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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. Here 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 (MZ) 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 (IAA), 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 patients with T1D is associated with the loss of B-lymphocyte tolerance to a range of islet-specific autoantigens . In the clinical setting the presence of islet specific autoantibodies serves as an important prognostic marker of autoimmune diabetes susceptibility . In addition to their value for risk stratifying susceptible patients, islet specific autoantibodies, including insulin autoantibodies (IAA), are important participants in the pathogenesis of autoimmune diabetes . Indeed, B-lymphocytes are required for the progression of islet inflammation [4, 5] and act as antigen presenting cells responsible for the activation of islet specific T-cells [6-11]. By virtue of their ability to produce islet-specific autoantibodies, B-cells are capable of efficient autoantigen uptake and presentation . Several studies have demonstrated defects in peripheral negative selection of autoreactive B-lymphocyte clonotypes [11-13]. This defect is associated with a lymphopenic transitional (TR) B-cell compartment, which reduces the competitive constraint on follicular entry [12, 14]. It is well established that entry of newly emerging TR B-cells into the follicle is a competitive process dominantly regulated by the TNF-related cytokine, BLyS [15-24]. Therefore, it has been suggested that the level of inter-clonal competition for BLyS at the TR stage dictates the probability of autoreactive B cells entering into the pre-immune B cell repertoire .
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.
C57BL/6J and NOD/ShiLtJ mice were obtained from Jackson Laboratories. All animals were housed in specific pathogen free conditions at the University of Pennsylvania Medical Center. 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 measurement with Accu-Check Advantage test strips (Boehringer Mannheim, Indianapolis, IN) greater than 250 mg/dl (13.9 mmol/l) for 2 consecutive readings.
The antibodies used in this study were as follows: PerCP conjugated anti-CD45R (B220, RA3-6B2), APC conjugated anti-IgM (II/41), FITC/PE conjuagated anti-CD21/35 (7G6) PE conjugated anti-AA4.1, biotinylated λ-1, biotinylated λ-2+3 (all purchased from BD Biosciences, San Diego, CA), Biotinylated mAb were detected by streptavidin-allophycocyanin (BD Bioscences). Intracellular staining for Foxp3 expression was performed by following the manufacturer’s protocol (eBioscience, San Diego, CA). A total of 1-2 × 106 lymphoid cells were surface stained in 96-well microtiter plates with various combinations of the previously described antibodies. FACS analysis was performed using a FACSCalibur (BD Biosciences, San Jose, CA) and the data were analyzed using FlowJo Software (Version 8, Tree Star, Inc, Ashland, OR).
The hamster anti-mouse BLyS monoclonal antibody (mAb), 10F4, was injected intra-peritoneally (i.p.). Isototype control ChromPure Hamster IgG (Jackson ImmunoResearch, West Grove, PA) was injected i.p. in control mice. Two 100μg 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 weeks following injection. Long-term in vivo BLyS neutralization, also termed “maintenance dose” throughout this paper, was achieved by injecting 15μg of 10F4, biweekly.
Serum BLyS levels were measured by ELISA using mBAFFR-Fc (Alexis) as a capture reagent and an anti-murine BLyS hamster monoclonal antibody (16D7, Human Genome Sciences, Inc., Rockville, MD) 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, which 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 antibody is 16D7 and is totally non-cross-reactive with the BLyS epitope bound by 10F4.
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 minutes at 3200 rpm. Polyvinyl 96-well plates were coated overnight at 4°C with goat monoclonal anti-mouse IgG (reacts with IgG1, IgG2a, IgG2b, IgG3; Southern Biotechnology Associates, Inc) 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 antibody was detected using an alkaline phosphatase-conjugated goat anti-mouse IgG (diluted 1:1000 PBS and 1% BSA plus azide; Southern Biotechnology Associates, Inc, Birmingham, AL). Plates were washed and developed using 1mg/ml p-nitrophenyl phosphate (Southern Biotechnology Associates, Inc) in a developing buffer of 0.1 M sodium bicarbonate, 1mM magnesium chloride, pH 9.8. Absorbances were read at 405 nm using a microplate reader.
Pancreatic tissue was fixed in formalin, paraffin embedded, and stained with Hematoxylin and Eosin (H&E) and Aldehyde Fuchsin (AF), (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.
