Treatment with CD20-specific antibody prevents and reverses autoimmune diabetes in mice

Abstract

The precise roles of B cells in promoting the pathogenesis of type 1 diabetes remain undefined. Here, we demonstrate that B cell depletion in mice can prevent or delay diabetes, reverse diabetes after frank hyperglycemia, and lead to the development of cells that suppress disease. To determine the efficacy and potential mechanism of therapeutic B cell depletion, we generated a transgenic NOD mouse expressing human CD20 (hCD20) on B cells. A single cycle of treatment with an antibody specific for hCD20 temporarily depleted B cells and significantly delayed and/or reduced the onset of diabetes. Furthermore, disease established to the point of clinical hyperglycemia could be reversed in over one-third of diabetic mice. Why B cell depletion is therapeutic for a variety of autoimmune diseases is unclear, although effects on antibodies, cytokines, and antigen presentation to T cells are thought to be important. In B cell-depleted NOD mice, we identified what we believe is a novel mechanism by which B cell depletion may lead to long-term remission through expansion of Tregs and regulatory B cells. Our results demonstrate clinical efficacy even in established disease and identify mechanisms for therapeutic action that will guide design and evaluation of parallel studies in patients.

Figure 1. 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.

Figure 2. 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).

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.

Figure 4. 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.

Figure 5. 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.

Figure 6. 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.

Figure 7. 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.

Figure 8. 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.

Figure 9. 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.

Figure 10. 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.