THE ROLE OF CD4+CD25+ REGULATORY T CELLS IN OPERATIONAL TOLERANCE AFTER LIVING DONOR LIVER TRANSPLANTATION

Abstract

O179 Aims: Recent evidence suggests that CD4+CD25+ regulatory T cells (Tregs) regulate immune responses, including responses to alloantigen in organ transplantation. However, their roles in human liver transplantation (LTx) remain unclear. We have followed a group of liver allograft recipients with good liver graft function who have been weaned off any immunosuppression. We reported that the number of CD4+CD25high T cells in these recipients was higher than that found in healthy age-matched volunteers. The purpose of this study was to determine whether Tregs contribute to the mechanisms of graft acceptance after liver transplantation. Methods: (1) For the functional assay, PBMCs were taken from LTx recipients free from immunosuppression. Whole CD4+ T cells and CD4+ T cells in the absence of CD4+CD25high cells were collected by FACS cell sorter. We confirmed that CD4+CD25high population contained T cells with regulatory activity in suppression assays and by determining the level of expression of FOXP3 mRNA; a transcription factor specific to Tregs. The proliferation of the CD4+ populations against donor and the 3rd party stimulators was examined by [3H]-thymidine uptake (n=7). To determine antigen-specificity of Tregs, serial diluted doses of CD4+CD25+ T cells were co-cultured with CD4+CD25- T cells, and the proliferation responses against donor and 3rd party were examined. (2) The phenotype of PBMCs was analyzed by flow cytometry. The expression level of FOXP3 mRNA was analyzed by real-time PCR. Results: (1) CD4+ T cells from all of the LTx recipients free from immunosuppression, showed hyporesponsiveness to the donor but not 3rd party stimulators. However, even after depletion of the CD4+CD25high population, hyporesponsiveness to the donor was still observed. In 1 out of 3 cases, the suppression exhibited by CD4+CD25+ T cells was more specific to donor antigen (Figure 1). (2) The expression level of FOXP3 mRNA in whole PBMC, and CD4+ T cells was not significantly different between two groups.FigureConclusions: The findings from this study, using in vitro proliferation assays to determine the functional activity and specificity of CD4+CD25high T cells, suggest that donor alloantigen specific regulation by Tregs may contribute to the maintenance of liver graft survival in the absence of immunosuppression in some patients but that in the majority of recipients multiple mechanisms are involved.