B lymphocytes are required for the pathogenesis of autoimmune diabetes in NOD mice. Previous studies established that a lymphopenic transitional (TR) B cell compartment reduces the competitive constraint on the entry of newly emerging TR B cells into the splenic follicle (FO), thereby disrupting a peripheral negative selection checkpoint in NOD mice. Thus, development of clinically feasible immunotherapeutic approaches for restoration of appropriate negative selection is essential for the prevention of anti-islet autoimmunity. In this study we hypothesized that in vivo neutralization of the B lymphocyte stimulator (BLyS/BAFF) may enhance the stringency of TR→FO selection by increasing TR B cell competition for follicular entry in NOD mice. This study demonstrated that in vivo BLyS neutralization therapy leads to the depletion of follicular and marginal zone B lymphocytes. Long-term in vivo BLyS neutralization caused an increased TR:FO B cell ratio in the periphery indicating a relative resistance to follicular entry. Moreover, in vivo BLyS neutralization: 1) restored negative selection at the TR→FO checkpoint, 2) abrogated serum insulin autoantibodies, 3) reduced the severity of islet inflammation, 4) significantly reduced the incidence of spontaneous diabetes, 5) arrested the terminal stages of islet cell destruction, and 6) disrupted CD4 T cell activation in NOD mice. Overall, this study demonstrates the efficacy of B lymphocyte-directed therapy via in vivo BLyS neutralization for the prevention of autoimmune diabetes.
Autoimmune diabetes in the NOD mouse and in patients with type 1 diabetes (T1D)3 is associated with the loss of B lymphocyte tolerance to a range of islet-specific autoantigens (1). In the clinical setting, the presence of islet-specific autoantibodies serves as an important prognostic marker of autoimmune diabetes susceptibility (2). In addition to their value in risk stratifying susceptible patients, islet-specific autoantibodies, including insulin autoantibodies (IAA), are important participants in the pathogenesis of autoimmune diabetes (3). Indeed, B lymphocytes are required for the progression of islet inflammation (4, 5) and act as APCs responsible for the activation of islet-specific T cells (6, 7, 8, 9, 10, 11). By virtue of their ability to produce islet-specific autoantibodies, B cells are capable of efficient autoantigen uptake and presentation (11). Several studies have demonstrated defects in the peripheral negative selection of autoreactive B lymphocyte clonotypes (11, 12, 13). This defect is associated with a lymphopenic transitional (TR) B cell compartment that reduces the competitive constraint on follicular entry (12, 14). It is well established that the entry of newly emerging TR B cells into the follicle (FO) is a competitive process dominantly regulated by the TNF-related cytokine called the B lymphocyte stimulator (BlyS) (15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Therefore, it has been suggested that the level of interclonal competition for BLyS at the TR stage dictates the probability of autoreactive B cells entering into the preimmune B cell repertoire (14).
In this study, we hypothesized that in vivo BLyS neutralization restores the competitive constraint necessary for appropriate negative selection to occur at the TR→FO checkpoint in NOD mice. The results demonstrate that in vivo BLyS neutralization therapy promotes B lymphocyte depletion, restores appropriate negative selection at the TR→FO checkpoint, abrogates serum IAA titers, impairs the activation potential of CD4 T cells, and prevents the progression of autoimmune diabetes in NOD mice. As such, BLyS may be a logical and novel target of immunotherapy for the prevention of islet β-cell destruction in T1D patients.
Materials and Methods
C57BL/6J and NOD/ShiLtJ mice were obtained from The Jackson Laboratory. All animals were housed in specific pathogen-free conditions at the University of Pennsylvania Medical Center (Philadelphia, PA). Animal procedures were in accordance with the Animal Welfare Act. NOD/ShiLtJ mice were observed for diabetes development biweekly by screening for glycosuria. Diabetes was confirmed by blood glucose measurements with Accu-Check Advantage test strips (Boehringer Mannheim) >250 mg/dl (13.9 mmol/L) for two consecutive readings.
mAbs and flow cytometric analysis
The Abs used in this study were as follows: PerCP-conjugated anti-CD45R (B220, RA3-6B2), allophycocyanin-conjugated anti-IgM (II/41), FITC/PE-conjugated anti-CD21/35 (7G6), PE conjugated anti-AA4.1, biotinylated λ-1, and biotinylated λ-2 plus λ-3 (all purchased from BD Biosciences). Biotinylated mAbs were detected by streptavidin-allophycocyanin (BD Bioscences). Intracellular staining for Foxp3 expression was performed by following the manufacturer’s protocol (eBioscience). A total of 1–2 × 106 lymphoid cells were surface stained in 96-well microtiter plates with various combinations of the previously described Abs. FACS analysis was performed using a FACSCalibur flow cytometer (BD Biosciences) and the data were analyzed using FlowJo software (version 8; Tree Star).
