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The Journal of Immunology, 2007, 179: 1004-1012.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Constitutive Expression of B7-1 on B Cells Uncovers Autoimmunity toward the B Cell Compartment in the Nonobese Diabetic Mouse1

Hélène Bour-Jordan*, Benoit L. Salomon{dagger}, Heather L. Thompson*, Rex Santos*, Abul K. Abbas{ddagger} and Jeffrey A. Bluestone2,*

* University of California, San Francisco Diabetes Center, Department of Medicine, University of California, San Francisco, CA 94143; {dagger} Centre National de la Recherche Scientifique/Université Pierre et Marie Curie Unité Mixte de Recherche 7087, Hôpital Pitié-Salpêtrière, Paris, France; and {ddagger} Department of Pathology, University of California, School of Medicine, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The NOD mouse is an invaluable model for the study of autoimmune diabetes. Furthermore, although less appreciated, NOD mice are susceptible to other autoimmune diseases that can be differentially manifested by altering the balance of T cell costimulatory pathways. In this study, we show that constitutively expressing B7-1 on B cells (NOD-B7-1B-transgenic mice) resulted in reduced insulitis and completely protected NOD mice from developing diabetes. Furthermore, B7-1 expression led to a dramatic reduction of the B cell compartment due to a selective deletion of follicular B cells in the spleen, whereas marginal zone B cells were largely unaffected. B cell depletion was dependent on B cell specificity, mediated by CD8+ T cells, and occurred exclusively in the autoimmune-prone NOD background. Our results suggest that B cell deletion was a consequence of the specific activation of autoreactive T cells directed at peripheral self Ags presented by maturing B cells that expressed B7-1 costimulatory molecules. This study underscores the importance of B7 costimulatory molecules in controlling the amplitude and target of autoimmunity in genetically prone individuals and has important implications in the use of costimulatory pathway antagonists in the treatment of human autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The NOD mouse strain spontaneously develops autoimmune diabetes in 60–80% of females and 20–30% of males (1, 2). Often underappreciated is the fact that mice with this genetic background are susceptible to other autoimmune syndromes targeting a variety of distinct tissues. In addition to diabetes, NOD mice are prone to develop autoimmune peripheral neuropathy (3, 4), autoimmune sialadenitis (5), autoimmune thyroiditis (6), prostatitis in male mice (7), and some features of organ-nonspecific autoimmune diseases such as late-onset anti-nuclear Abs, a systemic lupus erythematosus-like disease if exposed to killed mycobacterium, and hemolytic anemia (8, 9). In this regard, the NOD mouse has also been considered as a model for other autoimmune diseases such as Sjogren’s syndrome, Guillain-Barre syndrome, and multiple sclerosis (3, 10, 11).

CD28 is one of the primary costimulatory pathways involved in the control of autoimmunity. On the pathogenic side, CD28 engagement with its ligands, B7-1 (CD80) and B7-2 (CD86), has been shown to be a critical costimulatory signal for T cell proliferation, differentiation, and trafficking (12). In contrast, the CD28 pathway controls negative regulatory pathways, including expression of CTLA-4 and PD-1 as well as the development and survival of regulatory T cells (Tregs)3 (13, 14). Thus, it is not surprising that CD28 costimulation appears to play important roles in controlling the development of different autoimmune diseases in the NOD background. For instance, elimination of CD28 or both B7-1 and B7-2 results in the exacerbation of a host of autoimmune diseases in large part due to loss of Tregs (15). By contrast, individual blockade of B7-2 costimulation protects NOD mice from diabetes, whereas altering B7-1 costimulation results in accelerated incidence of diabetes, suggesting that the individual ligands have distinct roles based on tissue distribution and differential interaction with CD28 vs CTLA-4 (3, 16, 17). In this regard, we have demonstrated previously that the CD28/B7 pathway not only controls the severity of autoimmune diabetes, but can alter the target specificity of the autoimmune response. As an example, whereas B7-2-deficient NOD mice are protected from diabetes and sialadenitis, they develop an autoimmune peripheral neuropathy (3). Together, these studies suggest that autoimmune-prone individuals have immune dysfunctions that can manifest as distinct disease entities at least in part dependent upon the costimulatory milieu.

Autoimmune lesions in NOD mice are characterized by complex mononuclear infiltrates composed of CD4+ T cells, CD8+ T cells, B cells, NK cells, dendritic cells, and macrophages (2, 18, 19). NOD diabetes is primarily dependent on CD4+ and CD8+ T cells, as evidenced by the ability to transfer disease with purified CD4+ and CD8+ T cells and individual T cell clones derived from NOD islets (20, 21, 22). However, the disease is characterized by the early appearance of islet-specific autoantibodies that target major antigenic epitopes, including glutamic acid decarboxylase and insulin (23). Furthermore, although diabetes cannot be transferred by autoantibodies, B cells are clearly important for the development of the disease. Indeed, NOD mice rendered genetically deficient in B cells or depleted of B cells after treatment with anti-IgM Abs developed reduced insulitis and were protected from diabetes (24, 25, 26, 27). In addition, the important role of B cells as APCs for islet Ag-specific autoreactive T cells has been clearly established (28, 29, 30, 31).

