Treatment with CD20-specific antibody prevents and reverses autoimmune diabetes in mice (original) (raw)
Characterization of hCD20/NOD mice. To test the efficacy of anti-hCD20 in prevention and treatment of autoimmune diabetes, we generated a bacteria artificial chromosome–transgenic NOD mouse that expresses hCD20 on B cells (Figure 1A). The expression of hCD20 did not have any obvious effect on general mouse development. No gross abnormality was found in development of the immune system in the transgenic mice (data not shown). Similar to the expression of endogenous mouse CD20 (28–30), the expression of transgenic hCD20 occurred from the pre–B cell stage to mature B cells (Figure 1B). The expression of hCD20 was restricted to B cells only (B220+CD19+) (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI32405DS1). B cell development and the expression of other B cell markers including MHC class II (I-Ag7) and the costimulatory molecule (CD86) in hCD20/NOD mice were similar to nontransgenic NOD mice (Figure 1C).
Phenotypic expression of transgenic hCD20 on B cells. (A) Splenocytes from hCD20 mice were gated on B220+ B cells and stained with anti-hCD20 mAbs (2H7). All mature B cells expressed the transgenic hCD20. (B) Expression of hCD20 in BM cells of transgene-positive and control NOD mice. (C) Phenotypic characterization of hCD20 transgene-positive and control NOD mouse spleen cells. The splenocytes were gated on B220+ cells prior to analyzing for the different B cell markers.
Expression of hCD20 did not obviously alter the production of natural immunoglobulins (Supplemental Figure 1B), anti-insulin autoantibodies (Supplemental Figure 1C), or immune responses to foreign antigens, such as OVA, after immunization (data not shown). Expression of hCD20 also did not alter the upregulation of costimulatory molecules by B cells stimulated with anti-Ig or anti-CD40 (Supplemental Figure 1D). Most importantly, the hCD20 transgene did not affect the natural history of diabetes development, as the incidence of diabetes was very similar in both transgenic hCD20/NOD and nontransgenic groups (Figure 2). However, expression of hCD20 allowed us to use 2H7, a mouse anti-hCD20 mAb that targets the same epitope as rituximab, to specifically deplete B cells. We were able, therefore, to investigate the role of B cells in diabetes development at different stages and to test the efficacy of anti-hCD20 treatment in the disease.
Expression of hCD20 does not affect spontaneous diabetes in NOD mice. The incidence of diabetes in transgenic hCD20 mice was compared with nontransgenic mice (A, female mice; and B, male mice). No differences were seen in diabetes incidence (P = 0.47 when female incidence curves compared by log-rank test and P = 0.84 when male incidence curves compared).
Kinetics of B cell depletion in hCD20/NOD mice and the function of repopulated B cells. To optimize the depletion protocol, we performed pilot experiments using different doses of 2H7 (2 mg, 1 mg, 0.5 mg, and 0.25 mg per mouse) and different injection routes (i.p. or i.v.). Our results showed that a 9-day cycle of 4 i.v. injections with a 3-day interval provided the most effective B cell depletion in hCD20/NOD mice (data not shown). In peripheral blood, depletion was observed as early as 1 hour after the first injection (Supplemental Figure 2), and B cells were almost completely depleted within 1 week after the last anti-hCD20 antibody injection (Figure 3). B cells started to repopulate 3 weeks after the last injection and reached normal levels by 12 weeks after the last injection (Figure 3).
Depletion of B cells following initiation of a course of 4 2H7 anti-hCD20 or IgG control injections. Peripheral blood cells, after removing erythrocytes, were costained with anti-CD22 and anti-B220 and analyzed by flow cytometry. B cell (CD22+B220+) numbers are shown at various time points after the last injection with 2H7 (circles) or control IgG (triangles). Mean values of at least 10 observations ± SE are presented.