In vivo BLyS neutralization
The hamster anti-mouse BLyS mAb 10F4 was injected i.p. Isotype control ChromPure hamster IgG (Jackson ImmunoResearch Laboratories) was injected i.p. into control mice. Two 100-μg doses of 10F4 were administered on days 0 and 5. This regimen resulted in a peak level of FO and MZ B cell depletion by 3 wk following injection. Long-term in vivo BLyS neutralization, also termed “maintenance dose” throughout this article, was achieved by injecting 15 μg of 10F4 biweekly.
BLyS and anti-BLyS ELISA
Serum BLyS levels were measured by ELISA using mBAFFR-Fc (Alexis) as a capture reagent and an anti-murine BLyS hamster mAb (16D7; Human Genome Sciences) as a detector. Samples were diluted to a final concentration of 10% matrix on the assay plate. The limit of detection of this assay is 0.8 ng/ml in undiluted matrix. Serum anti-BLyS (10F4) levels were measured by ELISA; the limit of quantification of this assay is 0.075 μg/ml and the limit of detection is 0.039 μg/ml. Of note, this is designed to measure free BLyS levels. 10F4 is a mAb that effectively blocks BLyS binding to its receptor and, therefore, the mBAFFR-Fc capture reagent. We have determined that the BLyS-10F4 complexes are not captured in this assay. The secondary developing Ab is 16D7 and is totally non-cross-reactive with the BLyS epitope bound by 10F4.
Total serum IgG ELISA
ELISA was used to analyze total serum IgG in NOD and anti-BlyS-treated NOD mice. After cardiac puncture, samples were collected and centrifuged for 10 min at 3200 rpm. Polyvinyl 96-well plates were coated overnight at 4°C with goat monoclonal anti-mouse IgG (reacts with IgG1, IgG2a, IgG2b, and IgG3; Southern Biotechnology Associates) diluted 1/1000 in PBS plus azide. The plates were washed and blocked with PBS and 1% BSA plus azide. Plates were washed and serum samples were diluted in triplicate. Bound Ab was detected using an alkaline phosphatase-conjugated goat anti-mouse IgG (diluted 1/1000 PBS and 1% BSA plus azide; Southern Biotechnology Associates). Plates were washed and developed using 1 mg/ml p-nitrophenyl phosphate (Southern Biotechnology Associates) in a developing buffer of 0.1 M sodium bicarbonate and 1 mM magnesium chloride (pH 9.8). Absorbances were read at 405 nm using a microplate reader.
Histology and insulitis scoring
Pancreatic tissue was fixed in formalin, paraffin embedded, stained with H&E and aldehyde fuchsin (which stains islet β-cells dark blue), and examined for the presence of mononuclear cell infiltration. Slides were examined by light microscopy and photographed using a digital camera system. The insulitis score was graded as follows: 0, no insulitis; 1, peri-insulitis/insulitis involving <25% of islet; 2, insulitis involving 25%–75% of islet; 3, >75% islet infiltration.
CFSE labeling and in vitro stimulation of CD4 T cells
Splenocytes and lymph nodes were harvested from NOD/ShiLtJ and C57BL/6J mice 3 wk following anti-BLyS or isotype IgG treatment. Lymphocytes were labeled with CFSE (Molecular Probes). Briefly, a 5 mM stock solution of CFSE was prepared in DMSO. Lymphocytes isolated from spleens and lymph nodes of mice were resuspended at a concentration of 20–30 × 106 cells/ml in serum-free IMDM (Invitrogen) at 37°C. An equal volume of a 1/250 dilution of the 5 mM CFSE stock in 37°C IMDM was added to the cell preparation. After a 5-min incubation period at 37°C, the excess CFSE was quenched by adding an equal volume of heat-inactivated FCS. The CFSE-labeled cells were then washed once and resuspended at the desired concentration in IMDM containing 10% heat-inactivated FCS. CFSE-labeled cells were plated in 24-well plates at a density of 1 × 106 total cells/ml in medium containing 10% heat-inactivated FCS and varying amounts of anti-CD3 (2C11) (0–2 μg/ml). Loss of CFSE intensity upon stimulation was used as a sensitive and reproducible measure of cell division.
