallo, et
al. 2013. Attenuating homologous recombination stimulates an
AID-induced antileukemic effect. J. Exp. Med. 210: 10211033.
24. Serreze, D. V., E. H. Leiter, M. S. Hanson, S. W.
Christianson, L. D. Shultz,
R. M. Hesselton, and D. L. Greiner. 1995. Emv30null NOD-scid
mice. An im- proved host for adoptive transfer of autoimmune
diabetes and growth of human lymphohematopoietic cells.
Diabetes 44: 13921398.
25. Hill, J. T., B. L. Demarest, B. W. Bisgrove, Y. C. Su,
M. Smith, and H. J. Yost.
2014. Poly peak parser: method and software for
identification of unknown indels using sanger sequencing of
polymerase chain reaction products. Dev. Dyn. 243: 16321636.
26. Morgan, H. D., W. Dean, H. A. Coker, W. Reik, and S. K.
Petersen-Mahrt. 2004.
Activation-induced cytidine deaminase deaminates 5-
methylcytosine in DNA and is expressed in pluripotent
tissues: implications for epigenetic reprogram- ming. J. Biol.
Chem. 279: 5235352360.
27. Cartwright, R., A. M. Dunn, P. J. Simpson, C. E. Tambini,
and J. Thacker. 1998.
Isolation of novel human and mouse genes of the recA/RAD51
recombination- repair gene family. Nucleic Acids Res. 26: 1653
1659.
28. Johnson, E. A., P. Silveira, H. D. Chapman, E. H.
Leiter, and D. V. Serreze. 2001.
Inhibition of autoimmune diabetes in nonobese diabetic mice
by transgenic restoration of H2-E MHC class II expression:
additive, but unequal, involvement of multiple APC subtypes.
J. Immunol. 167: 24042410.
 et al. 2016. High-quality full-length immunoglobulin 46. Kaku, H., K. F. Cheng, Y. Al-Abed, and T. L. Rothstein.
profiling with unique molecular barcoding. Nat. Protoc. 11: 2014. A novel mech- anism of B cell-mediated immune
suppression through CD73 expression and adenosine production.
15991616. J. Immunol. 193: 59045913.
34. Shugay, M., O. V. Britanova, E. M. 47. Kleffel, S., A. Vergani, S. Tezza, M. Ben Nasr, M. A.
Merzlyak, M. A. Turchaninova, Niewczas, S. Wong,
I. Z. Mamedov, T. R. Tuganbaev, D. A. R. Bassi, F. DAddio, T. Schatton, R. Abdi, et al. 2015.
Bolotin, D. B. Staroverov, Interleukin-10+ regu- latory B cells arise within antigen-
experienced CD40+ B cells to maintain tol- erance to islet
E. V. Putintseva, K. Plevova, et al. 2014. Towards error- autoantigens. Diabetes 64: 158171.
free profiling of immune repertoires. Nat. Methods 11: 653 48. Colwell, R. K., A. Chao, N. J. Gotelli, S.-Y. Lin, C. X.
655. Mao, R. L. Chazdon, and
35. Bolotin, D. A., S. Poslavsky, I. Mitrophanov, M. J. T. Longino. 2012. Models and estimators linking
Shugay, I. Z. Mamedov, individual-based and sample- based rarefaction, extrapolation
and comparison of assemblages. J. Plant Ecol. 5: 321.
E. V. Putintseva, and D. M. Chudakov. 2015. MiXCR: software 49. Britanova, O. V., E. V. Putintseva, M.
for compre- hensive adaptive immunity profiling. Nat. Methods Shugay, E. M. Merzlyak,
12: 380381. M. A. Turchaninova, D. B. Staroverov, D. A.
36. Shugay, M., D. V. Bagaev, M. A. Turchaninova, D. A. Bolotin, S. Lukyanov,
Bolotin, O. V. Britanova, E. A. Bogdanova, I. Z. Mamedov, et al. 2014. Age-related
E. V. Putintseva, M. V. Pogorelyy, V. I. Nazarov, I. V. decrease in TCR repertoire diversity measured with deep
Zvyagin, V. I. Kirgizova, et al. 2015. VDJtools: unifying
post-analysis of T cell receptor repertoires. PLoS Comput. and normalized sequence profiling. J. Immunol. 192: 2689
Biol. 11: e1004503. 2698.
37. Luzina, I. G., S. P. Atamas, C. E. Storrer, 50. Bono, M. R., D. Fernandez, F. Flores-Santibanez, M.
L. C. daSilva, G. Kelsoe, Rosemblatt, and D. Sauma.
J. C. Papadimitriou, and B. S. Handwerger. 2001. Spontaneous 2015. CD73 and CD39 ectonucleotidases in T cell
formation of germinal centers in autoimmune mice. J. Leukoc. differentiation: beyond im- munosuppression. FEBS Lett. 589:
Biol. 70: 578584. 34543460.
