LOSS OF DONOR ISLET GRAFTS IN DIABETIC NOD MICE WITH A LOW LEVEL OF DONOR CHIMERISM IS MEDIATED BY AUTOIMMUNITY

Abstract

P1207 Aims: A substantial level of donor lymphocyte chimerism is required to protect donor-specific islet grafts in the mouse model with autoimmune diabetes. Donor islet grafts can be rejected in diabetic NOD mice with a low level of donor lymphocyte chimerism. In this study, we investigated whether donor islet graft rejection in diabetic NOD mice with a low level of donor lymphocyte chimerism is mediated by autoimmune or alloimmune response. Methods: Allogeneic BALB/c (H-2d) splenocytes were injected into diabetic NOD mice (H-2g7) at day -3, followed by cyclophosphamide at day -1. BALB/c bone marrow and islets were transplanted simultaneously at day 0. Anti-CD154 mAb and rapamycin were given from day 0 to day 14. Mixed chimerism was measured by flow cytometric analysis. Islet graft function was monitored by measuring blood glucose. After donor islet grafts were rejected, anti-islet autoantigens-specific autoimmune response was determined by adoptive transferring splenocytes from chimeric NOD mice that had rejected donor-specific islet grafts into NOD.scid mice. Alloimmune response was tested by MLR for lymphocyte proliferation and ELISPOT for INF-γ producing cells in vitro.. Results: Mixed chimerism was achieved in all recipient mice. At 8 weeks post-bone marrow transplantation, the level of donor-derived CD3+ T cells was 11.4±7.9%. After donor-specific islet grafts were rejected, 5x106 splenocytes from these chimeric NOD mice with at least over 1% donor CD3+ T cells were adoptive transferred into each NOD.scid mouse. 12 of 15 NOD.scid mice developed diabetes 12 weeks after adoptive transfer. All 8 NOD.scid mice that received splenocytes from control diabetic NOD mice also developed diabetes 5 weeks after adoptive transfer. Proliferation of splenocytes from chimeric NOD mice against donor BALB/c stimulators was markedly reduced, as compared with proliferation of splenocytes from naïve NOD mice (P<0.01). However, the response of chimeric mice to third-party C3H/He was not significantly different from the response of naïve NOD mice. The number of IFN-γ secreting splenocytes significantly increased in the splenocytes of control NOD mice under the stimulation of BALB/c mouse splenocytes. In contrast, the level of IFN-γ-secreting splenocytes in the chimeric NOD mice did not increase and it was the same as the background level under the stimulation of BALB/c mouse splenocytes (P<0.01). The number of IFN-γ-secreting splenocytes in both the chimeric NOD mice and control NOD mice significantly increased when third-party (C3H/He) splenocytes were used as the stimulators, as compared with the naïve splenocytes as the stimulators. Conclusions: These data demonstrate that islet antigen-specific autoreactive T cells exist in diabetic NOD mice with donor lymphocyte chimerism and they are responsible for islet graft loss.