Ninety-six-well filtration plate micro-IAA assay
IAA was measured using a 96-well filtration plate micro-IAA assay. Briefly, 125I-insulin (Amersham) of 20,000 cpm was incubated with 5 μl of serum with and without cold human insulin, respectively, for 3 days at 4°C in buffer A (20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1% BSA, 0.15% Tween 20, and 0.1% sodium azide). Fifty μl of 50% protein A and 8% protein G-Sepharose (Amersham) were added to the incubation in a MultiScreen-NOB 96-well filtration plate (Corning) that was precoated with buffer A. The plate was shaken for 45 min at 4°C followed by two cycles of four washes, each cycle with cold buffer B (buffer A with 0.1% BSA added). After washing, 40 μl of scintillation liquid (Microscint-20; Packard Instruments) was added to each well and radioactivity was determined directly in the 96-well plate with a TopCount beta counter (Packard). The IAA level was calculated based on the difference (Δ) in cpm between the well without cold insulin and the well with cold insulin and expressed as an index, where index = (sample Δcpm − human negative control Δcpm)/(human positive control Δcpm − human negative control Δcpm). The limit of normal (0.010) was chosen by the analysis of IAA in nondiabetic strain mice including 23 BALB/c and C57BL/6 mice.
Glucose tolerance test
Mice were fasted overnight with access to water before i.p. injection with glucose (1.5 mg/g body weight). Blood glucose was measured with Accu-Check Advantage test strips at 15–60 min intervals following glucose challenge.
Statistical analyses were performed using the Student’s t test and χ2 with Microsoft Excel software; differences were considered significant at p < 0.05.
B lymphocyte depletion following in vivo BLyS neutralization in NOD mice
We first determined that two 100-μg i.p. injections of the anti-BLyS mAb 10F4 caused depletion of the following: 1) mature/recirculating B cells in the bone marrow (BM) and peripheral blood; 2) FO B cells in the LN and spleen; and 3) splenic MZ B cells (Fig. 1). Fig. 1,a demonstrates the gating strategy used to resolve the various B cell subsets in the BM and periphery. This gating strategy is similar to that used in our previous study (14). Maximal depletion was observed at 2–3 wk following injection, at which time the majority of mature B cells in the periphery were depleted (Fig. 1, b and c). Notably, BLyS neutralization effectively depleted the MZ B cell compartment, which is highly BLyS dependent (25). In contrast, the pro-/pre-B cells and immature B cells in the BM as well as the TR B cells in the periphery were spared from depletion following BLyS neutralization. As such, the TR B cell compartment in NOD mice treated with anti-BLyS was proportionally expanded but remained stable with respect to absolute numbers. At 10 days following treatment, the depleting regimen of anti-BLyS rendered serum BLyS undetectable (Fig. 1,d). As expected, we found an inverse correlation between the detectable serum levels of BLyS and the serum concentration of anti-BLyS (Fig. 1,d). Interestingly, at 3 wk following treatment when peak B cell depletion was observed, serum BLyS levels were found to be at a supraphysiological high (Fig. 1 d). We attribute this latter finding to B cell compartment lymphopenia causing a decreased level of in vivo BLyS utilization, leading in turn to a higher detectable systemic BLyS concentration. Indeed, such a phenomenon has been reported in patients undergoing B cell depletion therapy (26). Finally, by 7 wk after anti-BLyS injection, the peripheral B lymphocyte compartment and systemic BLyS levels re-equilibrated to the pretreatment steady state.
Impact of in vivo BLyS neutralization on diabetes incidence in NOD mice
We treated two cohorts of 8-wk-old prediabetic female NOD mice with two 100-μg i.p. doses of anti-BLyS (n = 19) or an isotype control IgG (n = 13). The kinetics of B cell depletion and re-emergence was confirmed in all of these mice via peripheral blood screening using flow cytometry and was identical with that shown in Fig. 1. In the anti-BLyS treated group, diabetes incidence was significantly delayed for up to 30 wk of age (Fig. 2,a). However, by 45 wk of age the overall incidence of diabetes was comparable to that of the control IgG-treated group (Fig. 2,a). It is important to note that the kinetics of diabetes incidence in the hamster IgG-treated control mice is identical to that of untreated female NOD mice in our Jackson-derived colony. These results indicate that a one-time depleting dose of anti-BLyS significantly protracted the kinetics of diabetes incidence without ultimately changing overall diabetes incidence at 45 wk of age. Histological examination of pancreata from isotype control and anti-BLyS treated mice at 12–18 wk of age demonstrated a significantly reduced severity of insulitis in the anti-BlyS-treated cohort (Fig. 2 b).
