B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model
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Type 1 diabetes is a T cell-mediated autoimmune disease characterised by the destruction of beta cells in the islets of Langerhans, resulting in deficient insulin production. B cell depletion therapy has proved successful in preventing diabetes and restoring euglycaemia in animal models of diabetes, as well as in preserving beta cell function in clinical trials in the short term. We aimed to report a full characterisation of B cell kinetics post B cell depletion, with a focus on pancreatic islets.
Transgenic NOD mice with a human CD20 transgene expressed on B cells were injected with an anti-CD20 depleting antibody. B cells were analysed using multivariable flow cytometry.
There was a 10 week delay in the onset of diabetes when comparing control and experimental groups, although the final difference in the diabetes incidence, following prolonged observation, was not statistically significant (p = 0.07). The co-stimulatory molecules CD80 and CD86 were reduced on stimulation of B cells during B cell depletion and repopulation. IL-10-producing regulatory B cells were not induced in repopulated B cells in the periphery, post anti-CD20 depletion. However, the early depletion of B cells had a marked effect on T cells in the local islet infiltrate. We demonstrated a lack of T cell activation, specifically with reduced CD44 expression and effector function, including IFN-γ production from both CD4+ and CD8+ T cells. These CD8+ T cells remained altered in the pancreatic islets long after B cell depletion and repopulation.
Our findings suggest that B cell depletion can have an impact on T cell regulation, inducing a durable effect that is present long after repopulation. We suggest that this local effect of reducing autoimmune T cell activity contributes to delay in the onset of autoimmune diabetes.
KeywordsB cell depletion B cells Insulitis NOD mice Type 1 diabetes
Regulatory B cell
Pancreatic lymph node
Simplified presentation of incredibly complex evaluations
Regulatory T cell
Type 1 diabetes, an organ-specific autoimmune disease with a multifactorial aetiology, is characterised by the immune-mediated destruction of beta cells in pancreatic islets, resulting in insufficient insulin production . Although relatively few immunotherapeutic strategies delay the loss of islet beta cell function, depleting B cells using anti-CD20 monoclonal antibody (rituximab) has delayed C-peptide loss within the first year . Follow-up studies demonstrated that during depletion there was a decreased antibody response to new and recall antigens  but that rituximab suppressed anti-insulin autoantibodies more than anti-GAD, -IA-2 and -ZnT8 autoantibodies . Interestingly, during depletion, T cell proliferative responses to islet antigens increased, particularly in responders to B cell depletion therapy . In addition, the frequencies of autoreactive B cells, judged by islet autoantibody-producing cell clones and by polyreactive B cells, as shown by Hep-2 cell reactivity, were unchanged a year after rituximab treatment and reconstitution .
In NOD mice, agents effecting timed depletion of B cells prevent diabetes [7, 8, 9, 10, 11] and reverse disease after onset [7, 8, 12]. These include anti-human CD20 antibody in human CD20 (hCD20)/NOD transgenic mice (in which the human gene MS4A1, encoding hCD20, is expressed), anti-mouse CD20 antibody, anti-CD22 antibody coupled to immunotoxin, B-Lys/BAFF neutralisation and BCMA-Fc chimerised protein [7, 8, 12]. While these strategies all influence development of diabetes, there are important differences in the effector mechanisms. The many factors determining therapeutic efficacy are not fully characterised. For example, the duration of B cell depletion may be important: monoclonal anti-CD20 that depletes B cells transiently (repopulation by 5 weeks) was not effective in preventing or protecting against diabetes . Thus, it is important to understand kinetics and immunological effects following anti-B cell treatment with such agents.
In a mouse model, using a strategy and antibody similar to rituximab, responses of pathogenic CD4+ T cells were greater in the short term . This recapitulated the finding in humans where T cell responses increased . However, some potentially regulatory subsets of cells, including transitional-zone 2 (T2) B cells, T cells and Gr1+ cells, were increased following repopulation after depletion . Thus, the protection conferred by treatment with anti-CD20 relates not only to the depletion of effector B cells but also to the increase of regulatory populations. The combination of anti-CD20 and oral administration of anti-CD3 had a synergistic effect in hCD20/NOD mice, also related to increased frequency and function of regulatory T cells .