The efficiency of B cell depletion varied in different organs (Supplemental Figure 3). Four days after the last injection of anti-CD20 mAbs, more than 90% of B cells were depleted in peripheral blood and mesenteric lymph nodes. The depletion was also effective in axillary lymph nodes and BM, but less complete in spleen, ranging from 50% to 70% depletion compared with controls (Supplemental Figure 3). Consistent with recent studies by other investigators using anti-mouse CD20 (28, 29), B cells in the peritoneal cavity of hCD20/NOD mice were more resistant to depletion (31) (data not shown).
To examine the function of the repopulated B cells, we immunized mice 2 months after anti-hCD20 treatment with OVA, using alum as adjuvant, when approximately 60% to 70% of B cells had been repopulated. IgG-treated mice were used as controls. OVA-specific antibody responses 2 weeks after immunization were comparable between mice that had been treated with anti-hCD20 and mice without treatment (Supplemental Figure 4).
Effect of anti-CD20 treatment on spontaneous diabetes development in hCD20/NOD mice. To test the role of B cells in the preclinical stages of diabetes development, we treated 4- and 9-week-old female hCD20/NOD mice with 2H7 or control IgG using 1 treatment cycle (9-day period with 4 injections at 3-day intervals). These ages were chosen because, in our NOD colony, 4-week-old mice had little or no pancreatic infiltration while almost all 9-week-old mice displayed insulitis in females. Mice received 0.5 mg antibody in the first injection and 0.25 mg for the subsequent 3 injections. Diabetes development was monitored to 35 weeks of age. Figure 4, A and B, shows that temporary depletion of B cells by a single cycle of anti-hCD20 treatment significantly delayed the progression of diabetes in both 4- and 9-week-old female mice (P = 0.0002 and P = 0.016, respectively) and also reduced the overall incidence of diabetes in female mice treated at 9 weeks of age. Delay in diabetes in the 4-week-old mice suggests that B cells are important in initiation of islet β cell destruction. The results from the prediabetic 9-week-old mice indicate that B cells play an important role in diabetes progression. Similar results were also obtained in male hCD20/NOD mice, but these were not statistically significant (P = 0.06 and P = 0.07, respectively) as a result of the general phenomenon of later diabetes onset in male NOD mice (data not shown). As expected, anti-hCD20 treatment reduced the production of anti-insulin autoantibody either 2 or 7 months after treatment compared with the control group (Supplemental Figure 5).
Diabetes incidence following 2H7 or control IgG treatment at different ages. (A) Four-week-old and (B) 9-week-old hCD20 female mice were treated 4 times within 10 days with 2H7 (closed squares; n = 12) or control IgG (open squares; n = 12) antibodies and monitored for diabetes.
Effect on established clinical diabetes. To determine whether B cells play a continuing role in established clinical diabetes and are thus a good treatment target, we treated new-onset diabetic (blood glucose ranging from 250 to 500 mg/dl) hCD20/NOD mice within 6 days of diagnosis with anti-hCD20 (n = 14) or control IgG (n = 10) using the same treatment protocol. Diabetic hCD20/NOD mice were given a subtherapeutic dose of insulin soon after diagnosis in order to maintain the animals in a hyperglycemic state but in relatively good general health. Their blood glucose was monitored every 24 (± 1 to 2) hours, and the subtherapeutic dose of insulin was withdrawn if blood glucose was less than 250 mg/dl. Of 14 of the anti-CD20–treated mice, 5 (36%) demonstrated declining blood glucose and required no further insulin treatment, remaining euglycemic for over 2 months after 2H7 treatment (Figure 5A). Four of these 5 mice remained euglycemic for over 130 days. In contrast, none of the IgG-treated mice had a sustained decline of blood glucose, and all required subtherapeutic doses of insulin treatment (Figure 5B, comparison of 5A with 5B; P = 0.03). To investigate the function of islet β cells in the euglycemic mice, we challenged them with a glucose tolerance test via i.p. injection (ipGTT) (at 1.5 mg/g body weight) after fasting overnight. We used newly diagnosed diabetic female NOD mice (n = 5) as controls for dysfunctional islet β cells and young nondiabetic female NOD mice (n = 5) as controls for functional islet β cells. As shown in Figure 5C, the 2H7-treated euglycemic mice were able to regulate their blood glucose to below 250 mg/dl (the diagnostic level) 30 minutes after glucose challenge albeit less effectively than young NOD mice. In contrast, blood glucose in newly diabetic NOD mice remained at high levels 2 hours after glucose challenge, as expected (Figure 5C).