Impact of maintenance anti-BLyS treatment on the efficiency of follicular entry and systemic BLyS levels in NOD mice
Our next objective was to increase competition for BLyS at the level of follicular entry during B cell compartment reconstitution following depletion to increase selection stringency at the TR→FO checkpoint. Therefore, following administration of the B cell-depleting dose of anti-BLyS (i.e., two doses at 100 μg each), we implemented a low-dose maintenance anti-BLyS treatment regimen to maintain systemic BLyS levels at a lower level than that found at steady state. Four weeks following administration of the depleting dose (two doses at 100 μg each), we initiated a biweekly maintenance regimen of 15 μg of anti-BLyS in a cohort of 8-wk-old female NOD mice. Control mice were treated with an identical dose of the isotype control IgG. This maintenance regimen was continued for 7 additional weeks. During this period, we monitored the distribution of TR and mature/FO B cells, as well as, serum BLyS levels. Serum BLyS levels remained stable over the course of this treatment period, ranging from 4 to 8 ng/ml in control IgG-treated mice (Fig. 3,a). In contrast, in mice receiving an identical regimen of anti-BLyS, serum BLyS was undetectable at 2 wk following treatment and remained in the 1–3 ng/ml range for up to 13 wk following initiation of treatment (Fig. 3,b). The maintenance course of anti-BLyS caused a marked and persistent elevation in the ratio of TR:FO B cells in the periphery that lasted for up to 16 wk following initiation of treatment, as compared with that found in IgG-treated controls (Fig. 3,b). This persistently elevated ratio of TR:FO B cells in anti-BlyS-treated NOD mice indicates a relative inefficiency of TR B cell entry into the FO, resulting in a slower reconstitution rate of follicular B cells to a normal steady state. We observed a marked increase in the frequency of TR B lymphocytes in the setting of in vivo BLyS neutralization that persisted at steady state (Fig. 3,c). This phenotype is in contrast to the profound degree of TR B cell lymphopenia normally seen in control and untreated NOD mice at steady state (Fig. 3,c and Ref. 12). Fig. 3 d demonstrates the absolute numbers of TR, MZ, and FO B cells in cohorts of three mice per time point up to 16 wk following initiation of the maintenance anti-BLyS treatment. It is important to note that the BLyS-limited condition achieved with the maintenance treatment does not increase the production rate of TR B cells, as evident from the constant absolute number of TR B cells. Rather, it increases the stringency of FO entry, thereby leading to protracted reconstitution kinetics of the FO compartment to a steady state size that lasts beyond the 16-wk end point of this experiment.
Impact of maintenance anti-BLyS treatment on the stringency of negative selection at the TR→FO checkpoint in NOD mice
We next assessed whether the proportional expansion of the TR B cell subset induced by the long-term BlyS-neutralizing regimen led to a restoration of negative selection stringency at the TR→FO peripheral B cell tolerance checkpoint in NOD mice. In our previous studies we used an established readout of clonotype negative selection in the Igλ L chain-bearing B cell clonotypes (12, 27). Autoreactive Igλ L chain-bearing B cells are sufficiently frequent in the TR compartment to permit visualization of a stepwise reduction in their frequency as they pass through the various B cell selection checkpoints in the development of a preimmune B cell repertoire (27). We previously demonstrated that in NOD mice a lymphopenic TR B cell compartment is associated with a striking defect in the negative selection of Igλ L chain-bearing B cells at the BlyS-dependent TR→FO checkpoint in NOD mice (12). Indeed, at 20 wk following initiation of anti-BLyS treatment, the NOD B cell compartment exhibited a graded decrease in the frequency of Igλ L chain-bearing B cells at the TR→FO checkpoint (Fig. 4,a); this is unlike control counterparts in which the TR→FO checkpoint remained relaxed. This result suggested that long-term in vivo BLyS neutralization increases the stringency of peripheral B cell negative selection at the TR→FO checkpoint. Consistent with this finding, we found both significantly reduced serum IAA titers and a larger proportion of IAA negative mice in the anti-BlyS-treated cohort of NOD mice for up to 30 wk of age (Fig. 4,b). This abrogation of IAA titers is not related to a global decrease in total IgG serum titers (Fig. 4 c).
