of December 22, 2015.This information is current as

Diabetic MiceCell Antigens in Diabetes-Prone Nonobese

βSpreading of T Cell Autoimmunity among B Cells Are Crucial for Determinant

KaufmanJide Tian, Dan Zekzer, Yuxin Lu, Hoa Dang and Daniel L.

http://www.jimmunol.org/content/176/4/2654doi: 10.4049/jimmunol.176.4.2654

2006; 176:2654-2661; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/176/4/2654.full#ref-list-1

, 23 of which you can access for free at: cites 45 articlesThis article

Subscriptionshttp://jimmunol.org/subscriptions

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on Decem

ber 22, 2015http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on D

ecember 22, 2015

http://ww

w.jim

munol.org/

Dow

nloaded from

B Cells Are Crucial for Determinant Spreading of T CellAutoimmunity among � Cell Antigens in Diabetes-ProneNonobese Diabetic Mice1

Jide Tian,2 Dan Zekzer, Yuxin Lu, Hoa Dang, and Daniel L. Kaufman

The determinant spreading of T cell autoimmunity plays an important role in the pathogenesis of type 1 diabetes and in theprotective mechanism of Ag-based immunotherapy in NOD mice. However, little is known about the role of APCs, particularlyB cells, in the spreading of T cell autoimmunity. We studied determinant spreading in NOD/scid or Ig��/� NOD mice reconsti-tuted with NOD T and/or B cells and found that mice with mature B cells (TB NOD/scid and BMB Ig��/� NOD), but not micethat lacked mature B cells (T NOD/scid and BM Ig��/� NOD), spontaneously developed Th1 autoimmunity, which spreadsequentially among different � cell Ags. Immunization of T NOD/scid and BM Ig��/� NOD mice with a � cell Ag could primeAg-specific Th1 or Th2 responses, but those T cell responses did not spread to other � cell Ags. In contrast, immunization of TBNOD/scid and BMB Ig��/� NOD mice with a � cell Ag in IFA induced Th2 responses, which spread to other � cell Ags.Furthermore, we found that while macrophages and dendritic cells could evoke memory and effector T cell responses in vitro, Bcells significantly enhanced the detection of spontaneously primed and induced Th1 responses to � cell Ags. Our data suggest thatB cells, but not other APCs, mediate the spreading of T cell responses during the type 1 diabetes process and following Ag-basedimmunotherapy. Conceivably, the modulation of the capacity of B cells to present Ag may provide new interventions for enhancingAg-based immunotherapy and controlling autoimmune diseases. The Journal of Immunology, 2006, 176: 2654–2661.

T ype 1 diabetes (T1D)3 is thought to be a T cell-mediatedautoimmune disease (reviewed in Refs. 1–4). Previousstudies have suggested that the determinant spreading of

T cell autoimmunity plays an important role in both the pathogen-esis of T1D and in the protective mechanisms of Ag-based immu-notherapy (5–9). Studies of the T1D process in NOD mice haveshown that spontaneous proinflammatory Th1 responses sequen-tially spread among � cell Ags, such as glutamic acid decarbox-ylase (GAD), the dominant determinant of heat shock protein(HSP) 277, and insulin, creating a hierarchy of determinant spread-ing (5, 6, 10). Ag-based immunotherapies, by treatment with a �cell Ag to prime Th2 responses, result in the rapid spread of Th2responses to other � cell Ags and the inhibition of disease pro-gression in NOD mice (7–9, 11, 12). Presumably, these spreadingprocesses are due to the cytokines produced by the first wave ofautoreactive T cells, which create a microenvironment in the pan-creatic islets and/or lymph nodes, leading to recruitment of secondwave autoreactive T cells toward similar phenotype by positivefeedback mechanisms (7, 13, 14). However, the role of APCs,

particularly B cells, in the spreading of T cell autoimmunity inNOD mice has not been well-defined.

Notably, B cells appear to be necessary components contribut-ing to the T1D process in NOD mice as few Ig��/� NOD micedisplay spontaneous autoimmunity and develop diabetes (15, 16).Furthermore, B cells have been suggested to be the crucial APCsfor the development of proinflammatory T cell responses to � cellAgs (17–19). Importantly, B cells have Ag-specific receptors, al-lowing them to concentrate Ag and present it much more effi-ciently to T cells than other APCs (20). Based on these properties,B cells may be the mediator of the determinant spreading of T cellautoimmunity to � cell Ags during the natural development of TIDand following Ag-based immunotherapy. However, a recent reportshowed that dendritic cells educated by activated T cells couldaffect naive T cell responses to unrelated Ags (21). Apparently,educated dendritic cells may contribute to the determinant spread-ing of T cell immunity. Given that B cell-deficient NOD mice havedendritic cells, can they be experimentally primed to mount Th1and Th2 responses to � cell Ags? Once primed experimentally,could dendritic cells as well as macrophages mediate the spreadingof Th1 and Th2 responses to other � cell Ags in B cell-deficientNOD mice? As characterization of T cell responses in vitro relieson APC activity to present � cell Ags, how does APC activity,particularly that of B cells, affect the detection of spontaneouslyprimed and experimentally induced T cell autoimmunity in vitro?

We studied the role of APCs in determinant spreading in twoindependent experimental models. In the first, we transfused NOD/scid mice with naive T and B cells isolated from 2- to 3-wk-oldNOD mice to generate TB NOD/scid mice, or with naive T cellsalone to generate B cell-deficient T NOD/scid mice. In the second,we reconstituted lethally irradiated Ig��/� NOD mice with syn-genic bone marrow cells and mature B cells isolated from 2- to3-wk-old NOD mice to establish BMB Ig��/� NOD mice, or withbone marrow cells alone to establish B cell-deficient BM Ig��/�

Department of Molecular and Medical Pharmacology, University of California, LosAngeles, CA 90095

