Aim: We aim to identify markers that can differentiate activated alloantigen-specific Treg from naïve CD4+CD8−FOXP3+ tTreg. We previously reported that tTreg cultured with alloantigen and IL-2 are induced to express IFN-gamma receptor (IFNGR) and IL-12 receptor β2 and we call these Ts1 cells. Ts1 cells have increased alloantigen-specific suppression of rejection and specific MLC responses. Only a small portion of the tTreg cultured with IL-2 have specific receptor for alloantigen. Here, we examined if the alloantigen-specific Treg were those induced to co-express CD8.
Methods: Naïve CD4+CD8−CD25+Treg from DA rats were cultured with PVG stimulator cells and IL-2 for 3-4d to induce Ts1 cells. 10-30% of cells were induced to co-express CD8. We compared the phenotypes and function of the CD4+CD8+CD25 + Treg to the CD4+CD8−CD25+T cells in vitro and in vivo.
Results: RT-PCR showed an increase in IFNGR and IL-12Rβ2 that characterizes Ts1 cells and was mainly in the CD8+ fraction as was IRF4, a transcription factor induced by TCR-activation. In MLC, the proliferation to alloantigen and IL-2 measured by CSFE was higher in CD4+CD8+CD25+T cells compared to CD4+CD8−CD25+T cells. In MLC, the CD8+ fraction suppressed response to PVG at 1:1012, and to third-party Lewis at 1:32. The CD4+CD8−CD25+T cells had no enhanced suppression to PVG. Unfractionated CD4+CD25+T cells suppressed at 1:32–1:64. In an adoptive transfer assay, IL-2 and alloantigen-activated Treg suppressed graft rejection mediated by naïve CD4+T cells at 1:10 and induce tolerance, whereas CD4+CD8−CD25+FOXP3+T cells at 1:10 did not suppress rejection or induce tolerance. DA rats rejecting PVG grafts or treated to induce tolerance were examined 10d post-transplant, 12-18% of CD4+CD25+T cells expressed CD8 vs <5% in naïve DA. Whole CD25+T cells from both rejecting and tolerance-induced rats suppressed MLC to PVG at 1:256–1:512, and to Lewis at 1:16–1:32, while enriched CD4+CD8+CD25+Treg suppressed PVG at 1:1000 and did not suppress third-party. The CD8− fraction did not suppress.
Conclusions: Our study indicated that CD8 expression on alloactivated CD4+CD8−CD25+FOXP3+Treg is a marker of antigen-specificity. The dual markers (CD4 and CD8) expressing Treg are potent and suppress responses to specific-alloantigen in vivo at <1:100 and in vitro at <1:1000. Sorting CD8 expressing Treg may allow selective expansion of potent alloantigen-specific Treg for immune therapy and transplant tolerance induction.