Diabetes reversal following 2H7 treatment. Diabetic hCD20 mice were treated within 6 days of diagnosis with (A) 2H7 or (B) IgG as described in the legend for Figure 3, and their blood glucose was monitored daily. A subtherapeutic dose of insulin was administered, and this was discontinued when the blood glucose level was reduced to less than 250 mg/dl. Seven of the 14 mice treated with 2H7 are represented in A. Five of these mice recovered (3 of which remained euglycemic for more than 120 days and 1 of which was euglycemic for more than 150 days), and 2 mice remained diabetic. Five of the 10 mice treated with IgG are represented in B, none of which recovered from diabetes. The transient reduction in blood glucose seen in some mice was likely to be related to exogenous insulin treatment. The difference between 2H7- and IgG-treated mice was statistically significant (P = 0.03). (C) Three long-term anti-hCD20–treated euglycemic mice in A were challenged with glucose (1.5 mg/g body weight) i.p. Five newly NOD mice used as controls for dysfunctional islet β cells also had an ipGTT performed, and their blood glucose levels before fasting were all above 300 mg/dl. Five young NOD mice were used as controls for normal functional β cells, and their blood glucose levels before fasting were between 93 and 121 mg/dl.
Effect of anti-CD20 treatment on cellular infiltrate of pancreatic islets. Randomly selected mice were sacrificed 1 or 2 months after the end of the treatment period in both the groups initially treated at 4 weeks and 9 weeks. Representative histology for each of these groups is shown in Figure 6A, and insulitis scores in these mice are shown in Figure 6B. There was significantly less infiltration in mice treated with 2H7 compared with the IgG-treated groups at both the 1- and 2-month time points after treatment. At the termination of the experiments, when the mice were over 8 months of age, the histology in the small number of nondiabetic IgG-treated mice was similar to that seen in the nondiabetic 2H7-treated mice. As expected, no obvious differences were detected in the classic NOD histology among frankly diabetic mice in any group (data not shown).
Histology and insulitis scores following 2H7 or IgG treatment. (A) Sections of pancreas illustrating islets taken from euglycemic hCD20/NOD mice at 2 months after treatment with 2H7 (top panels) or IgG antibody (lower panels) administered (Rx) at 4 weeks (left panels) or 9 weeks (right panels) of age. Magnification, ×100. (B) The graph illustrates different insulitis scores following treatment: I, treatment at 4 weeks, observation 1 month after treatment; II, treatment at 4 weeks, observation 2 months after treatment; III, treatment at 9 weeks, observation 1 month after treatment; and IV, treatment at 9 weeks, observation 2 months after treatment. Islets were examined from at least 3 euglycemic mice in each group and insulitis scored in 16–56 islets. When the insulitis scores were compared between 2H7 and IgG treatment within each of the 4 groups, we found P < 0.0001 for each group, which was statistically significant, correcting for multiple comparisons.
Despite euglycemia, insulitis in the cured mice, which were euglycemic for over 4 months after anti-hCD20 treatment, was not strikingly different compared with mice that remained hyperglycemic after anti-hCD20 or control IgG treatment (Supplemental Figure 6). It is possible that the reversal was not permanent and the mice might have been on the edge of “relapsing” to clinical diabetes, as insulitis is likely to be a dynamic process. Alternatively, as insulitis is not necessarily synonymous in other settings with β cell destruction, it is possible that the quality of the insulitis had been altered or that the process was being actively regulated (see below).