In this study, we set out to examine the relationship of CD28 costimulation and the critical role of B cells in the development of autoimmunity in the NOD mice by altering B7 costimulation specifically on B cells. Our results showed that NOD mice constitutively expressing high levels of B7-1 on B cells were protected from autoimmune diabetes and displayed reduced insulitis. However, autoimmunity was not completely abrogated, but instead redirected toward the B cell compartment as evidenced by a selective deletion of mature B cells in all lymphoid tissues. We propose that B cell deletion resulted from cognate T-B interactions most likely reflecting defective B cell tolerance in these animals. Together the results confirm the critical role of B cells in the development of spontaneous diabetes and highlight the crucial role of costimulation in the expression of autoimmunity in genetically prone individuals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

FVB mice transgenic (Tg) for B7-1 have been described previously (32, 33). B7-1-Tg mice were backcrossed to NOD mice for >10 generations and maintained as hemizygous by further breeding to NOD mice. Polymorphic markers at known diabetes susceptibility/resistance (Idd) loci were verified to be NOD in origin (data not shown). B7-1-Tg NOD mice (NOD-B7-1B-Tg mice) were genotyped by flow cytometry after staining of PBLs with B7-1 and B220 mAbs. NOD and NOD-H-2b congenic mice were purchased from Taconic Farms. NOD-CD28–/– mice have been described previously (15, 34). NOD mice deficient in TCR {alpha}-chain (NOD-TCR-{alpha}KO) were purchased from The Jackson Laboratory and bred at our facility. NOD-Ig hen egg lysozyme (HEL)-MD4 Tg mice (31) were provided by D. Serreze (The Jackson Laboratory, Bar Harbor, ME). All mice were housed in a pathogen-free facility at University of California. All experiments complied with the Animal Welfare Act and the National Institutes of Health guidelines for the ethical care and use of animals in biomedical research and were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.

Assessment of diabetes and insulitis

Blood glucose levels were measured every week with a Lifescan glucose meter (One Touch II; Lifescan). Mice were considered diabetic after two consecutive measurements >250 mg/dl. For histological analysis, pancreases were snap frozen in OCT. Multiple 5-µm sections were stained with H&E and scored blindly for severity of insulitis. The insulitis was considered peri-insulitis when lymphocytes were found surrounding, but not infiltrating the architecture of the islets; moderate insulitis if less than half of the islet architecture was infiltrated with lymphocytes; and severe insulitis if more than half of the islet architecture was infiltrated with lymphocytes.

Abs and flow cytometry

For in vivo depletion of CD8+ T cells, NOD and NOD-B7-1B-Tg mice were genotyped at 2.5–3 wk of age for expression of the B7-1 transgene, and treatment with anti-CD8 mAbs (YTS-169, produced in our laboratory) was performed, as previously described (35). Briefly, individual mice received i.p. injections of 500 µg of anti-CD8 mAbs immediately after genotyping and every other week for the duration of the experiment. This regimen led to depletion of >90% of CD8+ T cells. For flow cytometry, the following Abs were used for staining: B7-1 (16.10A1) and Fc block (2.4G2) mAbs were produced in our laboratory; purified anti-I-Ag7 (10.2.16) was generously provided by A. Sant (University of Rochester Medical Center, Rochester, NY) and produced in our laboratory; CD11b-FITC, CD21-FITC, IgMb-FITC, B220-PerCP, IgMa-biotin, B220-allophycocyanin, streptavidin (SAV)-allophycocyanin, SAV-PerCP, and SAV-allophycocyanin-Cy7 were purchased from BD Pharmingen; B220-FITC, rat anti-mouse IgG2b-FITC, IgM-PE, CD4-biotin, and CD8-biotin were purchased from Southern Biotechnology Associates; CD23-biotin and CD23-allophycocyanin were obtained from Caltag Laboratories; and 7-aminoactinomycin D (BD Pharmingen) and 4',6'-diamidino-2-phenylindole, dilactate (DAPI; Invitrogen Life Technologies) were used to assess cell viability, according to the manufacturer’s instructions. Single-cell suspensions were prepared from blood, spleen, peritoneal lavage, and bone marrow using standard procedures; stained for 20–30 min on ice in staining buffer (2% FCS and 0.01% sodium azide); and analyzed on a BD FACSCalibur flow cytometer with CellQuest software or a BD LSR II flow cytometer with FACSDiva software (BD Pharmingen).

Other reagents

Serum Ig concentrations were measured by ELISA using isotype-specific mAbs from Southern Biotechnology Associates, according to the manufacturer’s instructions. CFSE was purchased from Molecular Probes.

Adoptive transfer experiments

Single-cell suspensions were prepared from the spleen of NOD and NOD-B7-1B-Tg mice. Spleen cells were stained with biotinylated anti-CD4 and anti-CD8 mAbs, followed by anti-biotin beads (StemCell Technologies) and negative selection on autoMACS (Miltenyi Biotec), according to the manufacturer’s instruction. Enriched B cells were labeled with 1.5 µM CFSE, and 2.5–3.5 x 106 B cells were transferred into NOD recipients via retro-orbital injection. Six to 8 days after adoptive transfer, recipient spleen cells were harvested and B cell proliferation was assessed by dilution of the CFSE dye in the B220+ population.