In this study, we focused on characterising the effects of B cell depletion on peripheral B cells and the characteristics of the cellular infiltrate in the islets of Langerhans in the hCD20/NOD mouse. We hypothesised that B cell depletion alters the islet immune infiltrate, contributing to protection from diabetes.
Human CD20 (hCD20) transgenic mice on a BALB/c background (in which the human gene MS4A1, encoding hCD20, is expressed)  were backcrossed to the NOD genetic background more than ten generations, and designated hCD20/NOD mice . Mice were maintained at Cardiff University in specific pathogen-free isolators or scantainers. Mice received water and food ad libitum and were housed in a 12 h dark–light cycle. Animal experiments were conducted in accordance with United Kingdom Animals (Scientific Procedures) Act, 1986 and associated guidelines.
Mice were monitored weekly for glycosuria (Bayer Diastix) from 12 weeks of age. Diabetes was confirmed by blood glucose levels >13.9 mmol/l.
Female hCD20/NOD mice, aged 6–8 or 12–15 weeks were chosen at random to receive anti-hCD20 antibody (clone 2H7; Bio-XCell [West Lebanon, NH, USA]) or control IgG2b antibody (clone MPC-11; Bio-XCell [7, 14, 15]). An i.v. injection of 500 μg of antibody in 200 μl of saline solution (154 mmol/l NaCl) was followed at 3 day intervals by three i.p. injections (modified from ).
Pancreatic lymph nodes (PLNs) were disrupted mechanically with a 30G needle. Bone-marrow cells were flushed out from the hind legs (femur and tibia). Spleens were homogenised and erythrocytes were lysed. Pancreases were inflated with collagenase P solution (Roche, Burgess Hill, UK) in Hanks’ Balanced Salt Solution (HBSS) via the common bile duct, followed by collagenase digestion with shaking at 37°C for 10 min. Islets were isolated by Histopaque density centrifugation (Sigma-Aldrich, Gillingham, UK), hand-picked under a dissecting microscope and trypsinised to generate single-cell suspensions. Islet cells were rested at 37°C, 5% CO2 in complete Iscove’s Modified Dulbecco’s Medium (IMDM) overnight, before stimulation for intracellular staining.
Single-cell suspensions were incubated with TruStain (anti-mouse CD16/32; Biolegend [London, UK]) for 10 min at 4°C, followed by fluorochrome-conjugated monoclonal antibodies against cell surface markers for 30 min at 4°C. Multivariable flow cytometry was carried out using monoclonal antibodies: CD4-FITC (GK1.5 [1:200]), CD8-PE-594 (53-6.7 [1:800]), CD103-BV510 (2E7 [1:100]), PD-1-BV785 (29F.1A12 [1:200]), IFN-γ-BV711 (XMG1.2 [1:100]), CD107a-PeCy7 (1D4B [1:200]), CD69-AF700 (H1.2F3 [1:100]), TGF-β-BV421 (TW7-16B4 [1:100]), CD38-FITC (90 [1:200]) and CD86-PeCy7 (PO3 [1:200]) (all from Biolegend); IL-10-APC (JES5-16E3 [1:200]), CD1d-BV510 (1B1 [1:200]), CD21-PE-594 (7G6 [1:1000]), CD23-BV711(B3B4 [1:800]), CD24-BV650 (M1/69 [1:400]), CD3-BV786 (145-2C11 [1:100]) and CD80-BV650 (16-10A1 [1:100]) (all from BD Biosciences, Reading, UK); CD19-efluor780 (eBio1D3 [1:800]), CD5-PeCy7 (53-7.3 [1:200]), IL-6-PerCP-Cy5.5 (MP5-20F3 [1:200]) and CD44-PerCP-Cy5.5 (IM7 [1:1600]) (all from eBiosciences, Waltham, MA, USA). All antibodies were titrated before use. Dead cells were excluded from the analysis by Live/Dead exclusion (Invitrogen, Paisley, UK). For intracellular cytokine analysis, splenocytes were either unstimulated or stimulated for 24 h with 5 μg/ml lipopolysaccharide (LPS) (Sigma-Aldrich) or 5 μg/ml anti-CD40 (Bio-XCell) and washed. After an overnight resting period, 3 h before antibody staining, phorbal 12-myristrate-13-acetate (PMA) (50 ng/ml), ionomycin (500 ng/ml) and monensin (3 μg/ml) (all from Sigma-Aldrich) were added to the cells. CD107a antibody was added prior to stimulation, as previously described . Fc receptors were blocked using TruStain and, after extracellular staining, cells were fixed using fixation/permeabilisation kit (BD Biosciences) according to the manufacturer’s instructions and then stained for intracellular cytokines or appropriate isotype controls. Cell suspensions were acquired on LSRFortessa (FACSDIVA software; BD Biosciences). All analysis was performed using Flowjo software (Tree Star, Ashland, OR, USA).