Splenocytes and lymph nodes were harvested from NOD/ShiLtJ and C57BL/6J mice 3 weeks following anti-BLyS or isotype IgG treatment. Lymphocytes were labeled with CFSE (Molecular Probes, Eugene, OR). 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 (Life Technologies, Gaithersburg, MD) 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 (HI-FCS). The CFSE-labeled cells were then washed once and resuspended at the desired concentration in IMDM containing 10% HI-FCS. CFSE-labeled cells were plated in 24-well plates at a density of 1 × 106 total cells/ml in media containing 10% HI-FCS, 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.
IAA was measured using a 96-well filtration plate micro IAA assay. Briefly, [125I]insulin (Amersham Pharmacia, Piscataway, NJ) of 20,000 counts per minute (c.p.m.) was incubated with 5 μL of serum with and without cold human insulin, respectively, for three days at 4 °C in buffer A (20mM Tris-HCl buffer pH 7.4 containing 150 mM NaCl, 1% BSA, 0.15% Tween-20 and 0.1% sodium azide). 50 μl of 50% Protein A/8% Protein G-Sepharose (Amersham) were added to the incubation in a MultiScreen-NOB 96-well filtration plate (Corning, Acton, MA) which was precoated with buffer A. The plate was shaken for 45 min at 4 °C followed by 2 cycles of 4 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, Meriden, CT) was added to each well and radioactivity determined directly in the 96-well plate with a TopCount beta-counter (Packard). The IAA level was calculated based on the difference (Delta) in c.p.m. between the well without cold insulin and the well with cold insulin and expressed as an index: index = (sample Delta cpm - human negative control Delta cpm)/(human positive control Delta cpm - human negative control Delta cpm). The limit of normal (0.010) was chosen by the analysis of IAA in non-diabetic strain mice including 23 BALB/c and C57BL/6 mice.
Mice were fasted overnight with access to water prior to i.p. injection with glucose (1.5 mg/g body weight). Blood Glucose was measured with Accu-Check Advantage test strips at 15-60 minute intervals following glucose challenge.
Statistical analyses were performed using the Student’s t test and χ2 in the Microsoft Excel software, with differences considered significant at p < 0.05.
We first determined that two 100 μg i.p. injections of the anti-BLyS mAb, 10F4, caused depletion of: 1) mature/recirculating B-cells in the bone marrow (BM) and peripheral blood (PBL), 2) FO B-cells in the LN and spleen, and 3) splenic marginal zone (MZ) B-cells (Figure 1). Figure 1 panel 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 . Maximal depletion was observed at 2-3 weeks following injection at which time the majority of mature B-cells in the periphery were depleted (Figure 1, panels b and c). Notably, BLyS neutralization effectively depleted the MZ B-cell compartment, which is highly BLyS dependent . On the other hand, the Pro-/Pre- and immature B-cells in the BM as well as 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 (figure 1 panel d). As expected, we found an inverse correlation between the detectable serum levels of BLyS and the serum concentration of anti-BLyS (figure 1, panel d). Interestingly, at 3 weeks following treatment when peak B-cell depletion was observed, serum BLyS levels were found to be at a supra-physiological high (figure 1, panel d). We attribute this latter finding to B-cell compartment lymphopenia causing a decreased level of in vivo BLyS utilization, in turn, leading to a higher detectable systemic BLyS concentration. Indeed, such a phenomenon has been reported in patients undergoing B-cell depletion therapy . Finally, by 7 weeks post anti-BLyS injection, the peripheral B-lymphocyte compartment and systemic BLyS levels re-equilibrated to the pre-treatment steady-state.
We treated two cohorts of 8 week-old pre-diabetic 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 these mice via peripheral blood screening using flow-cytometry and was identical to that shown in figure 1. In the anti-BLyS treated group, diabetes incidence was significantly delayed for up to 30 weeks of age (Figure 2, panel a). However, by 45 weeks of age, the overall incidence of diabetes was comparable to that of the control IgG-treated group (Figure 2, panel 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 weeks of age. Histological examination of pancreata from isotype control and anti-BLyS treated mice at 12-18 weeks of age demonstrated a significantly reduced severity of insulitis in the anti-BLyS treated cohort (Figure 2, panel b).
