RETENTION OF REGULATORY ACTIVITY IN CD4+CD25+ T-CELLS ISOLATED FROM RENAL TRANSPLANT RECIPIENTS FOLLOWING EX-VIVO EXPANSION:

O247* Aims: Emerging evidence suggests that donor alloimmune responses mediated via the indirect pathway contribute to ongoing tissue injury in chronic allograft nephropathy. Recently active regulation of the indirect pathway alloimmune response by a sub-population of CD4+ cells co-expressing the IL2 receptor α chain, CD25 has been demonstrated in experimental and clinical studies. We sought to develop a protocol for the expansion and characterization of CD4+CD25+ T-regulatory (Treg) cells ex vivo. Methods: CD4+CD25+ T cells were purified by magnetic bead positive selection from peripheral blood samples of renal transplant recipients. Cells from 9 patients were expanded ex vivo for three, two weekly, cycles in the presence of IL-2, plate-bound anti-CD3 and irradiated syngeneic feeder cells previously incubated with synthetic peptides corresponding to the hypervariable regions of the β chain of the donor HLA-DR molecule(s) to which the transplant recipient was mismatched. Following the first expansion cycle the cell population was further enriched for the Treg phenotype by flow sorting the cells to acquire cells co-expressing high levels of CD25. Results: Retention of the regulatory phenotype was confirmed at time points of up to six weeks. Triple color flow cytometry examining the expression of molecular markers of regulatory activity including the negative co-stimulatory molecule CTLA4, GITR, CD45RO and OX40 demonstrated a progressive increase in staining intensity that paralleled that of CD25 suggesting enrichment of the regulatory phenotype in the CD25hi population. Quantitative PCR confirmed higher expression levels of FoxP3, a key gene in the development of regulatory function in CD4 cells expressing high levels of CD25 compared to ex-vivo expanded cells that had either lost CD25 expression or expressed it only at low levels. CD4CD25hi cells efficiently suppressed the proliferative responses of PBMCs to a combination of anti-CD3 and anti-CD28 confirming retention of a regulatory activity following expansion ex-vivo. Conclusions: The successful expansion of CD4+CD25+ Treg will greatly enhance our ability to further define the cell phenotype and its functional characteristics in vitro and in vivo as a prelude to possible cellular therapy for the management of CAN, the induction of tolerance and/or the minimization of the immunosuppresive burden in renal transplant recipients.