No data were excluded from the analysis. Statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software, San Diego, CA, USA). For islet T cells, multivariable flow cytometric analysis was performed using SPICE (Simplified Presentation of Incredibly Complex Evaluations) version 5.1 (http://exon.niaid.nih.gov). Comparison of distributions was performed using the Mann–Whitney U test and a partial permutation test . For diabetes incidence, the Gehan–Breslow–Wilcoxon test was used. All other data were analysed by the Mann–Whitney U test.
Kinetics of B cell subset repopulation after anti-CD20 treatment
Kinetics of repopulation of B cell regulatory subsets after anti-CD20 treatment
B cell depletion does not enrich for B cells producing regulatory cytokines or reduce inflammatory B cells after repopulation
We analysed the TGF-β response, as B cell regulation can occur via TGF-β [21, 22]. In mice aged 6–8 weeks, the number of TGF-β+ B cells was significantly increased 8 weeks post depletion, albeit at small percentages overall (Fig. 3c, e). Differences were not maintained at 12 weeks post treatment in either of the age groups. Therefore, anti-CD20 treatment did not promote a sustained Breg phenotype either during or long after repopulation of the spleen, in either of the age groups of mice studied. We examined the proinflammatory cytokine-producing B cell populations. In the younger mice, 2H7 and control antibody treatment produced little change in the number of IL-6+ B cells, with or without LPS or anti-CD40 stimulation; these mice had significantly fewer IL-6+ B cells at 8 weeks post depletion but at 12 weeks this effect was lost and stimulation with anti-CD40 caused an increase in IL-6+ B cells (ESM Fig. 4).
B cell co-stimulatory markers are downregulated during B cell depletion
Effect of anti-CD20 on B cells in pancreatic islets
Anti-CD20 treatment influences T cell populations in the islet microenvironment
Multivariable analysis of CD4+ T cells revealed a shift in the local proinflammatory environment
B cell depletion affects islet-associated CD8+ T cells long after treatment
At 30 weeks post treatment, islet CD8+ T cell subsets were comparable; although there were fewer activated CD8+ T cells, this was not statistically significant (Fig. 8b, d). Interestingly, CD8+ T cells expressing CD103 and CD69, which indicate a tissue-resident memory (TRM) CD8 T cell phenotype and are associated with controlling tissue immunity after infection , were enriched in the islets of 2H7-treated mice (Fig. 8f).
In the current study, we observed changes in the inflammatory islet environment during B cell regeneration and these persisted long after anti-CD20 treatment. We demonstrated a significant decrease in effector function of CD4+ and CD8+ T cells, with a reduction in IFN-γ production, correlated with a downregulation of activation markers. These changes were more pronounced in mice aged 6–8 weeks vs 12–15 weeks, possibly reflecting the difference in lymphocyte behaviour recently demonstrated in islets in NOD mice before and after insulitis . B cell depletion also reduced the expression of CD80 and CD86 on peripheral B cells when stimulated during the B cell regeneration period in the hCD20/NOD mouse model. Moreover, B cells with a regulatory phenotype were depleted in the periphery and were not increased following regeneration.