In vitro killing assay

Single-cell suspensions were prepared from the spleen of NOD-B7-1B-Tg mice. CD4+ and CD8+ T cells were sorted on a Mo-Flo cytometer (DakoCytomation) or a FACSAria (BD Biosciences) to >99% purity. CD4+ and CD8+ T cells were stimulated with anti-CD3 and anti-CD28 coupled to 4.5-µm paramagnetic beads (provided by Xcyte Therapeutics) supplemented with 200 IU/ml human rIL-2 (Chiron) in complete medium, which consisted of DMEM supplemented with 10% heat-inactivated FBS (BioSource International), nonessential amino acids, 5 mM HEPES, 1 mM glutaMax I (all from Invitrogen Life Technologies), and 55 µM 2-ME (Sigma-Aldrich). After 7–10 days, the anti-CD3 and anti-CD28 beads as well as dead cells were removed using Ficoll gradient. Single-cell suspensions were prepared from the spleen of NOD-TCR{alpha}KO-B7-1B-Tg mice to examine B cells that were not affected by T cells in vivo. A total of 2 x 105 spleen cells was incubated with activated CD4+ or CD8+ T cells at a T:B ratio of 10:1. Cultures were harvested after 1–4 days and labeled with B220, CD21, CD23, annexin V, and DAPI to analyze B cell viability. Viable B cells were defined as annexin V and DAPI negative.

Statistical analyses

Statistical analyses were performed using Student’s t test. Values of p are indicated in the text or figure legend, and p values <0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Constitutive expression of B7-1 on B cells protects NOD mice from diabetes

FVB Tg mice expressing high levels of B7-1 constitutively on B cells (B-B7-1 line) (32, 33) were backcrossed to NOD mice for >10 generations. Consistent with the original Tg line on the FVB background, the B7-1 transgene was expressed at high levels on B cells, but not T cells, in the NOD-B7-1B-Tg mice (data not shown). As shown in Fig. 1, constitutive expression of B7-1 on B cells completely abrogated the development of diabetes in NOD female and male mice. In addition, histological examination of the pancreas revealed that mononuclear infiltration was greatly reduced in the pancreatic islets of NOD-B7-1B-Tg mice as compared with transgene-negative NOD littermates (Fig. 1B).


Figure 1
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  		FIGURE 1. Constitutive expression of B7-1 on B cells protects NOD mice from diabetes and insulitis. A, FVB mice Tg for B7-1 on B cells were backcrossed to the NOD background. We compared the cumulative incidence of diabetes in NOD (open symbols) and NOD-B7-1B-Tg (closed symbols) females (circles, n = 9 and 9, respectively) and males (squares, n = 6 and 8, respectively). B, We performed histological analysis of the pancreas and scored the severity of insulitis as no insulitis ({square}), peri-insulitis (light gray bar), moderate insulitis (dark gray bar), and severe insulitis ({blacksquare}) in 20- to 25-wk-old NOD and NOD-B7-1B-Tg mice (287 and 355 islets were scored, respectively).

 
B cell defect in NOD-B7-1B-Tg mice

The lack of autoimmune diabetes raised the possibility that expression of B7-1 on B cells was either altering B cell function or suppressing T cell function. Thus, we examined whether constitutive expression of B7-1 affected the development of the T and/or B cell compartment in NOD mice. There was no gross defect in T cell development because the number of CD4+ T cells, CD8+ T cells, and CD4+ CD25+ regulatory T cells, and the expression of activation markers in the CD4 and CD8 subsets were unaltered in the spleen and lymph nodes of NOD-B7-1B-Tg mice (data not shown). In sharp contrast, NOD-B7-1B-Tg mice displayed a dramatic reduction in the percentage of circulating B cells, with only 2–3% B cells in the blood of NOD-B7-1B-Tg adult mice compared with 20–30% in age-matched transgene-negative NOD littermates (Fig. 2A). Importantly, B7-1B-Tg mice on the FVB background had normal B cell numbers in the periphery (32, 33), suggesting that the B cell defect observed as a consequence of B7-1 constitutive expression was peculiar to the NOD background. The B cell defect occurred early in life in Tg mice with a 3- to 4-fold reduction in the percentage of B cells as compared with NOD mice by 2 wk of age. It was nearly complete by 5–6 wk of age with a 10-fold reduction compared with NOD mice and persisted throughout the life of the Tg animals. Furthermore, B cells were affected in the spleen of NOD-B7-1B-Tg mice, although not as completely as what was observed in the blood, with an average reduction of 2- to 2.5-fold as compared with NOD littermates (Fig. 2B). These results suggested that there may be a change in the developmental program of B cells in the bone marrow. Analysis of bone marrow in NOD-B7-1B-Tg mice revealed that the total percentage of B cells was greatly decreased. However, a total block of B cell development was not observed. The reduction in Tg B cells selectively affected the more mature B cell subsets (Fig. 2C). Indeed, whereas the percentage of pre/pro B cells (B220+ IgM–) in the bone marrow was reduced <2-fold in NOD-B7-1B-Tg mice compared with NOD (3.8 ± 4.2% in NOD-B7-1B-Tg vs 7.0 ± 3.6% in NOD; p > 0.05), the percentage of immature/mature B cells (B220+ IgM+) was reduced >10-fold in NOD-B7-1B-Tg (0.6 ± 0.6% in NOD-B7-1B-Tg vs 7.0 ± 1.4% in NOD; p < 1 x 10–8). In addition, the decreased percentage of immature/mature B cells in the bone marrow was associated with increased cell death in these IgM+ B cell subsets, but not the IgM– pre/pro B cells compared with NOD mice (data not shown). Finally, analysis of circulating Ig levels by ELISA demonstrated a striking reduction in the level of all Ig isotypes tested, including IgM, IgG2b, and IgG1 in the serum of NOD-B7-1B-Tg mice compared with NOD (Fig. 2D). Together, these results suggested that constitutive expression of B7-1 on B cells led to a dramatic defect in B cell number and function in NOD mice.


