Donor lymphocyte infusion induces long-term donor-specific cardiac xenograft survival through activation of recipient double-negative regulatory T cells

Previous studies have shown that pretransplant donor lymphocyte infusion (DLI) can enhance xenograft survival. However, the mechanism by which DLI induces xenograft survival remains obscure. Using T cell subset-deficient mice as recipients we show that CD4⁺, but not CD8⁺, T cells are necessary to mediate the rejection of concordant cardiac xenografts. Adoptive transfer of naive CD4⁺ T cells induces rejection of accepted cardiac xenografts in CD4^-/- mice. This rejection can be prevented by pretransplant DLI in the absence of any other treatment. Furthermore, we demonstrate that DLI activates αβ-TCR⁺CD3 ⁺CD4^-CD8^- double-negative (DN) regulatory T (Treg) cells in xenograft recipients, and that DLI-activated DN Treg cells can inhibit the proliferation of donor-specific xenoreactive CD4⁺ T cells in vitro. More importantly, adoptive transfer of DLI-activated DN Treg cells from xenograft recipients can suppress the proliferation of xenoreactive CD4⁺ T cells and their ability to produce IL-2 and IFN-γ in vivo. Adoptive transfer of DLI-activated DN Treg cells also prevents CD4 ⁺ T cell-mediated cardiac xenograft rejection in an Ag-specific fashion. These data provide direct evidence that DLI can activate recipient DN Treg cells, which can induce donor-specific long-term cardiac xenograft survival by suppressing the proliferation and function of donor-specific CD4⁺ T cells in vivo.