Received for publication May 10, 2005. Accepted for publication December 1, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the National Institutes of Health, the Ju-venile Diabetes Research Foundation International, and the Diabetes Action Researchand Education Foundation.2 Address correspondence and reprint requests to Dr. Jide Tian, Department of Mo-lecular and Medical Pharmacology, David Geffen School of Medicine, University ofCalifornia, Box 173517, 10833 Le Conte Avenue, Los Angeles, CA 90095-1735.E-mail address: jtian@mednet.ucla.edu3 Abbreviations used in this paper: T1D, type 1 diabetes; GAD, glutamic acid decar-boxylase; HSP, heat shock protein; �-gal, �-galactosidase; PPD, purified protein de-rivative; SFC, spot-forming cell.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

by guest on Decem

ber 22, 2015http://w

ww

.jimm

unol.org/D

ownloaded from

NOD mice. We then characterized their spontaneous and Ag-primed T cell immunity to a panel of Ags. We found that B cellswere essential for the spreading of spontaneous Th1 autoimmunityas well as induced Th2 responses to � cell Ags. Furthermore, TNOD/scid and BM Ig��/� NOD mice were fully competent tomount T cell responses to administered � cell Ags, but spreadingto other � cell Ags was not detected, even in the presence of Bcells in vitro. This suggests that other APCs in B cell-deficientNOD could not mediate determinant spreading of Th1 and Th2responses to � cell Ags. Interestingly, we found that although mac-rophages and dendritic cells are capable of evoking � cell Ag-specific T cell responses, B cells can significantly enhance detec-tion of T cell responses to � cell Ags in vitro. Our data from thesetwo independent experimental models suggest that B cells, but notother APCs, mediate the determinant spreading of spontaneousTh1 responses during the T1D process in NOD mice and the in-duced Th2 spreading among � cell Ags following Ag-basedimmunotherapy.

Materials and MethodsMice

NOD (H-2NOD), NOD/scid (Taconic Farms), NOR (The Jackson Labora-tory), and Ig��/� NOD mice (provided by Dr. D. V. Serreze, The JacksonLaboratory, Bar Harbor, ME) were bred under specific pathogen-free con-ditions. In our colonies, �85% of the female NOD mice, but not NOD/scidand Ig��/� NOD mice, spontaneously develop T1D by 35–40 wk of age.Only female mice were used in our studies. All experimental procedureswere approved by the University of California Los Angeles (UCLA) Chan-cellor’s Animal Research Committee.

Chemicals and Ags

Mouse GAD (65 kDa) and control �-galactosidase (�-gal) were preparedas previously described (5). HSP277 (11) was synthesized at �95% purityby Multiple Peptide Systems. Insulin B-chain was purchased from Sigma-Aldrich. The purified protein derivative (PPD) was prepared from Myco-bacterium tuberculosis H37 Ra (Difco). The islet lysate was prepared byisolating the islets from NOD/scid mice as previously described (22) andhomogenizing them in PBS.

Isolation of T and B lymphocytes

We isolated naive T cells from young NOD mice by negative selection.Splenic mononuclear cell suspensions were prepared from 2- to 3-wk-oldNOD mice. After lysis of RBC with osmolar buffer, we purified naive Tcells by eliminating other cell populations using two successive treatmentswith complement (Pel-Freez Biologicals) and anti-Br220, anti-Mac 1, andanti-CD11c Abs (BD Pharmingen). After washing twice with HL-1 me-dium, the remaining T cell-enriched populations were incubated withMACS microbeads (with anti-mouse CD25, CD69, and CD19; MiltenyiBiotec) and loaded on a MACS Separation LD column. To purify B cells,we depleted T cells, macrophages, and dendritic cells in a similar fashionusing anti-CD3, anti-Mac 1, and anti-CD11c Abs for cytotoxicity andMACS microbeads (with anti-mouse CD-3, CD-11c, and MAC-1) fol-lowed by a magnetic separation column. The naive T or B cell populationsin the flow-through were collected and characterized by FACS analysisusing fluorescently labeled anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-Br220, anti-MAC-1, anti-MHC class II, and anti-CD11c. The purified na-ive T or B cells (�95% purity as determined by FACS analysis) were usedfor transfusion or as APCs (the purified B cells) in vitro.

Establishment of mouse models

NOD/scid mice at 3 wk of age were transfused with 50 million naive Tcells or 50 million naive T cells and 30 million B cells, generating TNOD/scid or TB NOD/scid mice, respectively. As a second model, weisolated bone marrow cells from 3- to 4-wk-old Ig��/� NOD mice andremoved mature B and T cells by Ab/complement-mediated cytotoxicity.We then transfused 5 million bone marrow cells together with 30 millionB cells (isolated from 2- to 3-wk-old NOD mice) into lethally irradiated(1000 rad) 4-wk-old Ig��/� NOD mice to establish BMB Ig��/� NODmice. We also reconstituted lethally irradiated Ig��/� NOD mice withsyngenic bone marrow cells, but not mature NOD B cells, to generate BMIg��/� NOD mice.

Immunizations

To prime Th1 responses, groups of T NOD/scid or BM Ig��/� NOD micewere immunized s.c. with 100 �g of GAD, HSP277, insulin B-chain, or�-gal in 50% CFA at the base of the tail 3 days posttransfusion or 6 wkpostreconstitution, respectively. To induce Th2 responses, groups of TB orT NOD/scid mice were i.p. immunized with 100 �g of GAD, HSP277,insulin B-chain, or �-gal in 50% IFA 3 days posttransfusion. Similarly,groups of BM Ig��/� or BMB Ig��/� NOD mice were also immunizedwith one of those Ags in IFA 6 wk postreconstitution. Ten days later, themice were boosted with the same Ag in IFA.

ELISPOT analysis

Spontaneously primed and experimentally induced T cell responses werecharacterized using the modified ELISPOT assays as previously described(7). Briefly, 106 splenic mononuclear cells were added per well (in dupli-cate) of an ELISPOT plate that had been coated with cytokine capture Absand incubated with Ag (100 �g/ml for whole proteins, 7 �M for peptides)for 24 h for IFN-�, or 40 h for IL-4 and IL-5 detection. After washing,biotinylated detection Abs were added and the plates were incubated at 4°Covernight. Bound secondary Abs were visualized using HRP-streptavidin(Dako Cytomation) and 3-amino-9-ethylcarbazole. Abs R4-6A2/XMG 1.2-biotin, 11B11/BVD6-24G2-biotin and TRFK5/TRFK4-biotin (BD Pharm-ingen) were used for capture and detection of IFN-�, IL-4, and IL-5,respectively.

To determine whether the Ag-presenting activities of B cells affect thedetection of effector and memory T cell responses in the ELISPOT assays,we mixed the purified T cells (4 � 105/well) from T or TB NOD/scid micewith 6 � 105/well T-depleted APCs (containing B cells) or T/B-depletedAPCs (without B cells) from 2- to 3-wk-old NOD mice.