Impact of long-term in vivo BLyS neutralization on progression of autoimmune diabetes in NOD mice
We next treated a cohort of 3–4 wk old female NOD mice with a depleting dose of anti-BLyS (two doses at 100 μg each) followed by a maintenance dose (15 μg biweekly) starting 3 wk later. The maintenance regimen was continued until 25 wk of age. A cohort of control NOD mice was treated with an identical regimen of isotype control IgG. These NOD mice were monitored up to 40 wk of age for the development of autoimmune diabetes. We found that, as compared with the control IgG treated cohort, long-term in vivo BLyS neutralization led to a marked protection from autoimmune diabetes (Fig. 5,a). It is important to note that the kinetics of diabetes incidence in the long-term hamster IgG-treated control mice is identical to that of untreated female NOD mice in our Jackson-derived colony (Fig. 5,a). At 16 wk following initiation of treatment (19–20 wk of age), this protection from autoimmune diabetes was associated with a significantly reduced severity of insulitis in mice undergoing long-term in vivo BLyS neutralization (Fig. 5, b and c). For histological comparison, we treated an additional cohort of NOD mice with a 15-μg biweekly maintenance dose of anti-BLyS without a prior depleting dose (Fig. 5,c). These mice had significantly fewer islets with severe lymphocytic infiltration as compared with isotype IgG-treated controls but were in contrast with NOD mice that had received the maintenance dose in addition to the initial depleting dose 3 wk prior. NOD mice treated with the depleting dose followed by the maintenance dose were largely free of insulitis 16 wk following initiation of treatment (Fig. 5 c).
B cell depletion via in vivo BLyS neutralization arrests the terminal stages of islet destruction in NOD mice
We assessed the efficacy of B lymphocyte depletion via in vivo BLyS neutralization for the reversal of recent onset diabetes. We previously determined that when NOD mice are first found to have a nonfasting blood glucose level of 160–200 mg/dl, a definitive diabetic state (i.e., nonfasting blood glucose > 300 mg/dl) is established within 2 wk. This period is reminiscent of the “honeymoon” phase that patients with T1D experience at the time of diabetes onset (28). Blood glucose levels in a cohort of female NOD mice were monitored three times weekly starting at 10 wk of age to select a cohort of mice with nonfasting glucose levels of 160–200 mg/dl. Three consecutive daily doses of 100 μg of anti-BLyS were administered to 12 female NOD mice with nonfasting blood glucose levels in the range of 160–200 mg/dl. These mice were subsequently treated with a weekly maintenance dose of 50 μg of anti-BLyS for 6 wk and their nonfasting blood glucose levels were monitored on a daily basis. A cohort of control NOD mice was similarly selected and treated with an equivalent dose of isotype control IgG. Fig. 6,a demonstrates that in vivo BLyS neutralization prevented the progression of NOD “honeymooners” to a fulminant and stable diabetic state (i.e., blood glucose > 300 mg/dl), maintaining these mice at a nonfasting blood glucose range of 150–300 mg/dl. This was in contrast to isotype control-treated mice, 100% of which progressed to a stable diabetic state (i.e., >400 mg/dl) within 2 wk after the initial detection of a blood glucose level in the 160–200 mg/dl range. “Honeymooning” untreated NOD mice progress to a fully diabetic state with kinetics similar to that of our hamster IgG-treated controls (data not shown). To assess the functional capacity of residual pancreatic islets in the anti-BlyS-treated NOD “honeymooners” after 5 wk of treatment with anti-BLyS, these mice were subjected to an i.p. glucose tolerance test (Fig. 6 b). The NOD “honeymooners” treated with anti-BLyS uniformly exhibited a glucose tolerance capacity at an intermediate level between the nondiabetic and fully diabetic NOD mice. This result demonstrated that anti-BLyS therapy is capable of arresting the terminal stages of islet destruction in NOD mice.