38. Petro, J. B., R. M. Gerstein, J. Lowe, R. S. Carter, N. 51. Mauri, C., and A. Bosma. 2012. Immune regulatory
Shinners, and W. N. Khan. function of B cells. Annu. Rev. Immunol. 30: 221241.
2002. Transitional type 1 and 2 B lymphocyte subsets are 52. Ray, A., S. Basu, C. B. Williams, N. H. Salzman, and B. N.
differentially re- sponsive to antigen receptor signaling. Dittel. 2012. A novel
J. Biol. Chem. 277: 4800948019. IL-10-independent regulatory role for B cells in suppressing
39. Su, T. T., and D. J. Rawlings. 2002. Transitional B autoimmunity by maintenance of regulatory T cells via GITR
lymphocyte subsets operate as distinct checkpoints in murine
splenic B cell development. J. Immunol. 168: 21012110. ligand. J. Immunol. 188: 31883198.
40. Kuraoka, M., T. M. Holl, D. Liao, M. Womble, D. W. Cain, 53. Berland, R., L. Fernandez, E. Kari, J. H. Han, I. Lomakin,
A. E. Reynolds, and S. Akira, H. H. Wortis,
G. Kelsoe. 2011. Activation-induced cytidine deaminase J. F. Kearney, A. A. Ucci, and T. Imanishi-Kari. 2006. Toll-
mediates central tol- erance in B cells. Proc. Natl. Acad. Sci. like receptor 7-dependent loss of B cell tolerance in
USA 108: 1156011565. pathogenic autoantibody knockin mice. Immunity 25: 429440.
41. Dahlberg, C. I., M. He, T. Visnes, M. L. Torres, E. M. 54. Meyers, G., Y. S. Ng, J. M. Bannock, A. Lavoie, J. E.
Cortizas, R. E. Verdun, Walter, L. D. Notarangelo,
L. S. Westerberg, E. Severinson, and L. Stro m. 2014. A novel S. S. Kilic, G. Aksu, M. Debre, F. Rieux-Laucat, et al. 2011.
mouse model for the hyper-IgM syndrome: a spontaneous Activation-induced cytidine deaminase (AID) is required for
activation-induced cytidine deaminase mutation leading to B-cell tolerance in humans. Proc. Natl. Acad. Sci. USA 108:
complete loss of Ig class switching and reduced somatic 1155411559.
hypermutation. J. Immunol. 193: 47324738. 55. Henry-Bonami, R. A., J. M. Williams, A. B.
42. Tomayko, M. M., N. C. Steinel, S. M. Anderson, and M. Rachakonda, M. Karamali,
J. Shlomchik. 2010. Cutting edge: hierarchy of maturity of P. L. Kendall, and J. W. Thomas. 2013. B lymphocyte
murine memory B cell subsets. J. Immunol. 185: 71467150. original sin in the bone marrow enhances islet
43. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. autoreactivity in type 1 diabetes-prone nonobese diabetic
mice. J. Immunol. 190: 59926003.
Plebani, O. Sanal, N. Catalan, 56. Wei, M., R. Shinkura, Y. Doi, M. Maruya, S. Fagarasan,
M. Forveille, R. Dufourcq-Labelouse, A. Gennery, et al. 2000. and T. Honjo. 2011. Mice carrying a knock-in mutation of
Activation- induced cytidine deaminase (AID) deficiency Aicda resulting in a defect in somatic hypermutation have
causes the autosomal recessive form of the Hyper-IgM impaired gut homeostasis and compromised mucosal de- fense.
syndrome (HIGM2). Cell 102: 565575. Nat. Immunol. 12: 264270.
44. Antonioli, L., P. Pacher, E. S. Vizi, and G. Hasko. 57. Kriegel, M. A., E. Sefik, J. A. Hill, H. J. Wu, C.
Benoist, and D. Mathis. 2011. Naturally transmitted segmented
2013. CD39 and CD73 in immunity and inflammation. Trends Mol. filamentous bacteria segregate with diabetes pro- tection in
Med. 19: 355367. nonobese diabetic mice. Proc. Natl. Acad. Sci. USA 108: 1154811553.
45. Cekic, C., and J. Linden. 2016. Purinergic regulation 58. Matsushita, T., K. Yanaba, J. D. Bouaziz, M. Fujimoto,
and T. F. Tedder. 2008. Regulatory B cells inhibit EAE
of the immune system. Nat. Rev. Immunol. 16: 177192. initiation in mice while other B cells promote disease
progression. J. Clin. Invest. 118: 34203430.