Transfusion of ethylene carbodiimide–fixed donor splenocytes prolongs survival of vascularized skin allografts

Allograft rejection is a major obstacle to the widespread clinical application of vascularized composite allotransplantation. Recent studies revealed a noncytoreductive strategy to protect allografts by the transfusion of ethylene carbodiimide–fixed donor splenocytes (ECDI-SPs). To determine whether this approach offers advantages in protecting skin allografts, we examined the immunological protection of infusing ECDI-SPs with a 30-d administration of rapamycin on the skin allografts of mice. C57BL/6 recipient mice received BALB/c donor full-thickness skin or vascularized skin transplants at day 0, along with the infusion of donor ECDI-SPs 7 d before and 1 d after allotransplantation and a 30-d course of rapamycin. Recipients received ECDI-untreated splenocytes or C3H allografts as controls. In vitro allostimulatory activity of ECDI-SPs and donor-specific ex vivo hyporesponsiveness were tested. Production of related cytokines (TGF-β, IL-10, IL-1β, and TNF-α) and expression of CD4+Foxp3+ regulatory T cells (Tregs) were also examined. Transfusion of ECDI-SPs combined with rapamycin significantly prolonged survival of full-thickness skin (median survival time [MST]: 28 d) and full-thickness skin allografts (MST: 71 d) compared with untreated splenocytes (MSTs: 11 d and 30 d) or C3H allografts (MSTs: 11 d and 38 d). This effect was accompanied by increased production of IL-10 and TGF-β, decreased production of IL-1β and TNF-α, and expansion of Tregs in vitro and in vivo. ECDI-SP infusion combined with short-term rapamycin administration provides a promising approach to prolong the skin allograft survival.