Assessment of diabetes

Groups of T or TB NOD/scid mice, BM Ig��/�, or BMB Ig��/� NODmice were monitored twice weekly for the onset of diabetes by measuringtheir urine glucose levels by Clinistix-Strips (Bayer). After abnormal glu-cose levels in the urine were detected, the blood glucose levels were mea-sured every other day using OneTouch Ultra strips (Lifescan). Two con-secutive blood glucose levels �13 mM were considered as diabetes onset.T or TB NOD/scid mice were monitored up to 25 wk after transfusion.Ig��/� BM or Ig��/� BMB NOD mice were monitored up to 35 wkpostreconstitution.

Statistical analysis

Group comparisons were analyzed by using the Student t test. Repeatedmeasurements over time were assessed by Life Table analysis. Statisticalsignificance was set at p � 0.05.

ResultsB cells are crucial for the spreading of spontaneous Th1autoimmunity among � cell Ags

B cells are professional APCs and can effectively present Ag toAg-specific T cells. However, the role of B cells in the develop-ment of spontaneous T cell responses to � cell Ags remains indebate. Studies of Ig��/� NOD mice have shown that they fail todevelop spontaneous T cell autoimmunity (16–18). In contrast, arecent report showed that spontaneous T cell autoimmunity tosome � cell Ags could be detected in NOD.�MT�/� mice, sug-gesting that B cells are not absolutely required for the developmentof spontaneous T cell autoimmunity (23). Furthermore, whichAPCs mediate the spreading of spontaneous Th1 responses among� cell Ags is poorly understood.

To examine the role of B cells in the determinant spreading ofspontaneous T cell autoimmunity among � cell Ags, we estab-lished two independent experimental models. In the first model, wepurified naive T and B cells from 2- to 3-wk-old NOD mice andtransfused naive T cells with, or without, purified B cells to NOD/scid mice, generating TB NOD/scid mice or T NOD/scid mice,respectively. The second model was generated by reconstitutingIg��/� NOD mice with syngenic bone marrow cells alone (BMIg��/� NOD mice) or with bone marrow cells and mature B cellsisolated from 2- to 3-wk-old NOD mice (BMB Ig��/� NOD

2655The Journal of Immunology

by guest on Decem

ber 22, 2015http://w

ww

.jimm

unol.org/D

ownloaded from

mice). Following transfusion, we characterized splenic T cell au-toimmunity to a panel of � cell Ags using ELISPOT assays. Wefound that 4 wk posttransfusion, TB NOD/scid mice developedweak Th1-biased responses to GAD, but not to HSP277 or insulinB-chain (Fig. 1A). At 10 wk posttransfusion, we detected strongerTh1 responses to GAD, and found that spontaneous Th1 responseshad spread to HSP277 and insulin B-chain (Fig. 1B). Hence, fol-lowing transfusion of naive T and B cells to NOD/scid mice, thespreading hierarchy of spontaneous Th1 autoimmunity is similar tothat in unmanipulated NOD mice (7). In contrast, there was nodetectable T cell response to any of the tested � cell Ags in TNOD/scid mice even at 10 wk posttransfusion.

Similarly, we detected low frequency of GAD-reactive IFN-�-secreting T cells in BMB Ig��/� NOD mice, but not in BMIg��/� NOD mice, 6 wk after reconstitution (Fig. 1A). At 12 wkpostreconstitution, the frequency of GAD-reactive IFN-�-secreting

T cells increased in BMB Ig��/� NOD mice (Fig. 1B). We alsodetected HSP277- and insulin B-chain-reactive IFN-�-secreting Tcells, suggesting that the early spontaneous Th1 responses spreadto other � cell Ags in vivo. However, we did not detect any Th1responses to the tested � cell Ags in BM Ig��/� NOD mice aswell as unmanipulated Ig��/� NOD mice, even though all strainsof mice developed similar levels of Th1 responses after anti-CD3stimulation (data not shown). Collectively, these data indicate thatB cells are crucial for the determinant spreading of spontaneousTh1 autoimmunity among � cell Ags in NOD mice.

Immunization can prime a � cell Ag-specific Th1 response,which does not spread to other � cell Ags in B cell-deficientNOD mice

Next, we examined whether � cell Ag-specific Th1 responsescould be induced in B cell-deficient NOD mice. Immunizationwith Ag in CFA is known to induce Th1-polarized responses to theinjected Ag (24). T NOD/scid mice and BM Ig��/� NOD micewere immunized with a � cell or control Ag in CFA 3 days post-transfusion or 6 wk postreconstitution, respectively. Their splenicT cell responses were characterized using ELISPOT assays 6 wk

FIGURE 2. Induced Th1 responses to � cell Ags in B cell-deficientNOD mice. Groups of T NOD/scid (3 days posttransfusion) and BMIg��/� NOD mice (6 wk postreconstitution) were immunized with theindicated Ag in CFA, and 6 wk later, their splenic T cell responses to apanel of Ags were characterized using the ELISPOT assays in T NOD/scidmice (A) or BM Ig��/� NOD mice (B). Data are presented as the meannumber of IFN-� SFC per 106 splenic cells � SEM. Mice from each group(n � 4–5) were tested simultaneously in two independent experiments.There were no detectable IL-4- and IL-5-secreting T cells to all of thetested Ags (data not shown).

FIGURE 1. TB NOD/scid and BMB Ig��/�, but not B cell-deficient,NOD mice develop spontaneous Th1 autoimmunity. Splenic mononuclearcells were isolated from TB NOD/scid or T NOD/scid at 4 and 10 wkposttransfusion, or from BMB or BM Ig��/� NOD mice at 6 or 12 wkpostconstitution as well as unmanipulated Ig��/� NOD mice at 6 or 12wk of age. Their spontaneous T cell autoimmunity to a panel of � cell andcontrol Ags were characterized by the ELISPOT assays at 4–6 wk (A) or10–12 wk (B) postreconstitution to determine the frequency of � cell Ag-specific IFN-�-, IL-4-, and IL-5-secreting T cells. Data are presented as themean number of spot-forming colonies (SFC) per 106 splenic cells � SEM.Mice from B cell-deficient or B cell-potent groups were tested simulta-neously in two independent experiments (n � 4–6 for each group). All ofthe mice had undetectable levels of IL-4- and IL-5-secreting T cell re-sponses to all of the tested Ags and had a similar frequency of T cellsresponding to anti-CD3 (data not shown). There were no detectable T cellresponses to control Ag (�-gal, data not shown).