As reported previously, we demonstrated that anti-CD20 treatment, in this transgenic system, successfully targets the B cells in peripheral and local tissue . Although all B cell populations were depleted, transitional- and marginal-zone B cells were more susceptible (not due to different hCD20 expression, data not shown). Our work supports some previous findings  but differs from other work indicating that the follicular zone was targeted more readily [20, 27]. This may be due to different mouse genetic background  and the different monoclonal antibody used . Our data, consonant with other studies, demonstrated the depletion of regulatory B cell populations in the marginal zone and T2 compartments, CD1dhiCD5+ and CD24hiCD38hi subsets, along with other B cells (data not shown). B cells with regulatory potential were not spared after B cell depletion. However, TGF-β+ B cells were proportionally significantly increased during depletion, although the percentages were small, whereas IL-10 production from B cells was not enriched during B cell regeneration under our experimental conditions. Furthermore, Breg subsets were not enriched during regeneration. This corroborated our earlier findings in the BDC2.5 transgenic mouse model, showing that CD1d− B cells are more protective than CD1d+ B cells, dependent on cell–cell contact, but not IL-10 . Therefore, B cells may confer protection after B cell depletion, but not via a typical Breg IL-10-mediated mechanism, even though overall anti-CD20 depletion, including depletion of the IL-10-producing B cells, increases the T cell activation observed immediately after B cell depletion [5, 14]. However, B cells located in the peritoneum are spared from depletion  and it is known that these cells (B-1) can produce IL-10 . We did not examine peritoneal B cells, so the ability of these cells to contribute to regulation of the immune response cannot be ruled out.
We observed a reduction in both CD86 and CD80 co-stimulatory molecules upon stimulation, more strikingly with anti-CD40, during repopulation of B cells in 2H7-treated mice. Lack of CD86 expression can impair T cell activation, specifically in NOD mice . Others have shown that CD80/86 expression on B cells is essential for activating proteoglycan-specific autoreactive T cells in an arthritic mouse model . Anti-CD20 B cell depletion influenced the immunostimulatory environment in the secondary lymphoid organs, marked by a reduced expression of CD86/CD80 along with MHC class II, in a marmoset model of autoimmune encephalomyelitis . When we examined regenerated B cells after full repopulation (12 weeks after depletion), we found no functional difference in the ability of the B cells from 2H7-treated mice to present insulin-specific peptide to insulin-specific CD8+ T cells in the proliferative assay that we employed (data not shown). However, because cells were not tested at an earlier time point during regeneration, we cannot discount the possibility that lack of co-stimulatory molecules alters antigen presentation in vivo during the regeneration period. This is indicated, indirectly, by the effects observed in the islets.
In human clinical trials, beta cell function is temporarily preserved after B cell depletion  but the effects on pancreatic immune cell infiltrate during B cell repopulation cannot be studied. Our current study showed a 10 week delay in onset of diabetes, although following extended observation, the endpoint was not statistically significant, in keeping with the human observations that B cell depletion delays disease progression. Our current study gives an important insight into the treatment effects on pancreatic islets, which are ultimately the target of protection. Interestingly, we showed no enrichment of IL-10 or TGF-β from B cells in the islets of anti-CD20-treated mice. The IL-10+ B cell population was downregulated at 12 weeks post treatment but was comparable with that in the control mice long after repopulation. Overall, the regulatory phenotype of B cells in islets was not altered by anti-CD20 treatment. Previously, it has been reported that islet B cells become CD20−CD138+ plasma cells after anti-CD20 treatment . While murine CD20 expression was not studied here, we demonstrated significant B cell depletion in 12- to 15-week-old mice, which have established insulitis. CD86/80 expression has been described in the islet B cells previously, along with the production of TNF-α . Though not specifically addressing the levels of CD80/CD86 or TNF-α on islet B cells after anti-CD20 treatment, we did not observe any IFN-γ production from either control IgG- or 2H7-treated mice (data not shown).
