P230

Intragraft Treg cell-mediated prevention of xeno-islet graft rejection by short term CD40-CD154 blockade treatment

Yoon Il-Hee^1,2,3, Shin Jun-Seop², Kim Yong-Hee^1,2,3, Lee Jae-Il², Min Sang-Il^2,4, Kim Sang-Joon^2,4, Park Chung-Gyu^1,2,3

¹Microbiology and Immunology, Seoul National University College of Medicine; ²Xenotransplantation Research Center, Seoul National University Hospital; ³Cancer Research Institute, Seoul National University College; ⁴Surgery, Seoul National University Hospital; Seoul, Korea

The induction of xenoantigen-specific unresponsiveness remains an elusive goal in islet xenotransplantation. Treg cells are critically involved in the induction and maintenance of transplantation tolerance. In anti-CD154 mAb-induced transplant tolerance setting, Treg cells can be found in tolerant grafts but the relative importance of Treg cells in the grafts versus those in the lymphoid tissues is not known, particularly in the xenogeneic setting. In this study, we examined whether intra-graft Treg cells possess suppressive activity in anti-CD154 mAb-treated mice that exhibit long-term graft survival. Flow cytometry analysis showed that the frequency of intra-graft Foxp3+ Treg cells in anti-CD154 mAb-treated mice was higher than that of hCTLA4-Ig-treated mice. By contrast, in the peripheral lymphoid organs, Foxp3+ Treg cells in anti-CD154 mAb treated mice decreased compared with untreated mice. When CD25+ Treg cells were depleted with anti-CD25 antibody or Treg cells generation was blocked with TGF-beta neutralizing-Ab in anti-CD154 mAb treated mice, islet-graft did not survive and was eventually rejected. In addition, when the lymphocytes from anti-CD154 mAb treated GFP-Foxp3 KI mice were adoptively transferred to porcine islet pre-transplanted Rag2 KO recipients, they prolonged the graft survival. By contrast, the lymphocytes from naïve or hCTLA4-Ig treated mice had no effects on islet graft survival. Moreover, in these Rag2 KO recipients, the number of intra-graft GFP+ Treg cells increased in comparison to those of naive or hCTLA4-Ig treated splenocytes. Finally, to confirm whether intra-graft Treg cells in anti-CD154 mAb treated mice have antigen-specific suppressive function ex vivo, when intra-graft-GFP+ Treg cells were isolated and re-stimulated with porcine islets, they effectively suppressed proliferation of the sensitized splenocytes in antigen-specific manner. In conclusion, we suggest that anti-CD154 induced graft tolerance require intragraft Foxp3+ Treg cells in islet xenotransplantation setting.