Elimination of insulitis and augmentation of islet beta cell regeneration via induction of chimerism in overtly diabetic NOD mice

Abstract

Type 1 diabetes in both humans and nonobese diabetic (NOD) mice results from autoreactive T cell destruction of insulin-producing beta cells. Cure of type 1 diabetes may require both reversal of autoimmunity and regeneration of beta cells. Induction of chimerism via allogeneic hematopoietic cell transplantation has been shown to reestablish tolerance in both prediabetic and diabetic NOD mice. However, it is unclear whether this therapy augments beta cell regeneration. Furthermore, this procedure usually requires total body irradiation conditioning of recipients. The toxicity of total body irradiation conditioning and potential for graft-versus-host disease (GVHD) limit the application of allogeneic hematopoietic cell transplantation for treating type 1 diabetes. Here we report that injection of donor bone marrow and CD4+ T cell-depleted spleen cells induced chimerism without causing GVHD in overtly diabetic NOD mice conditioned with anti-CD3/CD8 and that induction of chimerism in new-onset diabetic NOD mice led to elimination of insulitis, regeneration of host beta cells, and reversal of hyperglycemia. Therefore, this radiation-free GVHD preventive approach for induction of chimerism may represent a viable means for reversing type 1 diabetes.

Fig. 1.

Induction of chimerism in anti-CD3/CD8-conditioned diabetic NOD recipients given donor BM and CD4⁺ T cell-depleted SPL cells. Twelve weeks after HCT, blood mononuclear cells of the recipients were stained with anti-H-2K^q (donor marker), anti-TCRαβ, anti-B220, and anti-Mac-1/Gr-1. One representative is shown of four examined recipients.

Fig. 2.

Induction of chimerism reversed diabetes in new-onset diabetic NOD mice. NOD mice with blood glucose levels higher than 300 mg/dl for 3 consecutive days were diagnosed as diabetic. The new-onset diabetic NOD mice were conditioned with anti-CD3 and anti-CD8 within 3 days after diagnosis, and the late-stage diabetic NOD mice were conditioned 14 days after diagnosis. The diabetic NOD mice were implanted with insulin pellets immediately after diagnosis to temporarily normalize blood glucose. Kinetic changes of blood glucose levels of 24 new-onset diabetic NOD mice given anti-CD3/CD8 conditioning only (A), 20 new-onset diabetic NOD mice given conditioning and HCT (B), and 12 late-stage diabetic NOD mice given conditioning and HCT (C) are shown. Mice with recurrence of hyperglycemia were usually killed by day 70.

Fig. 3.

Increased β cell insulin-secreting capacity and quantity in chimeric NOD recipients with reversal of diabetes. One hundred twenty days after anti-CD3/CD8 conditioning and/or HCT, chimeric NOD recipients and nonchimeric NOD mice with reversal of diabetes, new-onset diabetic NOD mice before treatment, and 3-week-old NOD mice were fasted overnight and then injected i.p. with glucose at a dose of 2 mg/g of body weight. Blood glucose levels were measured before injection and at serial time points after injection (A). Serum insulin levels were measured before injection and 5 min after injection (B). Total insulin-secreting β cell surface and total pancreatic tissue surface of five sections with 75-μm distance of new-onset diabetic NOD mice before (day −5) and after conditioning and/or HCT were measured longitudinally. The percentage of total β cell surface among total pancreatic tissue surface of each mouse was calculated. Mean ± SE of six mice at each time point are shown (C). The nonchimeric diabetic NOD mice had too few residual islets to be detected on days 45 and 60 after conditioning, so their β cell surface at those time points are not shown. Only 7 of 36 nonchimeric new-onset diabetic NOD mice showed reversal of hyperglycemia, and they were used for the day-120 time point; thus, no nonchimeric mice are shown for day 90 or day 200.

Fig. 4.

Induction of chimerism gradually eliminated insulitis in diabetic NOD mice. Severity of insulitis in chimeric recipients given anti-CD3/CD8 conditioning and HCT, or nonchimeric NOD mice given conditioning only, were evaluated longitudinally. (A) Representative histopathology patterns of islets at different time points. (B) Percentage of residual islets with different level of insulitis at different time points. The mean of six examined mice is shown.

Fig. 5.

Proliferation of host residual β cells in chimeric NOD recipients with reversal of diabetes. Chimeric new-onset diabetic NOD mice given anti-CD3/CD8 conditioning and HCT, or nonchimeric new-onset diabetic NOD mice given anti-CD3/CD8 conditioning alone, were daily injected i.p. with BrdU (50 μg/g of body weight) for 2 weeks, and the BrdU⁺insulin⁺ proliferating β cells were measured longitudinally. (A) Representative patterns of BrdU, DNA, and insulin staining 30 days after conditioning and/or HCT. Arrows point to a representative BrdU⁺insulin⁺ β cell. (B) Percentage of BrdU⁺insulin⁺ β cells at various time points. Mean ± SE of six mice in each group at each time point are shown. The residual islets in nonchimeric NOD mice on days 45 and 60 after conditioning were too few to be found, and no nonchimeric NOD mice were available on day 200. (C) Representative patterns of DNA, GFP, and insulin staining of islets from chimeric recipients given BM and CD4⁺ T cell-depleted SPL cells 90–120 days after HCT. Arrows point to GFP⁺insulin⁻ cells. One of six examined recipients is shown.