Effect of anti-CD20 treatment on induced diabetes development in NOD/SCID mice. The results from both prevention and treatment experiments shown above suggested that 2H7 treatment induced a long-lasting suppression of islet autoimmunity. It seems likely that temporary removal of B cells suppressed the development of diabetogenic T cells and reduced islet autoantibody production by newly generated B cells. It is also possible that short-term depletion of B cells induced immune tolerance, given the fact that diabetes protection and reversal were so durable. To test for development of dominant immune tolerance, we performed adoptive transfer experiments (2, 32). NOD/SCID mice were given 107 NOD splenocytes from diabetic mice i.v., alone or together with 107 splenocytes from 2H7-treated or IgG-treated nondiabetic hCD20/NOD mice, obtained at 35 weeks of age (after they had been fully repopulated with B cells for a few months). We also injected a fourth group of NOD/SCID mice with 107 splenocytes from 2H7-treated nondiabetic mice alone. Diabetes development was monitored for 12 weeks after adoptive transfer. As expected (2, 32), splenocytes from diabetic mice induced diabetes in NOD/SCID recipients around 3 weeks after transfer (Figure 7A). NOD/SCID mice that received splenocytes from diabetic mice together with splenocytes from IgG-treated nondiabetic mice also developed diabetes with similar kinetics (Figure 7A). Strikingly, in mice that received splenocytes from diabetic donors together with splenocytes from anti-hCD20–treated nondiabetic mice, diabetes was substantially delayed (P = 0.007). This indicated that splenocytes from anti-CD20–treated mice that remained nondiabetic had a regulatory effect on diabetogenic spleen cells. Thus, B cell depletion in NOD mice unexpectedly induced cells that can dominantly suppress diabetogenic effector T cells. The results from NOD/SCID recipients that received splenocytes from 2H7-treated nondiabetic mice alone also supported this notion, as none of these recipients developed diabetes (Figure 7A).
Adoptive transfer of diabetes. (A) NOD/SCID mice were injected intravenously with (a) 107 spleen cells from diabetic mice alone (closed circles); (b) 107 spleen cells from diabetic mice cotransferred with spleen cells (107) from nondiabetic IgG-treated mice (open circles); (c) 107 spleen cells from diabetic mice cotransferred with spleen cells (107) from nondiabetic 2H7-treated mice (closed triangles); or (d) spleen cells (107) from nondiabetic 2H7-treated mice alone (open squares). There was a significant delay in the onset of diabetes in the group cotransferred with cells from 2H7-treated mice (P = 0.007). The experiments were performed twice with similar results. Figure 7A shows results of 1 of the 2 experiments. (B). NOD/SCID mice were injected intravenously with (a) 107 spleen cells from diabetic mice alone (filled circles); (b) 107 spleen cells from diabetic mice cotransferred with purified CD4 T cells (3 × 106) from nondiabetic 2H7-treated mice (open triangles); (c) 107 spleen cells from diabetic mice cotransferred with purified splenic B cells (3 × 106) from nondiabetic 2H7-treated mice (open circles); (d) purified CD4 T cells (3 × 106) alone from nondiabetic 2H7-treated mice (closed squares); and (e) purified splenic B cells alone (3 × 106) from nondiabetic 2H7-treated mice (closed triangles). There was a significant delay in the onset of diabetes in the group cotransferred with CD4+ T cells from 2H7-treated mice compared with spleen cells from diabetic mice alone (P = 0.029) and in the group cotransferred with purified splenic B cells compared with spleen cells from diabetic mice alone (P = 0.029). There was no statistical significance between the groups cotransferred with CD4+ T cells or B cells (P = 0.28). The results shown in Figure 7B were from 1 of the 2 sets of experiments.