Figure 2
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  		FIGURE 2. B7-1 transgene expression results in B cell depletion. A, NOD ({circ}) and NOD-B7-1B-Tg (•) littermates were bled at indicated ages, and the percentage of B cells in the blood was analyzed by flow cytometry after staining with B220 mAbs. The percentage of B cells in the blood is indicated. B, Spleen cells of age-matched NOD and NOD-B7-1B-Tg were labeled with B220 mAbs, and the percentage of B cells in the spleen is indicated (t test, p < 1.10–9). C, Bone marrow cells from age-matched NOD and NOD-B7-1B-Tg were labeled with B220 and IgM mAbs. Representative dot plots are shown in the left panels. The percentages of pre/pro (B220+ IgM–) and immature/mature (B220+ IgM+) B cells are indicated in the right panels. Each circle represents an individual mouse, and horizontal bars represent the mean. D, Serum was collected from age-matched NOD and NOD-B7-1B-Tg mice, and serum Ig concentrations were measured by ELISA.

 
Constitutive expression of B7-1 induces selective depletion of follicular (FO) B cells in the periphery

Because the B cell defect in the bone marrow affected more severely the later stage of B cell development, we examined whether B cell subsets were differentially affected by expression of the B7-1 transgene in the periphery. As shown in Fig. 3A (top panels), expression of the B7-1 transgene resulted in a selective reduction in the CD23highCD21low FO B cell subset, whereas the CD23lowCD21high marginal zone (MZ) B cell subset remained unchanged. Similarly, the analysis of the expression of CD21 vs IgM in the B220+ cell population confirmed that FO mature B cells (CD21low IgMlow) were severely depleted in NOD-B7-1B-Tg mice, whereas MZ B cells (CD21high IgM+) were mostly unaffected (Fig. 3A, bottom panels). Furthermore, examination of B-1 B cells in the peritoneal cavity revealed that the percentage of FO B-2 B cells (B220high CD11b–) decreased by >5-fold in NOD-B7-1B-Tg mice compared with NOD mice, whereas the percentage of B-1 B cells (B220low CD11b+) did not significantly change (Fig. 3B). It is notable that these results could explain the greater extent of B cell deficiency observed in the blood compared with the spleen of NOD-B7-1B-Tg mice because circulating B cells are mainly composed of FO B cells. Thus, constitutive expression of B7-1 on B cells led to a selective defect in the FO B cell population in the periphery. In contrast, the MZ and B-1 B cell subsets, which are considered to provide innate B cell memory and have been proposed to be involved in autoimmune responses, were unaffected.


Figure 3
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  		FIGURE 3. FO B cells are selectively depleted in NOD-B7-1-B-Tg mice. A–C, Left panel, NOD; right panel, NOD-B7-1-B-Tg. A, Spleen cells of age-matched NOD and NOD-B7-1B-Tg mice were labeled with B220, CD21, CD23, and IgM. Dot plots were gated on B220+ cells, and representative dot plots are shown. The percentage of MZ (CD23low CD21high or CD21high IgM+) and FO (CD23high CD21low or CD21low IgMlow) B220+ B cell subsets in the total lymphocyte population is indicated. B, Peritoneal lavage cells were labeled with B220 and CD11b (Mac-1). Representative dot plots are shown. The percentage of B2 B cells (B220high CD11b–) and B1 B cells (B220low CD11blow) is indicated. C, Spleen cells were labeled with B220 and I-Ag7. Representative dot plots are shown. The mean fluorescence intensity of I-Ag7 in the B cell population is indicated. D, B cells were enriched from the spleen of NOD and NOD-B7-1B-Tg mice, labeled with CFSE, and transferred into NOD recipients. Six to 8 days after transfer, recipient spleen cells were harvested and the proliferation of transferred B cells was assessed by dilution of the CFSE dye in the B220+ population. Representative dot plots are shown.

 
We further examined the splenic B cell population in NOD and NOD-B7-1B-Tg mice and found that NOD-B7-1B-Tg B cells presented features of activated cells, such as expression of high levels of MHC class II (Fig. 3C). Indeed, the mean fluorescence intensity of MHC class II was >2-fold increased in NOD-B7-1B-Tg mice as compared with age-matched NOD mice. Importantly, this result was not only a consequence of the dysregulated ratio of FO to MZ B cells in Tg mice because MHC class II expression was significantly increased in residual FO B cells in Tg mice compared with non-Tg littermates (data not shown). To analyze whether this activated phenotype could reflect autoreactivity in the B cell compartment, B cells were enriched from the spleen of NOD or NOD-B7-1B-Tg mice, labeled with the fluorescent dye CFSE, and transferred into NOD recipients. The proliferation of transferred B cells in the spleen of recipient mice was examined after 6–8 days. As shown in Fig. 3D, B cells from NOD-B7-1B-Tg mice actively proliferated in NOD recipients. By comparison, CFSE-labeled B cells from NOD mice did not undergo any proliferation when transferred into NOD recipients. Importantly, whereas 15–25% of splenic B cells are derived from the MZ, we did not observe any B cell proliferation when we transferred NOD B cells into NOD recipients. This supports our conclusion that B cells from NOD-B7-1B-Tg mice, but not NOD mice, proliferate upon transfer into NOD recipients. These findings thus suggest that NOD-B7-1B-Tg B cells may have presented autoantigens to recipient T cells and proliferated as a result of this cognate interaction.