Impact of anti-BlyS-mediated B cell depletion on absolute numbers of T regulatory cells in NOD mice
We assessed the effects of anti-BlyS-mediated depletion on T regulatory cells (Tregs) in NOD mice treated with a depleting dose of anti-BLyS or control IgG followed 4 wk later with a maintenance dose of anti-BLyS or isotype IgG. Ten wk after the initiation of treatment, NOD mice were sacrificed and their CD4+CD25+FoxP3+ Tregs were analyzed (Fig. 7,a). We found no significant difference in the absolute numbers of CD4+CD25+FoxP3+ Tregs in the spleens or lymph nodes (pooled cervical, axillary, and inguinal) of NOD mice treated with anti-BLyS or isotype IgG (Fig. 7 b).
Impact of anti-BlyS-mediated B cell depletion on in vitro CD4 T cell activation
We previously demonstrated that the efficiency of NOD CD4 T cell activation is primarily reliant on B lymphocyte-mediated costimulation in vitro and in vivo (29, 30). This phenotype is due to a defect in the costimulatory capacity of non-B cell APCs in NOD mice. Furthermore, this defect is demonstrable using the APC-dependent soluble anti-CD3 stimulation assay in vitro (29). Thus, we predicted that CD4 T cell activation may be disrupted in both lymph node and splenic CD4 T cell populations in anti-BlyS-treated NOD mice. We thus assessed the soluble anti-CD3 dose responsiveness of CD4 T cells from anti-BlyS-treated and control NOD mice at 3 wk following anti-BLyS injection (Fig. 8). Consistent with our previous studies (29, 30), in vivo B lymphocyte depletion using anti-BLyS significantly impaired the activation profile of splenic CD4 T cells from NOD mice in response to soluble anti-CD3 (Fig. 8, a and b). In contrast, splenic CD4 T cells from anti-BlyS-treated B6 mice were not impacted to an appreciable degree (Fig. 8, a and b). A similar anti-BlyS-mediated impairment was seen in the case of lymph node CD4 T cells (Fig. 8,c). Importantly, lymph node CD4 T cells from anti-BlyS-treated NOD mice were not stimulated sufficiently to undergo division, even at the maximal 2 μg/ml concentration of soluble anti-CD3 (Fig. 8 c).
Over the last decade the key role of B lymphocytes in the pathogenesis of autoimmune diabetes in NOD mice has come to light (1). Two studies initially demonstrated that both B cell “knockout” (10) and Ab-mediated B lymphocyte depletion (5) strategies prevent insulitis and diabetes onset in NOD mice. These studies were recently confirmed using therapeutically relevant mAbs specific for CD20 (31, 32) to treat prediabetic adult NOD mice and pointed to a potential role for B cell depletion therapy in the prevention of T1D in humans (31, 32). Based on the basic studies on the role of B lymphocytes in the pathogenesis of NOD diabetes, a clinical trial of rituximab (i.e., anti-CD20) for the reversal of recent onset T1D is currently underway (www2.diabetestrialnet.org/anti).
In this study we hypothesized that the B lymphocyte stimulator cytokine BLyS may be a logical target of immunotherapy for the prevention of T1D. It is generally accepted that deletion of autoreactive B cells and their exclusion from entry into the B cell FO at the TR stage are conditional (14, 33, 34, 35). That is, certain microenvironmental cues are able to rescue autoreactive TR B cells from deletion. In particular BLyS and its family of receptors have been identified as the major regulators of B cell homeostasis by controlling survival, differentiation, and lifespan (14, 22, 36). Mice with a mutation in the BLyS receptor BR3 fail to generate long-lived mature B cells and exhibit a developmental block at the TR B cell stage (15, 16, 17, 18, 19). Additionally, two notable studies have demonstrated that BLyS is the limiting resource for which TR B cells compete and can rescue autoreactive TR B cells from deletion (20, 21). These studies demonstrated that although negative selection of autoreactive immature B cells in the BM is not influenced by BLyS, that of TR B cells in the periphery is elastic and dependent upon the available systemic BLyS level. Furthermore, transgenic BLyS overexpression leads to development of humoral autoimmunity (22, 23, 24). Therefore, it has been suggested that the level of interclonal competition for BLyS at the TR stage dictates the probability of autoreactive B cells entering the preimmune B cell repertoire (14).
