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Roles of B cells and Tregs in anti-CD45RB induced transplant tolerance to islet xenografts

Gaoping Zhao^1,2, James Kim¹, Kang Mi Lee¹, Matthew O’Connor¹, Maozhu Yang^1,2, Ji Lei¹, Christian Schuetz¹, James Markmann¹, Shaoping Deng^1,2

¹Surgery, Massachusetts General Hospital, Boston, MA, United States; ²Surgery, Sichuan Provincial People’s Hospital, Chengdu, Sichuan, People’s Republic of China

Background: Transplantation of xenogenic islet cells with induction of tolerance represents an attractive alternative for the treatment of diabetes. In thisstudy we investigated the ability of anti-CD45RB in inducing tolerance to islet xenograft and the rolesof B cells and T regulatory cells(Tregs)in tolerant induction and maintenance.

Methods: Isolated human islets were transplanted under the kidney capsule of B6 diabetic mice. The recipients were treated with anti-CD45RB(100μg ip. on days 0, 1, 3, 5, 7 post transplant). To assess whether anti-CD45RB-induced transplant tolerance requires B cells, some B6 recipients received additional anti-CD20 treatment or B6μMT^-/- micewere used. Adaptive Treg generation by anti-CD45RB treatment was examined in vivo and in vitro. Tregs were identified in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by FCM and immunohistochemistry. For some anti-CD45RB-treated B6μMT^-/- mice, additional anti-CD25 mAb was applied at the early stage or late stage post transplant.

Results: anti-CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice, however100% of xenografts were accepted in mice treated simultaneously with anti-CD20, and 8 of 9 xenografts accepted in μMT^-/- micetreated with anti-CD45RB. Grafts were removed from these tolerant μMT^-/- mice by nephrectomy and mice were re-transplanted with second transplantof islets from either another human donor or baboon donor: five of 5 human islets were accepted and 3 of 3 baboon islets were rejected rapidly. In vitro, CD4+CD25-Foxp3-T cells which were activated with anti-CD3/anti-CD28 beads and cultured with anti-CD45RB demonstrated significant increase in Foxp3+ expression. In vivo, an increase of Foxp3+ T cells was observed in treated mice and more significant increase in tolerant mice, compared with a negligible increase in rejected mice. Additional administration of anti-CD25 mAb at the time of transplantation resulted in 100% rejection, whereas none of grafts was rejected while the antibody was administrated at days 60 post transplant. Adoptive cell transfer showedthatthe CD4+CD25+ T cells from the mice tolerant to the first human islets prolonged survival ofthe second human islets(p < 0.05).

Conclusions: These data suggest that simultaneously targeting host B cells can facilitate the anti-CD45RB induced transplant tolerance to islet xenograft, andthis transplant tolerance is species-specific, likely estabolished by the formation of antigen-specific Tregs.