Our next objective was to increase competition for BLyS at the level of follicular entry during B-cell compartment reconstitution following depletion, in order to increase selection stringency at the TR→FO checkpoint. Therefore, following administration of the B-cell depleting dose of anti-BLyS (i.e., 100μg x2 doses), we implemented a low-dose maintenance anti-BLyS treatment regimen, in order to maintain systemic BLyS levels at a lower level than that found at steady-state. Four weeks following administration of the depleting dose (100μg x2 doses), we initiated a bi-weekly maintenance regimen of 15μg anti-BLyS in a cohort of 8 week-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 4-8 ng/ml in control IgG treated mice (Figure 3, panel a). On the other hand, in mice receiving an identical regimen of anti-BLyS, serum BLyS was undetectable at 2 week following treatment and remained in the 1-3 ng/ml range for up to 13 weeks following initiation of treatment (Figure 3, panel b). The maintenance course of anti-BLyS caused a marked and persistent elevation in the ratio of TR:FO B-cells in the periphery, which lasted for up to 16 weeks following initiation of treatment, as compared to that found in IgG treated controls (Figure 3, panel 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 follicle, 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, which persisted at steady state (Figure 3, panel 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 (Figure 3, panel c and ref. ). Figure 3, panel d demonstrates the absolute numbers of TR, MZ, and FO B-cells in cohorts of 3 mice per time point up to 16 weeks 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 a protracted reconstitution kinetics of the FO compartment to a steady-state size, which lasts beyond the 16 week end-point of this experiment.
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 have utilized an established readout of clonotype negative selection in the Igλ L-chain bearing B cell clonotypes [12, 27]. Autoreactive Igλ light 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 pre-immune B-cell repertoire . 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 light chain-bearing B cells at the BLyS dependent TR→FO checkpoint in NOD mice . Indeed, at 20 weeks 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 (figure 4, panel a); unlike control counterparts in whom 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 weeks of age (Figure 4 panel b). This abrogation of IAA titers is not related to a global decrease in total IgG serum titers (Figure 4, panel c).
We next treated a cohort of 3-4 week old female NOD mice with a depleting dose of anti-BLyS (100μg x2 doses) followed by a maintenance dose (15μg biweekly) starting 3 weeks later. The maintenance regimen was continued until 25 weeks 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 weeks of age for the development of autoimmune diabetes. We found that, as compared to the control IgG treated cohort, long-term in vivo BLyS neutralization led to a marked protection from autoimmune diabetes (Figure 5 panel 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 (Figure 5, panel a). At 16 weeks following initiation of treatment (19-20 weeks 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 (Figure 5, panels b and c). For histological comparison, we treated an additional cohort of NOD mice with a 15μg biweekly maintenance dose of anti-BLyS without a prior depleting dose (Figure 5, panel 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 weeks prior. NOD mice treated with the depleting dose followed by the maintenance dose were largely free of insulitis 16 weeks following initiation of treatment (Figure 5, panel c).
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 non-fasting blood glucose level of 160-200mg/dl, a definitive diabetic state (i.e., non-fasting BG>300mg/dl) is established within 2 week. This period is reminiscent of the “honeymoon” phase that patients with T1D experience at the time of diabetes onset . Blood glucose levels in a cohort of female NOD mice were monitored three times weekly starting at 10 weeks of age in order to select a cohort of mice with non-fasting glucose levels of 160-200mg/dl. Three consecutive daily doses of 100μg anti-BLyS were administered to 12 female NOD mice with non-fasting blood glucose levels in the range 160-200 mg/dl. These mice were subsequently treated with a weekly maintenance dose of 50μg of anti-BLyS for six weeks and their non-fasting 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. Figure 6 panel a demonstrates that in vivo BLyS neutralization prevented the progression of NOD “honeymooners” to a fulminant and stable diabetic state (i.e., BG>300mg/dl), maintaining these mice at a non-fasting blood glucose range of 150-300mg/dl. This was in contrast to isotype control treated mice, 100% of which progressed to a stable diabetic state (i.e., >400mg/dl) within two weeks after the initial detection of a blood glucose level in the 160-200mg/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). In order to assess the functional capacity of residual pancreatic islets in the anti-BLyS treated NOD “honeymooners”, after 5 weeks of treatment with anti-BLyS, these mice were subjected to an i.p. glucose tolerance test (GTT) (Figure 6, panel b). The NOD “honeymooners” treated with anti-BLyS uniformly exhibited a glucose tolerance capacity at an intermediate level between the non-diabetic 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.