2656 THE ROLE OF B CELLS IN DETERMINANT SPREADING

by guest on Decem

ber 22, 2015http://w

ww

.jimm

unol.org/D

ownloaded from

after immunization (Fig. 2). As expected, we did not detect anyIL-4- or IL-5-secreting Ag-specific T cells in either strains of Bcell-deficient mice (data not shown), consistent with previous ob-servations (24). Second, the frequency of IFN-�-secreting PPD-reactive T cells was indistinguishable among the tested groups ofB cell-deficient mice, and induced Th1 responses to �-gal weresimilar to that in immunocompetent mice (7), suggesting that Bcells are not necessary for priming strong Th1 responses to a for-eign Ag. Third, immunization with a � cell Ag (HSP277 or insulinB-chain) primed robust Th1 responses to the Ag, indicating thatmacrophages and dendritic cells are sufficient to mount Th1 auto-immunity to these Ags. Interestingly, immunization with GAD inCFA only induced weak Th1 responses, implying that B cells areimportant for the induction of strong Th1 responses to GAD.

We then evaluated whether experimentally primed Th1 re-sponses to a � cell Ag could spread to other � cell Ags in Bcell-deficient NOD mice. We found that the Th1 responses inducedby immunization with a single � cell Ag in CFA failed to spreadto other � cell Ags (Fig. 2). For example, immunization withHSP277 primed Th1 responses only to HSP277, but not to GAD orinsulin B-chain, while injection with insulin B-chain induced Th1responses only to insulin B-chain in B cell-deficient mice. Thus,even after the induction of a vigorous � cell Ag-reactive Th1 re-sponse, proinflammatory T cell autoimmunity was not transmittedto other naive � cell Ag-reactive T cells and did not induce T1Din B cell-deficient NOD mice (data not shown). Apparently, cyto-kines produced by the primed first wave of T cells and T-dendritic/macrophage interactions are insufficient for mediating infectious �cell Ag-specific T cell autoimmunity in the absence of B cells.Interestingly, we found that B-potent NOR mice treated with sin-gle Ag in CFA developed unipolar Th1 responses to the injectedAg, but not to other Ags (data not shown), as observed in BALB/cmice (7). These data suggest that B cells within the stronger reg-ulatory environment in NOR mice are incapable of mediating de-terminant spreading of T cell autoimmunity (25).

Macrophages and dendritic cells can evoke, and B cells cansignificantly enhance, detection of Th1 autoimmunity in vitro

As APC activity is critical for efficiently detecting T cell responsesby T cell proliferation and ELISPOT assays, we tested how theAg-presenting activity of APCs, particularly B cells, affects thedetection of autoreactive T cells in vitro. We purified T cells fromT NOD/scid mice or TB NOD/scid mice at 10 wk posttransfusionof T and B cells and mixed them with T-depleted APCs (contain-ing B cells) or T/B-depleted APCs (without B cells) isolated from2- to 3-wk-old NOD mice. We then characterized their spontane-ous T cell autoimmunity to � cell Ags by ELISPOT assays (Fig.3A). In the absence of B cells in vitro, we failed to detect anyIFN-�-secreting Th1 cells responding to the tested � cell Ags in TNOD/scid mice. In the presence of B cells, we did detect lowfrequency of IFN-�-secreting T cells (with tiny, but clear spots)from some T NOD/scid mice in response to the islet lysate, but notto the tested � cell autoantigens. This suggests that there is spon-taneous priming of some islet-specific Th1 cells, but detection ofthese T cell responses by in vitro functional assays requires B cells.Furthermore, splenic T cells from TB NOD/scid mice responded toall of the tested � cell Ags in the presence of T/B-depleted APCs,although the frequency was significantly lower than that when cul-tured with T-depleted APCs (containing B cells). GAD-specific Tcell responses, in particular, were enhanced by addition of B cells.These data indicate that determinant spreading of spontaneous Th1autoimmunity had occurred in these mice. The appearance of de-tectable Th1 responses to the islet lysate in T NOD/scid mice andincreased Th1 responses to � cell Ags in TB NOD mice in the

culture with B cells suggest that APC function of the B cell isrequired for effectively recalling memory and effector Th1 re-sponses to � cell Ags in vitro.

Given the observed deficiencies in functional in vitro assays indetecting activated T cells in the absence of B cells, we reassessedwhether addition of B cells in vitro could allow the detection ofinduced Th1 spreading to other � cell Ags. We immunized TNOD/scid with a � cell Ag in CFA. Ten weeks later, their splenicT cells were purified and their responses to a panel of � cell Agswere characterized using ELISPOT assays in the presence of T-depleted or T/B-depleted APCs (Fig. 3B). We observed that T cellresponses to PPD were similar in all groups, indicating that mac-rophages and dendritic cells are capable of evoking Th1 responsesto this foreign Ag in vitro. In the presence of B cells, we detectedsignificantly increased Th1 responses to the injected � cell Ag( p � 0.035 for GAD, p � 0.025 for both HSP277 and insulin Bchain), but we failed to detect Th1 responses to other uninjected �

FIGURE 3. B cells significantly enhance detection of memory and ef-fector Th1 responses to � cell Ags in vitro. A, Effects of B cells on de-tecting spontaneous Th1 responses. Splenic T cells (4 � 105/well) werepurified from TB or T NOD/scid mice and mixed with T-depleted (�B) orT/B-depleted (�B) splenic APCs (6 � 105) in ELISPOT assays in thepresence of the indicated Ag. B, Effects of B cells on detecting primed Th1responses. T NOD/scid mice were immunized with the indicated � cell Agin CFA, and 10 wk later, their splenic T cells were purified and mixed withT-depleted or T/B-depleted APCs in ELISPOT assays in the presence ofthe indicated Ag. Data are presented as the mean number of IFN-� SFC per4 � 105 splenic T cells � SEM from three independent experiments. Therewere no detectable IL-4 and IL-5 responses, and all mice displayed similarlevels of Th1 responses to anti-CD3 (data not shown).

2657The Journal of Immunology

by guest on Decem

ber 22, 2015http://w

ww

.jimm

unol.org/D

ownloaded from

cell Ags, indicating the lack of Th1 spreading in B cell-deficientmice. Together, these observations suggest that B cells, but notother professional APCs, mediate determinant spreading of Th1responses in vivo.

