Novel Consistently Ex-vivo Expanded CD8+CD25+FoxP3+Treg Cells Prolongs the Allogeneic Islet Survival

Abstract

Abstract In addition to CD4+ regulatory T cells(Tregs), adoptive transfer of CD8+CD25+FoxP3+T cells is emerging as an alternative adjunctive therapy to diminish current reliance on lifelong, nonspecific immunosuppression after transplantation. Here we evaluated the possible therapeutic application of consistently ex-vivo expanded mouse CD8+CD25+FoxP3+T cells to prevent the rejection of allogeneic islets. Around 80% of mouse CD8+CD25+T and CD4+CD25+T cells expressed FoxP3+T cells in spleen. Higher level of FoxP3 was expressed in CD8+Tregs compared to CD4+Tregs. Ex-vivo enriched CD8+CD25+Treg cells were successfully and consistently expanded to 600–1,000 times from the initial culture for 12 days with optimized culture conditions. High concentration of IL-2 (>500units) and TGF-beta together with aCD3-and aCD28 stimulation were essential to the massive ex-vivo expansion. Expanded Tregs showed 99% of FoxP3 expression and expressed high level of CD27, CD47 and Lag-3 in their surface. Very high amount of Granzyme B and minor amount of IFN-r and IL-17 were xpressed. Expanded CD8+Tregs successfully inhibits the proliferation and activation of CD4+T cells in vitro. In the allogenic islet transplantation model (Balb.Cà B6), Single injection of ex-vivo expanded CD8+Tregs prolonged islet allograft survival more than 100 days after the reconstitution with CD90.2+T cells in immunodeficient B6 rag2(−/−) mice by inhibiting antigen-specific IFN-r producing cell generation. In this study, we proved that ex-vivo expanded CD8+Treg retain regulatory activity and can protect islet allograft rejection by single administration. These implies that ex-vivo expanded CD8+Tregs has clinical relevance of an adjunctive cellular therapy.