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 four weeks later with a maintenance dose of anti-BLyS or isotype IgG. 10 weeks after the initiation of treatment, NOD mice were sacrificed and their CD4+ CD25+ FoxP3+ Tregs were analyzed (Figure 7, panel 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 (Figure 7, panel b).
We previously demonstrated that the efficiency of NOD CD4 T-cell activation is primarily reliant on B-lymphocyte mediated co-stimulation in vitro and in vivo [29, 30]. This phenotype is due to a defect in the co-stimulatory 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 . 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 weeks following anti-BLyS injection (Figure 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 (Figure 8, panels a and b). On the other hand, splenic CD4 T-cells from anti-BLyS treated B6 mice were not impacted to an appreciable degree (Figure 8, panel a and b). A similar anti-BLyS mediated impairment was seen in the case of lymph node CD4 T cells (Figure 8, panel c). Importantly, lymph node CD4 T cells from anti-BLyS treated NOD mice were not stimulated sufficiently to undergo division, even at the maximal 2μg/ml concentration of soluble anti-CD3 (Figure 8, panel c).
Over the last decade the key role of B-lymphocytes in the pathogenesis of autoimmune diabetes in NOD mice has come to light . Two studies initially demonstrated that both B-cell “knock-out”  and antibody mediated B-lymphocyte depletion  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 pre-diabetic 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 (http://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 follicle at the TR stage, is conditional [14, 33-35]. That is, certain micro-environmental 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 life-span [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 [15-19]. 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 while 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 over-expression leads to development of humoral autoimmunity [22-24]. Therefore, it has been suggested that the level of inter-clonal competition for BLyS at the TR stage dictates the probability of autoreactive B cells entering the pre-immune B cell repertoire .
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 . In line with these studies, we recently identified a homeostatic defect in the production of TR B-cells, which prevents appropriate negative selection at the TR→FO stages of NOD B-cell development . 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 ; as we found to be the case in NOD mice . 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 weeks. Both the FO and MZ B-cell compartment, which are known to be BLyS dependent subsets, were effectively depleted. By 7 weeks 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, though 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 2-5 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 . 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 . 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 weeks following initiation of treatment, as compared to that found in IgG-treated controls. This elevated ratio of TR:FO B cells in anti-BLyS treated NOD mice is: 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 remain 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, which our group has delineated as an aberrant characteristic of B-lymphocyte homeostasis in NOD mice . 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 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 . Here, we tested the efficacy of B-cell depletion via in vivo BLyS neutralization therapy for the reversal of recent onset diabetes. Though 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; defined as a non-fasting BG>300mg/dl. Despite their elevated baseline non-fasting blood glucose levels (i.e., 150-250mg/dl) at 6 weeks following initiation of anti-BLyS therapy, these mice exhibited a glucose tolerance test (GTT) profile intermediate between that seen in non-diabetic and fulminantly diabetic NOD mice. This contrasted to control “honeymooners” all of whom became diabetic within 2 weeks. 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 .
An accumulating body of evidence has indicated that B-lymphocytes are critical antigen presenting cells, whose MHC class II mediated antigen presentation and costimulatory functions are critical for the activation of autoreactive CD4 T cells in NOD mice . 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 . We and others previously demonstrated that the spleen of NOD mice contains a significantly expanded MZ compartment [12, 43, 44]). 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, which 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 .
A previous study suggested that anti-CD20 mediated B-cell depletion protects NOD mice by promoting an expansion of CD4+ Treg cells . It has also been suggested that a relative deficiency of CD4+ Treg cells in NOD mice contributes to diabetes susceptibility [37, 38]. 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, it will be important to look for abnormalities in human T1D patients in the TR B cell compartment as well as entry of TR B cells into the follicle. 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 [39-41]. The phase 2 component of this trial demonstrated that after 52 weeks 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.
The authors wish to 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.
1This study was supported by NIH grants KO8-DK064603 and RO3-DK080286 to HN and a grant from the Juvenile Diabetes Research Foundation (4-2005-351) to AN.