B cells are important for the development of spontaneous T1D

To further study B cell involvement in T1D pathogenesis, we mon-itored diabetes onset in our B cell-potent or B cell-deficient NODmouse models. Irradiated Ig��/� NOD mice that had been recon-stituted with syngenic bone marrow cells, or combined with ma-ture B cells from young NOD mice, were monitored for diabetesdevelopment up to 35 wk postreconstitution. BM Ig��/� NODmice that did not receive B cells remained euglycemic, while BMBIg��/� NOD mice that received B cells developed diabetes with arate similar to that of unmanipulated NOD mice (Fig. 4). Follow-ing reconstitution, BMB Ig��/� NOD mice began to develop di-abetes �15 wk postreconstitution and �83% mice became dia-betic by 28 wk postreconstitution. Furthermore, none of T NOD/scid mice developed diabetes over a 25 wk posttransfusionobservation period. In contrast, TB NOD/scid mice developed di-abetes in an accelerated fashion. They began to develop diabetes�11 wk posttransfusion and �92% of the mice were hyperglyce-mic by 20 wk of age. The association of B cells with diabetesdevelopment in our NOD models complements and extends pre-vious studies (15, 16, 18), indicating that B cells are important forthe T1D pathogenesis.

B cells are crucial for the spreading of inducible Th2autoimmunity

Our previous studies, and those of others, have shown that follow-ing autoantigen-based immunotherapy, inducible Th2 responsescan spread to other unrelated target-tissue autoantigens, which isassociated with the inhibition of disease progression (7, 26, 27). Arecent report showed that dendritic cells educated by Th2 cyto-kines could affect the naive T cell responses to unrelated Ags (21),which may contribute to infectious Th2 autoimmunity. To evaluatewhich population of APCs contributes to the determinant spread-

ing of Th2 responses, we transfused NOD/scid mice with T, or Tand B cells, and 3 days later, immunized them with a � cell Ag inIFA. Ten days later, the mice were reinjected with the same Ag,and 10 wk posttransfusion, their splenic T cell responses to a panelof � cell and control Ags were characterized by ELISPOT assays(Fig. 5). Both B cell-deficient and B cell-potent mice developedsimilar levels of Th2 responses to �-gal, which failed to spread toany � cell Ags. Theses data suggest that macrophages and den-dritic cells are sufficient to mount Th2 responses to this foreign Ag.Notably, T NOD/scid mice that had been immunized with a � cellAg developed Th2 responses only to the injected Ag (Fig. 5A). Forexample, HSP277-immunized mice displayed Th2 responses toHSP277, but not to insulin B-chain and vice versa. Apparently,educated dendritic cells and macrophages are incapable of medi-ating the spreading of Th2 autoimmunity in B cell-deficient NODmice. In contrast, TB NOD/scid mice that had been immunizedwith a � cell Ag developed Th2 autoimmunity not only to theinjected Ag, but also to other unrelated � cell Ags (Fig. 5C). Forexample, the mice treated with GAD in IFA displayed vigorousTh2 responses to GAD as well as to HSP277 and insulin B-chain.Similarly, mice immunized with HSP277 displayed IL-5-secretingT cells responding to HSP277, GAD, and insulin B-chain. In par-allel experiments, we observed that while Th2 responses inducedby immunization with a � cell Ag were limited to the injected Agin BM Ig��/� mice, induced Th2 responses spread to other � cellAgs in BMB Ig��/� mice (Fig. 5, B and D). In addition, immu-nization of B-potent NOR mice with single Ag in IFA induced Th2responses only to the injected Ag, which did not spread to otherAgs (data not shown). Together, our data suggest that macro-phages and dendritic cells are sufficient for priming Th2 responsesto � cell Ags, and B cells in NOD mice are unique for the spread-ing of induced Th2 autoimmunity among � cell Ags.

DiscussionPrevious studies have found that during T1D development in NODmice, spontaneous splenic Th1 responses spread among � cell au-toantigens, and are associated with disease progression (5, 6, 10).An understanding of this infectious T cell autoimmunity may beuseful for the development of new approaches to control autoim-mune diseases. In the present study, we systemically evaluated therole of B cells in spontaneous Th1 spreading among � cell Ags intwo independent T1D mouse models. We found that TB NOD/scidand BMB Ig��/� NOD mice, which have mature B cells, devel-oped spontaneous Th1 responses to GAD followed by Th1 re-sponses to HSP277 and insulin B-chain. These mice had a highincidence of diabetes, similar to that of NOD mice. In contrast,none of the B cell-deficient NOD mice (T NOD/scid and BMIg��/� mice as well as Ig��/� NOD mice) displayed spontaneousTh1 responses to the tested � cell Ags nor became hyperglycemiceven if they had a low level of infiltration in their islets (data notshown). The lack of spontaneous Th1 autoimmunity in B cell-deficient mice was not caused by deficiencies in � cell Ag-specificT cells as these mice could mount vigorous Ag-specific Th1 re-sponses after immunization with a � cell Ag. However, the � cellAg-primed Th1 responses never spread to unrelated � cell Ags anddid not induce T1D in B cell-deficient NOD mice. These obser-vations, together with the association of B cells with spontaneousspreading of Th1 autoimmunity among � cell Ags, strongly sug-gest that B cells mediate infectious Th1 autoimmunity, contribut-ing to T1D development in NOD mice. Given that blocking de-terminant spreading inhibits disease progression in theexperimental autoimmune encephalomyelitis model (28), the mod-ulation of B cells in ways that reduce their capacity to promotedeterminant spreading may provide novel interventive therapies

FIGURE 4. TB NOD/scid and BMB Ig��/�, but not B cell-deficient,NOD mice spontaneously develop T1D. Female NOD/scid mice, whichhad been transfused with T and B cells or T cells alone, were monitored forthe development of T1D up to 25 wk posttransfusion. Irradiated Ig��/�

NOD mice that had been reconstituted with syngenic bone marrow cellsalone or bone marrow cells and mature B cells from young NOD mice.They were monitored for the T1D onset up to 35 wk postreconstitution.Two consecutive blood glucose levels of �13 mM were considered asdisease onset (n � 16 for each group).

2658 THE ROLE OF B CELLS IN DETERMINANT SPREADING

by guest on Decem

ber 22, 2015http://w

ww

.jimm

unol.org/D

ownloaded from

for T1D in humans. Notably, T cells from newly diabetic NODmice and T cell clones recognizing � cell Ags can adoptivelytransfer T1D to NOD/scid mice (29–31). Accordingly, once thecascade of diabetogenic T cell autoimmunity has been established,B cells are not required for effector T cells to destroy � cells in thepancreatic islets.

