Biobreeding (BB) rats model type 1 autoimmune diabetes (T1D). BB diabetes-prone (BBDP) rats develop T1D spontaneously. BB diabetes-resistant (BBDR) rats develop T1D after immunological perturbations that include regulatory T cell (Treg) depletion plus administration of low doses of a TLR ligand, polyinosinic-polycytidylic acid. Using both models, we analyzed CD4+CD25+ and CD4+CD45RC- candidate rat Treg populations. In BBDR and control Wistar Furth rats, CD25+ T cells comprised 5-8% of CD4+ T cells. In vitro, rat CD4+CD25+ T cells were hyporesponsive and suppressed T cell proliferation in the absence of TGF-beta and IL-10, suggesting that they are natural Tregs. In contrast, CD4+CD45RC(-) T cells proliferated in vitro in response to mitogen and were not suppressive. Adoptive transfer of purified CD4+CD25+ BBDR T cells to prediabetic BBDP rats prevented diabetes in 80% of recipients. Surprisingly, CD4+CD45RC-CD25- T cells were equally protective. Quantitative studies in an adoptive cotransfer model confirmed the protective capability of both cell populations, but the latter was less potent on a per cell basis. The disease-suppressing CD4+CD45RC-CD25- population expressed PD-1 but not Foxp3, which was confined to CD4+CD25+ cells. We conclude that CD4+CD25+ cells in the BBDR rat act in vitro and in vivo as natural Tregs. In addition, another population that is CD4+CD45RC-CD25- also participates in the regulation of autoimmune diabetes.

Treatment of mice with a single donor-specific transfusion plus a brief course of anti-CD154 mAb uniformly induces donor-specific transplantation tolerance characterized by the deletion of alloreactive CD8+ T cells. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. To analyze the mechanisms underlying tolerance induction, maintenance, and failure in euthymic mice we created a new analytical system based on allo-TCR-transgenic hemopoietic chimeric graft recipients. Chimeras were CBA (H-2(k)) mice engrafted with small numbers of syngeneic TCR-transgenic KB5 bone marrow cells. These mice subsequently circulated a self-renewing trace population of anti-H-2(b)-alloreactive CD8+ T cells maturing in a normal microenvironment. With this system, we studied the maintenance of H-2(b) allografts in tolerized mice. We documented that alloreactive CD8+ T cells deleted during tolerance induction slowly returned toward pretreatment levels. Skin allograft rejection in this system occurred in the context of 1) increasing numbers of alloreactive CD8+ cells; 2) a decline in anti-CD154 mAb concentration to levels too low to inhibit costimulatory functions; and 3) activation of the alloreactive CD8+ T cells during graft rejection following deliberate depletion of regulatory CD4+ T cells. Rejection of healed-in allografts in tolerized mice appears to be a dynamic process dependent on the level of residual costimulation blockade, CD4+ regulatory cells, and activated alloreactive CD8+ thymic emigrants that have repopulated the periphery after tolerization.