The correlation between diabetes susceptibility and loss of B cell tolerance to islet autoantigens is a recognized feature of T1D. In the NOD model, several studies have demonstrated the inefficiency of B cell tolerance mechanisms in NOD mice (1). In line with these studies, we recently identified a homeostatic defect in the production of TR B cells that prevents appropriate negative selection at the TR→FO stages of NOD B cell development (12). The resultant lymphopenic TR B cell compartment would be expected to abrogate TR B cell competition for BLyS, leading to promiscuous selection and follicular entry (14) as we found to be the case in NOD mice (12). Thus, we hypothesized that in vivo neutralization of BLyS would increase the stringency of negative selection at the TR→FO checkpoint in NOD mice. We initially treated a cohort of NOD mice with a B lymphocyte-depleting regimen of anti-BLyS. This mAb effectively neutralized BLyS and promoted a profound state of selective B cell deficiency within 3 wk. Both the FO and MZ B cell compartment, which are known to be BlyS-dependent subsets, were effectively depleted. By 7 wk following treatment, the B lymphocyte compartment size had reconstituted to a comparable steady state found in control mice. This regimen of anti-BLyS significantly delayed the onset of spontaneous diabetes. This result confirmed that transient B cell depletion, although effective at disrupting the progression of destructive islet inflammation, does not completely reverse the susceptibility of NOD mice to autoimmune diabetes. Interestingly, we found that upon in vivo clearance of the depleting dose of anti-BLyS mAb, the serum of B lymphocyte-depleted mice contained a 2- to 5-fold elevated concentration of BLyS. This elevated BLyS level, is attributable to reduced BLyS “consumption” by the absent FO and MZ B lymphocyte compartments in the depleted mice. A similar spike in systemic BLyS level is observed following B lymphocyte depletion using rituximab in patients with rheumatoid arthritis (26). The re-emergence of TR B cells into such a “BLyS-rich” environment in B cell-depleted mice could abrogate the BlyS-dependent TR→FO tolerance checkpoint at which competition for BLyS regulates the stringency of negative selection (14). As such, we next hypothesized that long-term BLyS neutralization following B cell depletion will be required for the restoration of appropriate negative selection at the TR→FO checkpoint as the B cell compartment reconstitutes to a steady state. We next demonstrated that a maintenance course of anti-BLyS caused a persistent elevation in the ratio of TR:FO B cells for up to 16 wk following initiation of treatment, as compared with that found in IgG-treated controls. This elevated ratio of TR:FO B cells in anti-BLyS treated NOD mice is one or both of the following: 1) a manifestation of a relative inefficiency of TR B cell entry into the follicle; and/or 2) a slower reconstitution of follicular B cells to a normal absolute number at steady state. In either case, the capacity of the anti-BLyS maintenance therapy to maintain an elevated TR:FO ratio points to an increased level of competition for follicular entry on the part of newly emerging TR B cells. Our results indicate that the absolute number of TR B cells in NOD mice remains constant, irrespective of BLyS levels or the depletion status of the FO/MZ compartments. Therefore, the elevated frequency of TR B cells in the setting of BLyS neutralization is not a function of a corrected production rate of TR B cells in anti-BlyS-treated NOD mice. Rather, the capacity of BLyS neutralization to expand the frequency of TR B cells demonstrates more stringent follicular entry.
Long-term in vivo BLyS neutralization reduced IAA titers and the frequency of NOD mice with an IAA-positive status. This abrogation of autoantibody titers in anti-BlyS-treated NOD mice also correlated with a restoration of Igλ clonotype negative selection at the TR→FO checkpoint, a defect that our group has delineated as an aberrant characteristic of B lymphocyte homeostasis in NOD mice (12). These data collectively suggest that long-term in vivo BLyS neutralization is capable of correcting a defect in NOD B cell tolerance by increasing the stringency of negative selection at the TR→FO checkpoint. Finally, long-term in vivo BLyS neutralization therapy, while permitting reconstitution of the B lymphocyte compartment to a steady state under stringent selection, also significantly reduced the severity of insulitis and the incidence of spontaneous diabetes in NOD mice.