Autoantigen-based immunotherapies which induce regulatory Tcell responses can effectively inhibit organ-specific autoimmunediseases in animal models (22, 32–35). We observed that autoan-tigen-primed Th2 responses rapidly spread to unrelated target-tis-sue Ags (9). Apparently, the first wave of Ag-primed Th2 re-sponses promotes the development of Th2 responses to other � cellAgs, leading to infectious Th2 autoimmunity and inhibition of thepathogenic process (7, 24). This spreading of Th2 autoimmunityboth increases bystander suppression and helps exhaust Ag-spe-cific T cells that could give rise to pathogenic T cell responses (9).Recent studies have shown that activated T cells can educate den-dritic cells which in turn influence the functional development ofunrelated naive T cells toward a similar phenotype (21). Accord-ingly, educated dendritic cells may contribute to the process of Th2spreading. In our studies of B cell-deficient NOD mice, we ob-served that although vigorous Th2 responses to a � cell Ag couldbe induced, the induced Th2 responses never spread to other � cellAgs. Hence, dendritic cells educated by � cell Ag-primed Th2cells and macrophages were incapable of mediating the spreadingof Th2 responses to other � cell Ags. In B cell-potent TB NODmice and Ig��/� BMB NOD mice, � cell Ag-primed Th2 re-sponses spread to other uninjected � cell Ags. Thus, B cells arecrucial for transmitting regulatory T cell responses following Ag-based immunotherapy. Accordingly, the manipulation of the APCcapacity of B cells may enhance the efficacy of Ag-based immu-notherapies in inhibiting organ-specific autoimmune diseases.

We examined how the Ag-presenting activities of B cells affectthe detection of T cell responses in vitro. With T/B-depleted APCs,we detected spontaneous Th1 autoimmunity to multiple � cell Agsin B cell-potent NOD mice, as well as induced Th1 responses to �

cell Ags in B cell-deficient NOD mice. Apparently, once T cellresponses to � cell Ags have been induced in vivo, macrophagesand dendritic cells can elicit memory and activated T cells re-sponding to � cell Ags in vitro. Addition of B cells to theELISPOT cultures significantly increased the detected frequencyof � cell Ag-specific Th1 cells in B cell-potent NOD mice, andenhanced detection of T cell responses to an immunogen in Bcell-deficient NOD mice, consistent with a recent report (36).Interestingly, in the presence of B cells in vitro, T cells from a fewof B cell-deficient NOD mice displayed weak T cell responses tothe islet lysate, which may reflect the ability of macrophages anddendritic cells to activate naive T cells (37). Thus, in the absenceof mature B cells, dendritic cells as well as macrophages mayinitiate T cell autoimmunity to � cell Ags, while B cells appear tobe important for expansion and diversification of the autoimmunecascade, mediating determinant spreading. It will be of interest tofurther determine the role each type of APC plays in � cellAg-specific T cell autoimmunity and T1D development.

Homeostatic proliferation has been demonstrated to promoteself-reactive T cell expansion, contributing to the initiation of au-toimmunity (38–41). We found that there is no significant differ-ence in the total number of T cells, and their CD4 and CD8 pop-ulation between B-potent and B-deficient NOD mice 4 wk aftertransferring T or T/B cells, or 6 wk after bone marrow constitution(data not shown). Importantly, the frequency of spontaneouslyprimed Th1 responses in TB NOD/scid, BMB Ig��/� NOD miceand induced Th1 responses in T NOD/scid mice and BM Ig��/�

NOD mice is proportionally similar to those NOD mice. Thesedata suggest that naive T cells in NOD/scid mice underwent ho-meostatic proliferation, independent of B cell activity. Apparently,homeostatic proliferation of naive T cells did not preferentiallyincrease (or diminish) the frequency of any particular � cell Ag-reactive T cell pool. We also noticed that following transfusion andreconstitution, proportional levels of B cells existed long-term inthe recipients, suggesting that B cells also underwent homeostaticproliferation in the recipients. Indeed, IL-21R is expressed by B

FIGURE 5. Immunization with a� cell Ag promotes Th2 spreading inTB NOD/scid and BMB Ig��/�, butnot in B cell-deficient, NOD mice.Groups of T NOD/scid (A), TBNOD/scid (C), BM Ig��/� (B), orBMB Ig��/� NOD mice (D) weretreated with a � cell or control Ag inIFA. Their splenic T cell responses toa panel of Ags were characterized us-ing ELISPOT assays. Data shown arethe mean number of IL-5 SFC permillion splenic T cells � SEM fromfour to six mice in two independentexperiments. A similar pattern ofIL-4 responses was observed (datanot shown). All mice showed a sim-ilar level of T cell responses to anti-CD3 (data not shown).

2659The Journal of Immunology

by guest on Decem

ber 22, 2015http://w

ww

.jimm

unol.org/D

ownloaded from

cells and IL-21 can potently stimulate B cell proliferation (42).Thus, the expansion and homeostatic proliferation of T and B cellsmay contribute to B cell-mediated determinant spreading of T cellautoimmunity.

The spreading of T cell autoimmunity has been thought to bedue to cytokines, such as IFN-� or IL-4, released by the first waveof autoreactive T cells, creating a microenvironment in the targettissue which educates APCs, and promotes subsequent waves ofautoreactive T cells to development toward a similar phenotype (7,13, 14, 43). We found that although vigorous Th1 or Th2 responsesto � cell Ags could be induced in B cell-deficient NOD mice aswell as in B-potent NOR mice, the spontaneous Th1 and inducedTh2 responses could spread only in B cell-potent NOD mice. Ap-parently, B cells, but not other professional APCs or cytokinesproduced by the first wave of T cells, can effectively mediate thedeterminant spreading of T cell autoimmunity in NOD mice.

How do B cells mediate this spreading of T cell autoimmunity?Notably, B cells in NOD mice are deficient in tolerance to autoan-tigens, respond strongly to BCR stimulation by proliferation withrelative resistance to activation-induced cell death, and have anunique pattern of B7 expression, which contribute to proinflam-matory responses in the islets (44, 45). Furthermore, besides cap-turing and concentrating Ag for presentation, activated B cellsexpress high levels of MHC II, B7, CD40, and other second signal-associated molecules, making them much more efficient presentersof Ag than other APCs (20). It is likely that the first wave ofactivated T cells promote B cell activation, through both secretedcytokines and T:B interaction in Ag-specific and/or bystanderfashions, enhancing the APC function of the B cells. Subsequently,activated B cells, which should have diverse Ag receptors, effec-tively capture, and present different � cell Ags to T cells, mediat-ing the spreading of T cell autoimmunity during the T1D patho-genesis, and following Ag-based immunotherapy.