To further investigate which cell population(s) mediated the dominant suppression of diabetes induction in NOD/SCID mice, we performed the following additional sets of adoptive transfer experiments: (a) NOD/SCID mice were given 107 splenocytes from NOD mice alone or (b) together with purified splenic CD4+ T cells (3 × 106/mouse, n = 3) or (c) B cells (3 ×106/mouse, n = 3) from 2H7-treated nondiabetic mice. As controls, splenocytes from NOD mice were also administered with purified splenic CD4+ T cells (3 × 106/mouse, n = 3) or B cells (3 × 106/mouse, n = 3) from diabetic mice. As further controls, purified splenic B cells or CD4 T cells (3 × 106/mouse) from 2H7-treated nondiabetic mice were also transferred without splenocytes from diabetic mice (n = 3 each group). Since most IgG-treated mice developed diabetes, it was not possible to use nondiabetic purified B cell or CD4 T cell control groups. As expected, NOD splenocytes from diabetic mice alone induced diabetes in 100% of the recipients by 4 weeks after transfer (Figure 7B). In sharp contrast, none of the NOD/SCID recipients developed diabetes after infusion of purified B cells or CD4+ T cells from 2H7-treated nondiabetic mice (Figure 7B). Both CD4+ T cells and B cells from 2H7-treated nondiabetic mice significantly delayed diabetes development in NOD/SCID mice when cotransferred with diabetic NOD splenocytes (Figure 7B; P = 0.029 for both). It is intriguing that B cells expressed a stronger immunoregulatory effect compared with CD4+ T cells (Figure 7B). As expected, CD4 T cells or B cells from diabetic mice did not confer protection (data not shown). Thus, these experiments confirm our initial series of experiments and further establish that either B or T cells from 2H7-treated nondiabetic mice are sufficient to transfer the regulatory effect.
Anti-hCD20 treatment expanded CD4+CD25+Foxp3+ and CTLA4+ T cells. To investigate the immunosuppressive mechanism mediated by CD4+ T cells, we assessed whether the depletion of B cells affected subsets of T cells previously shown to have regulatory activity (Tregs). We used FoxP3 as a marker for Tregs (33–35) along with CD4 and CD25 and analyzed splenocytes of nondiabetic hCD20/NOD mice 6 months after 2H7 or control IgG treatment. There was an increase of CD4_+CD25+FoxP3+_ Tregs in anti-CD20–treated mice compared with CD4_+CD25+Foxp3+_ cells observed in control IgG–treated mice, with a representative plot shown in Figure 8A. We also examined the expression of CTLA4 on CD4_+_ T cells and showed that CD4_+CTLA4+_ cells were also increased in anti-CD20–treated mice compared with control IgG–treated mice (Figure 8B). Interestingly, a fraction of CD4_+CTLA4+_ cells were not positive for CD25 (data not shown), suggesting that temporary removal of B cells may have induced 2 nonoverlapping populations of Tregs.
2H7-treated hCD20 mice have increased CD4 T cells expressing regulatory markers. (A) Splenocytes from hCD20 mice, treated at 9 weeks of age with 2H7 or IgG, were stained with anti-CD4, anti-CD25, and anti-Foxp3 antibodies. A representative flow cytometric plot is illustrated, and percentages shown in the gate represent the Foxp3+CD25+ cells as a percentage of total CD4 T cells. The graph in the middle is a summary of the values obtained from a number of mice. The graph on the right is a summary of the absolute number of CD4+CD25+FoxP3+ T cells. (B). Splenocytes from hCD20 mice, treated at 9 weeks of age with 2H7 or IgG, were stained with anti-CD4 and CTLA4. The percentages shown in the gate represent the CTLA4+ cells as a percentage of total CD4 T cells. The graph in the middle illustrates the values obtained from a number of mice. The graph on the right is a summary of the absolute number of CD4+CTLA4+ T cells.
Increased frequency of transitional B cells in regenerated B cells after anti-hCD20 treatment. To investigate whether the repopulated B cells after anti-hCD20 treatment were different from the original B cells, we examined the phenotype of the regenerated B cells more than 4 months after anti-hCD20 treatment (when the B cell compartment was fully repopulated) compared with control mice. As shown in Supplemental Figure 7, newly generated B cell populations in anti-hCD20–treated mice had an increased frequency of T2 transitional B cells compared with age- and sex-matched control-treated mice. There was no striking difference in plasmablasts in the 2 groups (data not shown).
