T CELL-DEPLETED ALLOGENEIC BONE MARROW TRANSPLANTATION (BMT) INDUCES MIXED CHIMERISM AND DONOR - SPECIFIC TOLERANCE AND PREVENTS DIABETES IN NOD MICE RECEIVING LOW-DOSE (4 GY) TOTAL BODY IRRADIATION (TBI), CD8 T CELL DEPLETION, AND ANTI-CD40LMAB.

Abstract

P662 Background: BMT with sublethal irradiation and anti-CD40L mAb induces full donor chimerism and prevents autoimmune diabetes in NOD mice. We have shown that mixed chimerism can be achieved in NOD mice using non-myeloablative conditioning with low dose TBI, anti-CD40L, anti-CD4, CD8, and Thy 1.2 mAbs, and allogeneic BMT. Autoimmune diabetes is prevented in mixed chimeric NOD mice. We now sought to establish less lymphoablative regimens for the induction of mixed chimerism in NOD mice and examined effects on diabetes onset. Methods: NOD (H-2g7) mice received TBI, anti-CD40LmAb (2mg) and 30x106 B6 (H-2b) BM cells on day 0, and anti-CD8α mAb (0.33mg, 116-31-1; specific for the recipient Ly 2.1 allele) on Days –1, 0, 1, 6, 7, 8. Anti-CD4 mAb and anti-Thy 1.2 mAbs were given when indicated. TBI doses of 4 Gy and 3 Gy TBI were compared (n=5 in each group). Skin grafting (donor, B6; third party, B10.RIII) was performed one day after BMT to assess tolerance. Chimerism in peripheral blood was followed by FCM analysis. Blood sugar levels were monitored (animals were diabetic if 3 consecutive levels were >250 mg/dl). Results: 4 Gy TBI, anti-CD4, CD8α, and Thy 1.2 mAbs with anti-CD40L mAb and BMT allowed induction of high levels of stable (>4 months) multi-lineage mixed chimerism (donor TCR-α/β 97.2±2.5% at 22 wks) and donor-specific tolerance to skin grafts (>100 days) in 5 of 7 mice. Surprisingly, removal of anti-CD4 and Thy 1.2 mAbs led to the development of donor full chimerism and donor-specific tolerance to skin grafts (>100 days) in 3 of 4 mice. 3 Gy TBI with a similar regimen, including anti-CD8 and anti-CD40L mAb and BMT led to full donor chimerism in only 1 of 3 mice. The remaining 2 mice showed no donor chimerism. We then evaluated administration of B6 BMC depleted ex vivo of CD4 and CD8 T cells into NOD mice treated with depleting anti-CD8 mAb, 4 Gy TBI, and anti-CD40L mAb. Administration of untreated BMC led to full donor chimerism, whereas administration of T cell-depleted BMC resulted in stable, multilineage mixed chimerism lasting >6 months. Regardless of the regimen and regardless of whether or not BMT was given, none of the conditioned NOD mice developed diabetes within the period of follow-up. Conclusion: We have established a non-lymphoablative, non-myeloablative regimen (4 Gy TBI, anti-CD40L, and anti-CD8 mAb) in NOD mice that achieves mixed chimerism and donor-specific tolerance from T cell-depleted allogeneic marrow, which avoids the risk of GVHD and prevents autoimmune diabetes. T cell depletion of donor BMC is critical for the induction of mixed, rather than full donor chimerism