We have previously identified peripheral alphabetaTCR+CD3 +CD4-CD8- double negative (DN) T cells as a distinct Treg subset. DN Tregs obtained from allograft-tolerant mice can suppress/kill anti-donor T cells in vitro in an antigen specific manner and prolong donor-specific allograft survival in recipient mice following adoptive transfer. In this thesis, I studied the role of DN Tregs in the prevention of organ xenograft rejection. I used a rat-to-mouse cardiac xenotransplantation model, in which CD4+ T cells play a critical role in rejecting xenografts since CD4+ T cell-deficient (CD4-/-) mice failed to reject rat hearts. I demonstrate that a combination treatment of pretransplant donor lymphocyte infusion (DLI) and a short course of anti-CD4 depleting mAb, but not anti-CD4 mAb alone, induced permanent rat-heart survival in wild type mice. Adoptive transfer CD4+ T cells from DLI/anti-CD4-treated xenograft-accepted mice can induce rejection of rat hearts in CD4-/- mice, indicating the presence of functional anti-donor CD4 + T cells in xenograft-accepted mice. Interestingly, the number of DN Tregs was significantly increased in the secondary lymphoid organs of DLI/anti-CD4-treated xenografted mice when compared to that of anti-CD4-alone treated recipients. DN Tregs isolated from the secondary lymphoid organs of DLI/anti-CD4-, but not anti-CD4-, treated xenografted mice could suppress the in vitro proliferation of anti-donor CD4+ T cells in an antigen-specific manner. In addition, when the number and phenotype of graft-infiltrating cells were compared between anti-CD4- and DLI/anti-CD4-treated groups, I observed a significant increase in both the number and suppressive activity of DN Tregs in the xenografts of DLI/anti-lCD4-treated mice. Thus, DN Tregs may be involved in controlling anti-xenograft CD4+ T cell responses in both secondary lymphoid organs and xenografts. Furthermore, adoptive transfer DN Tregs from xenograft-accepted mice significantly inhibited the in vivo proliferation and cytokine production of transferred naive CD4+ T cells, and prevented transferred CD4+ T cell-mediated rejection of rat cardiac xenografts in CD4-/- mice. These data provide in vivo evidence that DN Tregs are able to control anti-xenograft CD4+ T cell responses. Taken together, I have shown that suppression of anti-donor CD4+ T cells by DN Tregs contributes to the maintenance of xenograft survival.