CD25+CD4+ REGULATORY T CELLS GENERATED IN VIVO PREVENT ISLET ALLOGRAFT REJECTION BY BLOCKING THE RECRUITMENT OF EFFECTOR CELLS INTO THE GRAFT

Abstract

O178* The concept of CD25+CD4+ regulatory T cells (T-reg) is well established in various autoimmune and transplantation models and a growing body of evidence suggests that operational transplantation tolerance depends on active T cell regulation mediated by these T-regs. Previously, we have demonstrated that T-reg generated in vivo prior to transplant by pre-treatment with donor-specific blood transfusion (DST) plus anti-CD4 monoclonal antibody can regulate donor-specific allograft rejection in a CTLA-4 and IL-10 dependent manner. Aim: The aim of this study was to ask whether T-reg generated by the same protocol are capable of preventing the rejection of a life-sustaining islet allograft, and if so, explore the mechanisms involved. Methods: T cell depleted CBA (H-2k) recipients were rendered diabetic by streptozotocin (blood glucose >20 mmol/L). These mice were then reconstituted with 1x105 CD45RBhigh CD4+ effector cells from naïve mice with or without co-transfer of 4x105 CD25+CD4+ from cell donors tolerized with nondepleting anti-CD4 antibody on days –28 and –27 plus a C57BL/6 (H-2b) DST on day –27. The naïve CD45RBhigh CD4+ cells used as effectors in this model co-express human CD52 as a transgene which allows them to be distinguished phenotypically from putative T-regs following adoptive transfer. On day 0, reconstituted, diabetic animals were transplanted with 400 C57BL/6 islets under the kidney capsule. Graft function was assessed by daily blood glucose measurements and rejection was defined as a blood glucose level ≥14.5 mmol/L on two consecutive days. Cellular infiltration was quantified by immunofluorescence using anti-hCD52-FITC. Four separate planes were analysed with an average of 4 high power fields (hpf) examined per plane. Results and Conclusion: Mice reconstituted with CD45RBhigh CD4+ effector cells alone rejected their grafts acutely (MST=18, n=7). This was completely prevented by co-transfer of CD25+CD4+ T cells isolated from tolerized animals (MST>70, n=10). These animals remained normoglycemic (8-13 mmol/L, normal range 8-12 mmol/L). In the absence of CD25+CD4+ T cells, CD45RBhigh CD4+ effector cells rapidly infiltrated the graft site and accumulated until day 15. However, in the presence of CD25+CD4+ T cells, at day 5 there was a 7-fold decrease in the number of infiltrating effector cells (CD45RBhigh only 3.66 ± 0.33; CD45RBhighplus CD25+ 0.47 ± 0.15 cells/hpf, p<0.0001; syngeneic control 0.57 ± 0.2). To our knowledge, this is the first demonstration that CD25+CD4+ T cells generated in vivo by immunomodulation can protect a life-sustaining, functional primary islet allograft. The effect of regulation on the recruitment of effector cells into the graft provides an insight into one of the mechanisms in which regulatory T cells can prevent allograft rejection and confirms that the generation of CD25+CD4+ regulatory cells should be considered as an important strategy for providing operational tolerance in clinical transplantation.