A previous study suggested that B cell depletion using anti-CD20 “reverses” the final stages of islet destruction in NOD mice (32). In this study, we tested the efficacy of B cell depletion via in vivo BLyS neutralization therapy for the reversal of recent onset diabetes. Although our results do not demonstrate a complete reversal of recent onset diabetes, we found that B lymphocyte depletion during the “honeymoon” phase of T1D pathogenesis arrests progression to a fully diabetic state, which is defined as a nonfasting blood glucose > 300 mg/dl. Despite their elevated baseline nonfasting blood glucose levels (i.e., 150–250 mg/dl) at 6 wk following initiation of anti-BLyS therapy, these mice exhibited a glucose tolerance test profile intermediate between that seen in nondiabetic and fulminantly diabetic NOD mice. This contrasted with control “honeymooners,” all of whom became diabetic within 2 wk. These results collectively demonstrate that B cell depletion via in vivo BLyS neutralization is capable of arresting the final stages of islet destruction in NOD mice and permitting the retention of a residual degree of glucose homeostasis. However, our data did not demonstrate that B cell depletion promotes full reversal of T1D, as was suggested in a previous study (32).
An accumulating body of evidence has indicated that B lymphocytes are critical APCs whose MHC class II-mediated Ag presentation and costimulatory functions are critical for the activation of autoreactive CD4 T cells in NOD mice (1). Our previous studies demonstrated that NOD CD4 T cell activation is severely impaired, both in vivo and in vitro, in the absence of B cells (29, 30). Therefore, it is not unexpected that B cell-deficient or -depleted NOD mice would be protected from T1D. Moreover, it is likely that B cell depletion therapy via in vivo BLyS neutralization prevented the progression of diabetes by abrogating islet-reactive CD4 T cell priming. In this regard, the MZ B cell subset has the ability to present insulin-derived peptide-MHCs to diabetogenic T cells and has been suggested to play a role in breaking tolerance to islet autoantigens (37). We and others have previously demonstrated that the spleen of NOD mice contains a significantly expanded MZ compartment (12, 38, 39). Therefore, the unique capacity of in vivo BLyS neutralization, unlike anti-CD20 directed therapy, to deplete the MZ B cell compartment may be instrumental in its capacity to ameliorate the anti-islet T cell response. This study also demonstrated that in vivo BLyS neutralization leads to a profound state of B cell deficiency that in turn impairs CD4 T cell activation in the costimulation-dependent soluble anti-CD3 stimulation assay. This result is consistent with our previous studies demonstrating the reliance of NOD CD4 T cell activation on B cell costimulation (29).
A previous study suggested that anti-CD20-mediated B cell depletion protects NOD mice by promoting an expansion of CD4+ Treg cells (32). It has also been suggested that a relative deficiency of CD4+ Treg cells in NOD mice contributes to diabetes susceptibility (40, 41). However, we did not find an expansion in the absolute number of these regulatory CD4 T cells in NOD mice B cell depleted via in vivo BLyS neutralization. Rather, our previous studies, as well as data presented here, support the contention that B cell depletion disrupts the progression of T cell-mediated autoimmunity by eliminating the costimulatory function of this critical subset of APCs.
On the translational front, a clinical trial of the human anti-BLyS mAb belimumab, currently in phase 3, demonstrated substantially reduced autoantibody titers in patients with systemic lupus erythematosus (42, 43, 44). The phase 2 component of this trial demonstrated that after 52 wk of therapy with monthly doses of belimumab, patients experienced a dramatic decrease in serum titers of lupus autoantibodies and significant clinical remission. This clinical outcome is reminiscent of our finding of reduced IAA titers in NOD mice, which results from restoration of stringent peripheral negative selection of autoreactive B cell specificities. Thus, a unique advantage of in vivo BLyS neutralization therapy may be its capacity to correct repertoire selection defects and prevent recurrence of B cell-mediated autoimmunity without the need for long-term B lymphocyte depletion. The present study demonstrates a mechanistic rationale for testing the efficacy of belimumab as a novel immunotherapeutic agent for the prevention or reversal of T1D in the clinical setting.
This study was funded by The Iacocca Foundation. The authors thank Matthew J. Deasey, William J. Quinn III, Raha Mozaffari, Amy J. Reed, Robert E. Roses, Daniel J. Trainer, and Anne Wang for their technical and scientific input.
The authors have no financial conflict of interest.
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.
This study was supported in part by the Iacocca Foundation and by National Institutes of Health Grants KO8-DK064603 and R03-DK080286 (to H.N.) and Juvenile Diabetes Research Foundation Grant 4-2005-351 to (A.N.).
Abbreviations used in this paper: T1D, type 1 diabetes; BLyS, B lymphocyte stimulator; BM, bone marrow; FO, follicular; IAA, insulin autoantibody; MZ, marginal zone; TR, transitional; Treg, T regulatory cell.