B cell depletion depends on cognate interaction with T cells

Because our data suggested that NOD-B7-1B-Tg B cells may be involved in productive interactions with T cells, we examined whether altering these interactions may affect the phenotype observed in these mice. We first analyzed whether B cell depletion depended on the interaction of B7-1 with CD28 by crossing NOD-B7-1B-Tg mice to mice deficient in CD28 (NOD-CD28KO) to generate NOD-B7-1B-Tg-CD28KO mice. As observed previously, NOD-B7-1B-Tg spleen cells were characterized by a sharp decrease in B cells, with 7.2% spleen cells expressing IgM vs 52.2% in NOD mice (Fig. 4A, left and middle panels). The remaining NOD-B7-1B-Tg B cells expressed high levels of surface IgM similarly to what was shown previously (Fig. 3A). In contrast, CD28 deficiency restored the percentage of NOD-B7-1B-Tg B cells and the expression level of IgM to levels similar to NOD mice (Fig. 4A, right panel), demonstrating that the interaction of B7-1 on NOD-B7-1B-Tg B cells with CD28 on T cells is necessary for the B cell depletion observed in these mice. Furthermore, the protection from diabetes observed in NOD-B7-1B-Tg mice was dependent on CD28/B7-1 interactions, because the introduction of the B7-1 transgene in B cells did not alter the development of disease in NOD-CD28KO mice previously shown to develop an exacerbated form of diabetes due to a Treg deficiency (15) (Fig. 4B).


Figure 4
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  		FIGURE 4. B cell depletion in NOD-B7-1B-Tg mice depends on CD28, H-2g7, and Ig specificity. A, NOD-B7-1B-Tg mice were crossed to NOD-CD28KO mice to generate NOD-B7-1B-Tg-CD28KO mice. Spleen cells from NOD, NOD-B7-1B-Tg, and NOD-B7-1B-Tg-CD28KO mice were labeled with IgM and B7-1. Representative dot plots are shown. The percentage of B cells is indicated. Note that the mean fluorescence intensity of IgM+ cells most likely reflects the distribution of MZ (IgMhigh) and FO (IgMlow) B cell subsets. B, We compared the cumulative incidence of diabetes in NOD-CD28KO (open symbols) and NOD-B7-1B-Tg-CD28KO (closed symbols) females (circles, n = 3 and 4, respectively) and males (squares, n = 4 and 6, respectively). C, NOD-B7-1B-Tg mice were crossed to NOD mice for the MHC region (NOD-H-2b/b) to generate NOD-H-2b-B7-1B-Tg mice. The percentage of B cells in the spleen is indicated in NOD-H-2g7/g7, NOD-B7-1B-Tg-H-2g7/g7, NOD-H-2b/b, and NOD-H-2b/b-B7-1B-Tg mice. D, NOD-B7-1B-Tg mice were crossed to NOD mice Tg for a HEL-specific IgM (MD4). The percentage of B cells in the blood is shown in single- and double-Tg mice, as indicated.

 
The cognate interaction of NOD-B7-1B-Tg B cells with autoreactive T cells would be expected to involve the presentation of antigenic peptides by MHC molecules on B cells to the TCR on T cells. We thus examined whether the NOD H-2g7 MHC region, which is strongly associated with the development of autoimmunity in the NOD strain, was important for the phenotype observed in NOD-B7-1B-Tg mice. NOD-B7-1B-Tg mice were bred to NOD mice congenic for the MHC region (NOD-H-2b/b). As shown in Fig. 4C, whereas B7-1B-Tg NOD-H-2g7 mice had reduced numbers of B cells, B7-1B-Tg NOD-H-2b mice had similar numbers of B cells as compared with non-Tg NOD-H-2b mice. Thus, B cell depletion in NOD-B7-1B-Tg mice depended on the expression of autoimmune-prone NOD H-2g7 MHC molecules.

Finally, previous results have demonstrated the central role of membrane-bound IgM in the capture of Ags by B cells for their presentation to T cells, especially when Ag is potentially dose limiting (36). To examine whether B cell specificity played a role in the B cell depletion observed in NOD-B7-1B-Tg mice, we crossed NOD-B7-1B-Tg mice to NOD mice Tg for HEL-specific IgM (MD4, hereafter referred to as IgHEL-Tg mice) (31). Because of efficient allelic exclusion in these mice, most B cells in NOD.IgHEL-Tg mice expressed HEL-specific Ig molecules, thus severely limiting the number of B cells capable of presenting autoantigens following Ig-mediated capture. As shown in Fig. 4D, whereas NOD-B7-1B-Tg mice were severely depleted of B cells in the peripheral blood, NOD-IgHEL-Tg mice expressing the B7-1 Tg had a similar percentage of B cells compared with B7-1 Tg-negative littermates. Taken together, these results support a direct role for B7-1 costimulatory activity on B cells and an active mechanism of B cell depletion via cognate interactions with autoreactive T cells.

CD8+ T cells induce deletion of NOD-B7-1B-Tg B cells

To analyze directly whether T cells were important for B cell depletion, we crossed NOD-B7-1B-Tg mice to NOD-TCR{alpha}KO mice and analyzed the effect of the B7-1B transgene on the B cell compartment in NOD-TCR{alpha}+/– (which have a normal T cell compartment) vs NOD-TCR{alpha}KO (which are devoid of T cells) mice. As expected, NOD-TCR{alpha}+/–.B7-1B-Tg mice displayed a 2-fold reduction in pre/pro B cells and a 10-fold reduction in immature/mature B cells compared with NOD-TCR{alpha}+/– mice (Fig. 5A, upper panel). In contrast, there was no depletion of either pre/pro B cells or immature/mature B cells in the bone marrow of NOD-TCR{alpha}KO-B7-1B-Tg mice as compared with NOD-TCR{alpha}KO mice. Similarly, whereas FO B cells were selectively depleted in NOD-TCR{alpha}+/–.B7-1B-Tg mice compared with NOD-TCR{alpha}+/– mice (Fig. 5A, bottom panel), the relative distribution of FO vs MZ B cells was unchanged in NOD-TCR{alpha}KO-B7-1B-Tg spleen compared with NOD-TCR{alpha}KO. These results confirm that B cell development was not intrinsically affected by expression of the B7-1 transgene because B cells developed normally in NOD-B7-1B-Tg-TCR{alpha}KO mice. Furthermore, these results showed that T cells were necessary for the B cell deletion observed in the bone marrow and spleen of NOD-B7-1B-Tg mice.