B cell depletion affects T cell regulation [5, 14, 35]. No obvious differences in total T cells were observed, supporting the human anti-CD20 depletion studies . However, we did demonstrate that both effector CD4+ and CD8+ islet-infiltrating T cells were downregulated, including inflammatory IFN-γ production. This supports the notion that B cell depletion modulates T cell responses [5, 14]. While regulatory T cells are enriched during B cell repopulation [7, 8], we did not observe enrichment of IL-10 or TGF-β in islet-infiltrating cells; in fact IL-10+CD4+ T cells were downregulated. Thus, local Tregs may operate through IL-10- and TGF-β-independent mechanisms. In mice, peripheral T cells have decreased effector cytokines  and macrophages from 2H7-treated mice do not present antigen to T cells as efficiently as untreated macrophages . Furthermore, CD11b+GR1+ myeloid-derived suppressor cells are induced post B cell depletion, dependent on a cell–cell contact mechanism independent of IL-10 . Here, we show that B cell depletion directly affects the T cell composition of the islet infiltrate, long after the repopulation of B cells, possibly as a result of B cell–macrophage or B cell–dendritic cell crosstalk occurring directly in the pancreas.
We speculate that other mechanisms may play a role in the pancreatic islets. CD44 is an important mediator in inflammation  and anti-CD44 antibody successfully delays the onset of diabetes in the NOD mouse . Long after B cell depletion, decreased CD44 expression on CD8+ T cells may contribute to the protection seen in B cell-depleted animals, especially as the ligand for CD44 (hyaluronic acid) is expressed in islets during inflammation . Furthermore, we observed an increase in CD69+CD103+CD8+ T cells, which may be a regulatory CD8+ T cell population in the islets, long after antibody treatment and B cell repopulation. This specialised CD8+ T cell (TRM) population, found in tissues after viral infections, can control local immunity . It is possible that these cells may also play a role in inflammatory disease  and autoimmunity. Interestingly, this TRM population has recently been identified in individuals newly diagnosed with type 1 diabetes .
Our data is consonant with findings made in a small number of participants in the TrialNet study, showing maintenance of increased frequencies of autoreactive and polyreactive B cells before and after anti-B cell therapy (1 year) . Thus, therapeutic efficacy for the limited time studied was not related to maintaining depletion of autoreactive B cells or increase in regulatory B cell subsets. Anti-B cell therapy with rituximab is one of the few immunotherapeutic strategies trialled thus far in humans that has shown transient efficacy in delaying the decline of C-peptide, along with anti-CD3, anti-LFA1 and CTLA4-Ig [40, 41, 42, 43]. It is likely that more than one therapeutic strategy will be required for effective immunotherapy of type 1 diabetes. Ideally, this would encompass agents with differing mechanisms to complement each other (e.g. depletion of autoreactive lymphocytes, while increasing endogenous regulation). Our results, indicating that islet-targeting CD8+ T cells may be more affected by the B cell treatment, suggest that a potential adjunct therapy to rituximab could be one that maintains these changes in CD8+ T cells.
In conclusion, we show potential new mechanisms in the local tissue that may contribute to delay in diabetes onset following B cell depletion therapy at an early age. Further investigation is ongoing to dissect these mechanisms, both during and after B cell depletion. However, a key point is that removal of B cells alters effector T cells, either directly or through an antigen-presenting cell population, in the pancreatic islets.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
LCDR, JB, LW and FSW designed the study and wrote and edited the manuscript. LCDR, JB, JD and EDL performed the experiments and analysed the data. All authors reviewed and approved the manuscript. FSW is the guarantor of this work.
This study was funded by the Medical Research Council (MR/K021141/1) and CNPq (Conselho Nacional de Pesquisa, Brazil, grant no. 245609/2012-1).
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