SPECIFICITY AND FUNCTION OF CD4+ CD25+ REGULATORY T CELLS IN A NOVEL CD4+ TCR-TRANSGENIC MODEL OF ALLOIMMUNITY

Abstract

O180 Aims: CD4+ CD25+ regulatory T cells (Tregs) play a critical role in controlling peripheral allograft tolerance. However, it remains controversial whether Tregs can account for the alloantigen-specificity of the tolerant state. In addition, the pathways through which Tregs mediate immunosuppressive effects remain uncertain. Methods: We used the ABM TCR-transgenic (tg) system, a model of direct alloantigen presentation in which >90% of CD4+ T cells carry a tg TCR that directly recognizes the mutated MHC-II molecule I-Abm12. 5-6% of ABM TCR-tg CD4+ T cells constitutively express CD25, as well as high levels of Foxp3, CTLA-4 and CD103 transcripts, and exhibit anergy and suppressive effects upon stimulation with bm12 splenocytes in vitro. Results: To examine the immunosuppressive properties of ABM Tregs in vivo, ABM CD4+ CD25- (Teffs) were adoptively transferred together with either wild type polyclonal (wt) Tregs or ABM Tregs at a 1:1 ratio into syngeneic recipients of bm12 skin grafts. The median survival time (MST) of bm12 skin grafts after adoptive transfer of 1 x 105 ABM Teffs alone was 10 days. Wt Tregs did not prevent rapid rejection of bm12 skin grafts by ABM Teffs (MST=12 days), whereas ABM Treg significantly prolonged skin graft survival (MST=40, p=0.02). Next, to examine the alloantigen-specificity of ABM Tregs in vivo, wt Teffs were adoptively transferred with ABM Tregs at a 1:1 ratio into syngeneic recipients of either bm12 or BALB/c skin grafts. The MSTs of bm12 and BALB/c skin grafts after adoptive transfer of 1 x 105 wt Teffs were 13 and 26 days respectively. ABM Tregs suppressed rejection of bm12 skin grafts by wt Teffs (MST=20 days, p=0.02). In contrast, ABM Tregs did not prevent rejection of BALB/c skin grafts by wt Teffs (MST=30 days). ABM Tregs demonstrated higher expression levels of PD-L1 compared with ABM Teffs. To examine the role of PD-L1 on ABM Tregs in vivo, ABM TCR-tg T cells containing both ABM Teffs and Tregs populations were adoptively transferred into syngeneic recipients of bm12 skin grafts, and PD-L1 was blocked using anti-PD-L1 mAb. After PD-L1 blockade the number of TCR-tg T cells present in the draining lymph nodes was markedly increased (p=0.02 vs untreated controls). In addition, apoptosis of TCR-tg T cells was inhibited (p=0.03 vs. untreated controls). However, the administration of anti-PD-L1 mAb had no effect when only ABM Teffs were transferred. Conclusions: Our results indicate that the regulatory function of CD4+ CD25+ T cells is dependent on TCR-specific stimulation. These data favor a model in which the preferential activation of alloantigen-specific CD4+ CD25+ T cells during transplantation tolerance induction would account, at least in part, for the striking specificity of the tolerant state. In addition, our data indicate that Tregs may mediate their immunosuppressive effects at least in part through PD-L1.