Figure 5
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  		FIGURE 5. B cell deletion is mediated by CD8+ T cells. A, NOD-B7-1B-Tg mice were crossed to NOD-TCR{alpha}KO mice to generate T cell-deficient NOD-B7-1B-Tg mice (NOD-TCR{alpha}KO-B7-1B-Tg). The following B cell subsets are shown for the different strains: pre/pro (Figure 5) and immature/mature ({square}) B cells in the bone marrow (top panel), and FO (Figure 5) and MZ ({square}) B cells in the spleen (bottom panel). B, CD4+ and CD8+ T cells were sorted from the spleen of NOD-B7-1B-Tg mice and activated with anti-CD3- and anti-CD28-coated beads. Activated CD4+ and CD8+ T cells were cultured with splenic B cells from NOD-TCR{alpha}KO-B7-1B-Tg mice, and B cell death was analyzed after 1–4 days in culture by flow cytometry. The percentage of live cells in the total B cell population (top panel) or MZ and FO B cell subsets (day 3) (bottom panel) is shown. C, NOD-B7-1B-Tg (circles) and NOD (triangles) littermates were genotyped at 2–3 wk of age and immediately treated with anti-CD8 mAbs (open symbols) or left untreated (closed symbols). Top and middle panels, The percentage of B cells in the blood is shown before (3 wk of age) and after (5 and 7 wk of age) anti-CD8 treatment. Bottom panel, Untreated and anti-CD8-treated mice were euthanized at 8 wk of age and examined for FO (Figure 5) and MZ ({square}) B cell subsets in the spleen. Results are shown as percentage of each subset in B220+ B cells. Results from two independent experiments with the following number of mice per group were pooled: NOD, n = 7; NOD + anti-CD8, n = 6; NOD-B7-1B-Tg, n = 4; NOD-B7-1B-Tg + anti-CD8, n = 5.

 
We examined which T cell subset was responsible for B cell deletion in vitro and in vivo. Unaffected B cells from NOD-TCR{alpha}KO-B7-1B-Tg mice were incubated with activated CD4+ or CD8+ T cells from NOD-B7-1B-Tg mice and analyzed for viability after 1–4 days in culture. NOD-B7-1B-Tg CD8+ T cells, but not CD4+ T cells, induced B cell death in vitro (Fig. 5B, top panel) by selectively eliminating the FO B cell subset (Fig. 5B, bottom panel). Specifically, whereas the percentage of live B cells was similar in the FO and MZ subsets in the absence of T cells, the percentage of live B cells was reduced to 15% of FO B cells compared with 60% of MZ B cells when B cells were incubated with CD8+ T cells. This result suggested that the selective deletion of the FO B cell subset in vivo may result not only from the location of these B cell subsets relatively to T cells, but also from a higher sensitivity of FO B cells to CD8+ T cell killing.

To analyze whether CD8+ T cells could be directly responsible for B cell deletion in vivo, NOD-B7-1B-Tg mice were treated with depleting anti-CD8 mAbs starting at 3 wk of age. CD8+ T cell depletion resulted in a dramatic and stable increase in the percentage of B cells in the blood of NOD-B-7-1B-Tg mice as early as 2 wk after anti-CD8 mAb treatment (Fig. 5C, top panel). In contrast, it had little effect on the percentage of circulating B cells in NOD mice (Fig. 5C, middle panel), demonstrating that the restoration of B cell levels in NOD-B7-1B-Tg mice reflected a direct effect of CD8+ T cells on the B cell population rather than a nonspecific compensatory increase in the percentage of B cells. Examination of splenic B cell subsets demonstrated that CD8+ T cell depletion led to a restoration of the FO B cell subset (Fig. 5C, bottom panel) and the total percentage of B cells (data not shown) in NOD-B7-1B-Tg mice. Furthermore, analysis of circulating Ig levels by ELISA demonstrated a significant increase in the level of total Ig as well as IgM, IgG2b, and IgG1 in the serum of anti-CD8 mAb-treated NOD-B7-1B-Tg mice compared with untreated mice (data not shown). Taken together, our results thus demonstrate that CD8+ T cells are directly responsible for B cell deletion in NOD-B-7-1B-Tg mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The NOD mouse has long been recognized has a valuable tool to study autoimmune diabetes. In this study, we showed that altering expression of B7-1 costimulatory molecules specifically on B cells prevented the development of diabetes while leading to a T cell-dependent deletion of FO B cells. NOD mice constitutively expressing B7-1 on B cells (NOD-B7-1B-Tg) were completely protected from diabetes and displayed reduced insulitis. Furthermore, the B cell compartment displayed a selective deletion of the more mature B cell subsets in the bone marrow, peritoneal cavity, spleen, and blood, leading to a striking inversion of the MZ to FO B cell ratio in the spleen. The requirement for T cells and NOD MHC H-2g7 molecules for B cell deletion in NOD-B7-1B-Tg mice suggested that B cell deletion occurred as a consequence of T cell-mediated elimination of the FO B cell compartment due to the presentation of autoantigens by mature Tg B cells to T cells in the context of high level of the costimulatory molecule B7-1. This conclusion was further supported by the demonstration that B cell specificity was critical for B cell deletion in NOD-B7-1B-Tg mice because B7-1-Tg B cells expressing a HEL-specific Ig transgene were not deleted, suggesting that autoantigen uptake by surface Ig was a important step in the presentation of autoantigens and cognate B-T interactions in NOD-B7-1B-Tg mice. Furthermore, CD8+ T cells could kill NOD-B7-1B-Tg B cells in vitro and were crucial for B cell depletion in vivo. Thus, we propose a model in which autoreactive T cells normally reactive to self Ags expressed on B cells are hyperactivated due to the presence of B7-1 on those B cells. The subsequent activation of the B cell-directed autoimmune response results in killing of FO B cells, leading to an attenuation of B cell-dependent autoimmune diabetes. These results have important implications for the regulation of autoimmunity, not just in NOD mice, but potentially in individuals prone to multiple autoimmune syndromes. Specifically, altering T cell costimulation in these individuals through therapies such as CTLA4Ig may alter the tissue specificity of the autoimmunity and divert the tissue destructive process.