In summary, we have shown that B cell-potent TB NOD/scidand BMB Ig��/� NOD mice, but not B cell-deficient T NOD/scidand BM Ig��/� NOD mice, developed spontaneous Th1 autoim-munity, which spread among � cell Ags, and were associated witha high incidence of T1D. Treatment of B cell-potent NOD micewith a � cell Ag promoted Th2 responses, which spread to unre-lated � cell Ags. Although treatment with a � cell Ag could induceTh1 and Th2 responses to the injected Ag in B cell-deficient NODmice, these T cell responses failed to spread to other � cell Ags.Finally, B cells as professional APCs in vitro can significantlyenhance detection of Th1 responses that have been primed in vivo.Hence, our data suggest that B cells mediate the spreading of spon-taneous Th1 autoimmunity during the T1D development and thespreading of induced Th2 autoimmunity following Ag-based im-munotherapy in NOD mice. Our findings provide new insights intothe mechanism(s) underlying the process of determinant spreadingof T cell autoimmunity. Given that interference with determinantspreading has resulted in the inhibition of disease progression inthe experimental autoimmune encephalomyelitis model (28), themodulation of the capacity of B cells to present Ag may providenew interventions for enhancing Ag-based immunotherapy andcontrolling autoimmune diseases.

DisclosuresThe authors have no financial conflict of interest.

References1. Delovitch, T. L., and B. Singh. 1997. The nonobese diabetic mouse as a model

of autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7:727–738.

2. Atkinson, M. A., and G. S. Eisenbarth. 2001. Type 1 diabetes: new perspectiveson disease pathogenesis and treatment. Lancet 358: 221–229.

3. Tisch, R., and H. McDevitt. 1996. Insulin-dependent diabetes mellitus. Cell 85:291–297.

4. von Herrath, M. G. 2004. Pathogenesis of type 1 diabetes: a viewpoint. Adv. Exp.Med. Biol. 552: 317–321.

5. Kaufman, D. L., M. Clare-Salzler, J. Tian, T. Forsthuber, G. S. Ting,P. Robinson, M. A. Atkinson, E. E. Sercarz, A. J. Tobin, and P. V. Lehmann.1993. Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase inmurine insulin-dependent diabetes. Nature 366: 69–72.

6. Tisch, R., X. D. Yang, S. M. Singer, R. S. Liblau, L. Fugger, and H. O. McDevitt.1993. Immune response to glutamic acid decarboxylase correlates with insulitisin non-obese diabetic mice. Nature 366: 72–75.

7. Tian, J., P. V. Lehmann, and D. L. Kaufman. 1997. Determinant spreading of Thelper cell 2 (Th2) responses to pancreatic islet autoantigens. J. Exp. Med. 186:2039–2043.

8. Tisch, R., B. Wang, M. A. Atkinson, D. V. Serreze, and R. Friedline. 2001. Aglutamic acid decarboxylase 65-specific Th2 cell clone immunoregulates auto-immune diabetes in nonobese diabetic mice. J. Immunol. 166: 6925–6936.

9. Tian, J., A. P. Olcott, and D. L. Kaufman. 2002. Antigen-based immunotherapydrives the precocious development of autoimmunity. J. Immunol. 169:6564–6569.

10. Tian, J., S. Gregori, L. Adorini, and D. L. Kaufman. 2001. The frequency of highavidity T cells determines the hierarchy of determinant spreading. J. Immunol.166: 7144–7150.

11. Elias, D., and I. R. Cohen. 1994. Peptide therapy for diabetes in NOD mice.Lancet 343: 704–706.

12. Daniel, D., and D. R. Wegmann. 1996. Intranasal administration of insulin pep-tide B: 9–23 protects NOD mice from diabetes. Ann. NY Acad. Sci. 778:371–372.

13. Paul, W. E., and R. A. Seder. 1994. Lymphocyte responses and cytokines. Cell76: 241–251.

14. Lehmann, P. V., E. E. Sercarz, T. Forsthuber, C. M. Dayan, and G. Gammon.1993. Determinant spreading and the dynamics of the autoimmune T-cell reper-toire. Immunol. Today 14: 203–208.

15. Serreze, D. V., H. D. Chapman, D. S. Varnum, M. S. Hanson, P. C. Reifsnyder,S. D. Richard, S. A. Fleming, E. H. Leiter, and L. D. Shultz. 1996. B lymphocytesare essential for the initiation of T cell-mediated autoimmune diabetes: analysisof a new “speed congenic” stock of NOD.Ig � null mice. J. Exp. Med. 184:2049–2053.

16. Noorchashm, H., N. Noorchashm, J. Kern, S. Y. Rostami, C. F. Barker, andA. Naji. 1997. B-cells are required for the initiation of insulitis and sialitis innonobese diabetic mice. Diabetes 46: 941–946.

17. Serreze, D. V., S. A. Fleming, H. D. Chapman, S. D. Richard, E. H. Leiter, andR. M. Tisch. 1998. B lymphocytes are critical antigen-presenting cells for theinitiation of T cell-mediated autoimmune diabetes in nonobese diabetic mice.J. Immunol. 161: 3912–3918.

18. Falcone, M., J. Lee, G. Patstone, B. Yeung, and N. Sarvetnick. 1998. B lympho-cytes are crucial antigen-presenting cells in the pathogenic autoimmune responseto GAD65 antigen in nonobese diabetic mice. J. Immunol. 161: 1163–1168.

19. Noorchashm, H., Y. K. Lieu, N. Noorchashm, S. Y. Rostami, S. A. Greeley,A. Schlachterman, H. K. Song, L. E. Noto, A. M. Jevnikar, C. F. Barker, andA. Naji. 1999. I-Ag7-mediated antigen presentation by B lymphocytes is criticalin overcoming a checkpoint in T cell tolerance to islet � cells of nonobese dia-betic mice. J. Immunol. 163: 743–750.

20. Baird, A. M., and D. C. Parker. 1996. Analysis of low zone tolerance inductionin normal and B cell-deficient mice. J. Immunol. 157: 1833–1839.

21. Alpan, O., E. Bachelder, E. Isil, H. Arnheiter, and P. Matzinger. 2004. ‘Educated’dendritic cells act as messengers from memory to naive T helper cells. Nat.Immunol. 5: 615–622.