Anti-hCD20 treatment suppressed antigen presentation of macrophages and splenic DCs. DCs and macrophages are APCs that play an important role in adaptive immune responses. To determine whether the depletion of B cells led to the functional modulation of these 2 types of APCs, we harvested peritoneal macrophages and splenic DCs and generated BM-derived DCs (BMDCs) from anti-hCD20 or control IgG–treated mice 1 month after treatment. Peritoneal macrophages or splenic DCs were used directly ex vivo whereas BMDCs were used after a 5- to 6-day culture in IL-4 and GM-CSF. Islet antigen-specific proliferation counts of BDC2.5 CD4+ splenic T cells or insulin-specific CD8+ T cell clones were used as readouts. As shown in Figure 9, removal of B cells significantly reduced antigen presentation function of macrophages (Figure 9A) and splenic DCs (Figure 9, B and C) in 2H7-treated mice to both CD4+ and CD8+ T cells. The reduction of antigen presentation was also accompanied by a reduction of IFN-γ and IL-17 production (Figure 10, A and B). The suppression appeared to correlate with B cell depletion status, as the suppressed antigen presentation by macrophages or splenic DCs was no longer evident 6 months after anti-hCD20 treatment, when the mice had been completely repopulated with B cells for a long time (data not shown). As expected, removal of B cells by anti-hCD20 treatment did not alter the antigen presentation function of BMDCs (data not shown). The suppression appeared to be specific to antigen presentation function, as the same macrophages or DCs responded well to microbial stimuli (poly I:C, LPS, and CpG), upregulated costimulatory molecules, and produced similar amounts of proinflammatory cytokines compared with macrophages or splenic DCs from IgG-treated mice (data not shown).
Antigen presentation of antigenic peptides to islet autoantigen-specific T cells. (A) Peritoneal macrophages were harvested from hCD20/NOD mice 1 month after 2H7 or IgG treatment and used as APCs after irradiation in a proliferation assay with BDC2.5 CD4 cells responding to BDC2.5 mimotope or 6426 cloned CD8 T cells responding to 9-mer insulin B chain peptide of amino acid position 15–23 (B15–B23). 3H-thymidine incorporation is presented as Δ cpm (cpm in the presence of antigenic peptide minus cpm in the absence of antigenic peptide). Macrophages from 2H7-treated mice presented peptides poorly to both CD4 and CD8 T cells compared with macrophages from IgG-treated mice (P < 0.005). (B) One month after 2H7 or IgG treatment, splenic CD11c+ DCs of hCD20/NOD mice were used as APCs after irradiation. 6426 CD8 cloned T cells were cultured with irradiated DCs in the presence or absence of 9-mer insulin B chain peptide of amino acid position 15–23 (B15–B23) (at 3 μg/ml) in a 3H-thymidine incorporation proliferation assay. (C) Splenic CD11c+ DCs were purified from hCD20/NOD mice 1 month after 2H7 or IgG treatment and used as APCs after irradiation. Purified splenic BDC2.5 CD4 T cells were cultured with irradiated DCs in the presence or absence of BDC2.5 mimotope. IL-2 production was measured by cytotoxic T lymphocyte line (CTLL) assay in culture supernatants after a 48-hour incubation.
Suppressed IFN-γ and IL-17 production by diabetogenic CD4 or CD8 T cells. (A) Peritoneal macrophages were harvested from hCD20/NOD mice 1 month after 2H7 or IgG treatment and used as APCs after irradiation. Purified splenic BDC2.5 CD4 T cells or 6426 CD8 cloned T cells were cultured with irradiated macrophages in the presence or absence of BDC2.5 mimotope and 9-mer insulin B chain peptide of amino acid position 15–23 (B15–B23) (both at 3 μg/ml), respectively. IFN-γ was measured in culture supernatants after a 72-hour incubation. (B) Peritoneal macrophages or splenic CD11c+ DCs from hCD20/NOD mice 1 month after 2H7 or IgG treatment were purified as described. Purified splenic BDC2.5 CD4 T cells were cultured with irradiated macrophages or splenic DCs in the presence or absence of BDC2.5 mimotope. IL-17 was measured in culture supernatants after a 72-hour incubation.