The critical role of B cells in the development of autoimmune diabetes was established by the absence of disease in NOD mice rendered genetically deficient in B cells or depleted of B cells after treatment with anti-IgM Abs (24, 25, 26, 27). However, the precise function of B cells in the pathogenesis of the disease is still not fully elucidated. Although autoantibodies against pancreatic islet Ags can be found very early in NOD mice (23), their direct participation in the disease process has been controversial. Maternal Abs have been found to be important for the development of diabetes (37), but transfer of NOD serum into B cell-deficient NOD mice did not restore disease (38), suggesting that autoantibodies may not by themselves induce tissue damage to the pancreatic islets. Conversely, the important role of B cells as APCs for islet Ag-specific autoreactive T cells has been clearly established, and the expression of self-reactive Abs plays a critical role in this process by allowing efficient capture of autoantigens by surface Ig, followed by presentation of autoantigen peptides by MHC molecules to T cells (28, 29, 30, 31). Our model confirms the importance of B cells in autoimmune diabetes because B cell depletion in NOD-B7-1B-Tg mice was associated with protection from disease. Importantly, the loss of B cells early in NOD-B7-1B-Tg mice reduced, but did not eliminate insulitis similar to the low level of mononuclear infiltration observed in the pancreas of B cell-deficient NOD mice (25, 27). This suggests that B cells may not be essential to initiate the autoimmune process, but may be critical at later stages as autoantibody-mediated Ag uptake and presentation by memory B cells enhance autoreactivity and promote epitope spreading. These results have important implications for clinical research as the newly developed anti-CD20 mAb, Rituxan, is being tested clinically in patients newly diagnosed with type 1 diabetes.

Interestingly, we observed that FO B cells were selectively depleted in NOD-B7-1B-Tg mice, whereas the number of B cells in the MZ compartment was not affected. This result suggests that FO B cells may be the major B cell subset responsible for the APC function of B cells in NOD mice. However, it should be pointed out that B7-1 Tg B cells are functionally altered, as shown previously by Sethna et al. (32) and as evidenced in our studies by the low levels of IgM in the serum of NOD-B7-1B-Tg mice despite normal numbers of MZ and B-1 B cells. This was further confirmed by significantly decreased levels of serum IgM in NOD-IgHEL-Tg-B7-1B-Tg mice compared with NOD-IgHEL-Tg mice, although the percentage of B cells was similar in these two strains (data not shown). Finally, Noorchashm et al. (39) identified a defect in NOD T cell proliferation that led to resistance to activation-induced cell death and could be associated with the development of diabetes by contributing to the persistence of activated autoreactive T cells. They proposed that this defect was related to the poor costimulation capacity of B cells that made them suboptimal APCs and the otherwise general defect in Ag presentation by non-B cell APCs in NOD mice. Thus, an alternative model might be that the constitutive expression of B7-1 on B cells enhanced their capacity to present self Ags, and that this paradoxically contributed to diabetes prevention by leading to increased cell death of autoreactive T cells rather than an attenuation of autoreactive T cell activation.

The underlying mechanisms that lead to the selective deletion of FO B cells, but leave MZ B cells unaffected, have yet to be determined. Because our data suggested that B cell deletion was a direct consequence of B-T cognate interactions, the simplest explanation is that the anatomical location of the different subsets in the spleen plays a major role in the selective deletion. The specialized function of the different B cell subsets may be involved as well. Indeed, although MZ B cells can activate T cells, both MZ and B-1 B cells have been described as natural memory B cells that can provide a first line of defense against pathogens by providing responses to T-independent Ags (40, 41). The enrichment of B cells specific for T-independent Ags, particularly blood-borne particular Ags, in the MZ and B-1 B cell subsets (42) is most likely associated with a distinct Ig repertoire that may not favor interaction with autoreactive T cells. However, our data showing differential death induced by CD8+ T cells in MZ and FO B cells in vitro suggest that MZ and FO B cells may exhibit distinct molecular mechanisms and signaling pathways associated with cell survival and cell death, resulting in differential susceptibility to killing by autoreactive T cells. In this regard, it has been suggested that MZ and B-1 B cells may be distinct from FO B cells in their lifespan and self-renewal capabilities. Indeed, in studies examining the homeostasis of peripheral B cells, conditions of B cell deficiency and/or limited B cell influx from the bone marrow resulted in the preferential reconstitution of the peripheral B cell compartment with MZ and B-1 B cells (43, 44, 45). Thus, this phenomenon could be related to the preferential retention of these two B cell subsets in NOD-B7-1B-Tg mice. Finally, it is notable that the NOD mouse presents a relative increase in the MZ B cell population compared with nonautoimmune mouse strains (46). This relative enrichment in MZ B cells was genetically linked to chromosome 4 in NOD mice (46) and could participate to the skewed MZ/FO B cell ratio in NOD-B7-1B-Tg mice.