22. Tian, J., M. Clare-Salzler, A. Herschenfeld, B. Middleton, D. Newman,R. Mueller, S. Arita, C. Evans, M. A. Atkinson, Y. Mullen, et al. 1996. Modu-lating autoimmune responses to GAD inhibits disease progression and prolongsislet graft survival in diabetes-prone mice. Nat. Med. 2: 1348–1353.

23. Wong, F. S., L. Wen, M. Tang, M. Ramanathan, I. Visintin, J. Daugherty,L. G. Hannum, C. A. Janeway, Jr., and M. J. Shlomchik. 2004. Investigation ofthe role of B-cells in type 1 diabetes in the NOD mouse. Diabetes 53: 2581–2587.

24. Yip, H. C., A. Y. Karulin, M. Tary-Lehmann, M. D. Hesse, H. Radeke,P. S. Heeger, R. P. Trezza, F. P. Heinzel, T. Forsthuber, and P. V. Lehmann.1999. Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious di-chotomy defines the class of response. J. Immunol. 162: 3942–3949.

25. Prochazka, M., D. V. Serreze, W. N. Frankel, and E. H. Leiter. 1992. NOR/Ltmice: MHC-matched diabetes-resistant control strain for NOD mice. Diabetes41: 98–106.

26. Tisch, R., B. Wang, and D. V. Serreze. 1999. Induction of glutamic acid decar-boxylase 65-specific Th2 cells and suppression of autoimmune diabetes at latestages of disease is epitope dependent. J. Immunol. 163: 1178–1187.

27. Elias, D., A. Meilin, V. Ablamunits, O. S. Birk, P. Carmi, S. Konen-Waisman,and I. R. Cohen. 1997. Hsp60 peptide therapy of NOD mouse diabetes inducesa Th2 cytokine burst and downregulates autoimmunity to various �-cell antigens.Diabetes 46: 758–764.

28. Vanderlugt, C. L., W. S. Begolka, K. L. Neville, Y. Katz-Levy, L. M. Howard,T. N. Eagar, J. A. Bluestone, and S. D. Miller. 1998. The functional significanceof epitope spreading and its regulation by co-stimulatory molecules. Immunol.Rev. 164: 63–72.

29. Wegmann, D. R., N. Shehadeh, K. J. Lafferty, M. Norbury-Glaser, R. G. Gill, andD. Daniel. 1993. Establishment of islet-specific T-cell lines and clones from isletisografts placed in spontaneously diabetic NOD mice. J. Autoimmun. 6: 517–527.

2660 THE ROLE OF B CELLS IN DETERMINANT SPREADING

by guest on Decem

ber 22, 2015http://w

ww

.jimm

unol.org/D

ownloaded from

30. Wong, F. S., I. Visintin, L. Wen, R. A. Flavell, and C. A. Janeway, Jr. 1996. CD8T cell clones from young nonobese diabetic (NOD) islets can transfer rapid onsetof diabetes in NOD mice in the absence of CD4 cells. J. Exp. Med. 183: 67–76.

31. Zekzer, D., F. S. Wong, O. Ayalon, I. Millet, M. Altieri, S. Shintani,M. Solimena, and R. S. Sherwin. 1998. GAD-reactive CD4� Th1 cells inducediabetes in NOD/SCID mice. J. Clin. Invest. 101: 68–73.

32. Tisch, R., R. S. Liblau, X. D. Yang, P. Liblau, and H. O. McDevitt. 1998. In-duction of GAD65-specific regulatory T-cells inhibits ongoing autoimmune di-abetes in nonobese diabetic mice. Diabetes 47: 894–899.

33. Chen, Y., V. K. Kuchroo, J. Inobe, D. A. Hafler, and H. L. Weiner. 1994. Reg-ulatory T cell clones induced by oral tolerance: suppression of autoimmune en-cephalomyelitis. Science 265: 1237–1240.

34. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, andM. G. Roncarolo. 1997. A CD4� T-cell subset inhibits antigen-specific T-cellresponses and prevents colitis. Nature 389: 737–742.

35. Olcott, A. P., J. Tian, V. Walker, H. Dang, B. Middleton, L. Adorini,L. Washburn, and D. L. Kaufman. 2005. Antigen-based therapies using ignoreddeterminants of � cell antigens can more effectively inhibit late-stage autoim-mune disease in diabetes-prone mice. J. Immunol. 175: 1991–1999.

36. Dai, Y. D., K. P. Jensen, A. Lehuen, E. L. Masteller, J. A. Bluestone,D. B. Wilson, and E. E. Sercarz. 2005. A peptide of glutamic acid decarboxylase65 can recruit and expand a diabetogenic T cell clone, BDC2.5, in the pancreas.J. Immunol. 175: 3621–3627.

37. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu,B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu.Rev. Immunol. 18: 767–811.

38. King, C., A. Ilic, K. Koelsch, and N. Sarvetnick. 2004. Homeostatic expansion ofT cells during immune insufficiency generates autoimmunity. Cell 117: 265–277.

39. Baccala, R., and A. N. Theofilopoulos. 2005. The new paradigm of T-cell ho-meostatic proliferation-induced autoimmunity. Trends Immunol. 26: 5–8.

40. Gallegos, A. M., and M. J. Bevan. 2004. Driven to autoimmunity: the nod mouse.Cell 117: 149–151.

41. Marleau, A. M., and N. Sarvetnick. 2005. T cell homeostasis in tolerance andimmunity. J. Leukocyte Biol. 78: 575–584.

42. Parrish-Novak, J., D. C. Foster, R. D. Holly, and C. H. Clegg. 2002. Interleu-kin-21 and the IL-21 receptor: novel effectors of NK and T cell responses. J. Leu-kocyte Biol. 72: 856–863.

43. Tian, J., Y. Lu, L. Hanssen, H. Dang, and D. L. Kaufman. 2003. Memory andeffector T cells modulate subsequently primed immune responses to unrelatedantigens. Cell. Immunol. 224: 74–85.

44. Hulbert, C., B. Riseili, M. Rojas, and J. W. Thomas. 2001. B cell specificitycontributes to the outcome of diabetes in nonobese diabetic mice. J. Immunol.167: 5535–5538.

45. Silveira, P. A., E. Johnson, H. D. Chapman, T. Bui, R. M. Tisch, andD. V. Serreze. 2002. The preferential ability of B lymphocytes to act as diabe-togenic APC in NOD mice depends on expression of self-antigen-specific im-munoglobulin receptors. Eur. J. Immunol. 32: 3657–3666.

2661The Journal of Immunology

by guest on Decem

ber 22, 2015http://w

ww

.jimm

unol.org/D

ownloaded from