The results presented in this study on the fate of B cells in NOD-B7-1B-Tg mice differ significantly from other models of Tg expression of B7 molecules in B cells. Indeed, B7-1B-Tg mice were originally derived on the FVB background and backcrossed to the NOD background for our studies. FVB-B7-1B-Tg mice had a normal B cell population in the spleen (33), in agreement with our data showing an absence of B cell deletion in mice congenic for the MHC region or when NOD-B7-1B-Tg mice were crossed to nonautoimmune strains (data not shown). These differences suggest that B cell deletion in NOD-B7-1B-Tg mice depended directly on the NOD mouse genetic propensity to autoimmunity. In contrast, Fournier et al. (47) reported that expression of B7-2 Tg on B cells in C57BL/6 mice (hereafter referred to as B6-B7-2B-Tg mice) resulted in B cell depletion in the bone marrow and the periphery that depended on cognate interactions with T cells, similar to our observations in NOD-B7-1B-Tg mice. However, B cell deletion in B7-2-Tg mice depended on B cell interaction with Ag-nonspecific T cells, whereas our data suggest that deletion in NOD-B7-1B-Tg mice resulted from interactions between self-reactive T and B cells, implying fundamental differences in the underlying mechanisms of B cell deletion in the two models. Remaining B cells in B7-2-Tg mice expressed levels of surface Ig similar to non-Tg animals, suggesting that FO B cells were not selectively targeted. Furthermore, Fournier et al. (47) showed that serum Ig levels were not reduced in B6-B7-2B-Tg mice compared with their transgene-negative counterparts, whereas the concentration of all Ig isotypes was severely reduced in NOD-B7-1B-Tg mice compared with NOD mice. Finally, and most importantly, whereas CD8+ T cells appear to be the main mediators of B cell deletion in NOD-B7-1B-Tg mice, they do not seem to play a role in B6-B7-2B-Tg mice, strengthening the argument that the two seemingly similar phenotypes are due to different mechanisms.

In normal individuals, most autoreactive B cells are removed from the B cell repertoire at different stages of maturation in the bone marrow and in the periphery (reviewed in Refs. 48 and 49), thus precluding the potentially deleterious production of autoantibodies. In contrast, autoantibodies specific for islet self Ags as well as other tissues targeted by autoimmunity are found in the serum of NOD mice (23). Aged NOD mice develop anti-nuclear autoantibodies and can develop a lupus-like syndrome after treatment with CFA (8). In this regard, it has been shown in Ig Tg and other models that NOD mice were defective in several tolerogenic mechanisms in B cell development, including clonal deletion in the bone marrow and developmental arrest and anergy in the periphery (50, 51, 52, 53, 54), leading to the development and persistence in the periphery of self-reactive B cells that would be deleted in nonautoimmune background. Thus, the multiple defects in B cell tolerance in NOD mice could account for the presence of self-reactive B cells in NOD-B7-1B-Tg mice. Although NOD mice present multiple defects in B cell tolerance, it is unlikely that all the B cells that were deleted in NOD-B7-1B-Tg mice were autoreactive. We thus hypothesize that CD8+ T cells directly target and eliminate autoreactive B cells in NOD-B7-1B-Tg mice, whereas other nonautoreactive B cells are deleted as a result of bystander killing that could involve cytotoxic and/or cytokine pathways. Taken together, our data thus suggest that self-reactive NOD-B7-1B-Tg B cells presented self Ags to autoreactive T cells, leading to autoreactive T cell activation and B cell deletion. Importantly, B cells have recently been shown to be crucial for epitope spreading in NOD mice (55), and B7-1 costimulation is essential for epitope spreading in experimental autoimmune encephalomyelitis, the mouse model for multiple sclerosis (56, 57). Thus, we speculate that the physiological up-regulation of B7-1 on B cells upon activation (12) may result in the deletion of autoreactive B cells under physiological conditions and participate in epitope spreading in autoimmune diseases. This study reveals yet another facet of the underlying autoimmunity affecting the NOD mouse, and underlines the importance of B7 costimulatory molecules in controlling the amplitude and target of autoimmunity.


   Acknowledgments
 
We thank P. Wegfahrt for animal care; S. Jiang, M. R. Lee, and W. Liu for cell sorting; J. G. Cyster and M. S. Anderson for critical reading of the manuscript; and members of the Bluestone laboratory for discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by research grants from the National Institutes of Health (AI-50834, P30 DK63720, U19 AI056388) and an American Diabetes Association mentor-based award. Back

2 Address correspondence and reprint requests to Dr. Jeffrey A. Bluestone, University of California Diabetes Center, University of California, Box 0540, 513 Parnassus Avenue, San Francisco, CA 94143-0540. E-mail address: jbluest{at}diabetes.ucsf.edu Back

3 Abbreviations used in this paper: Treg, regulatory T cell; DAPI, 4',6'-diamidino-2-phenylindole, dilactate; FO, follicular; HEL, hen egg lysozyme; MZ, marginal zone; SAV, streptavidin; Tg, transgenic. Back

Received for publication April 18, 2007. Accepted for publication May 4, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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