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Mechanisms by which donor-specific blood transfusion (DSBT) promotes organ allograft acceptance are unclear. In a rat fully mismatched cardiac allograft model, we found that DSBT alone (without immunotherapy) induces the development of regulatory T cells (DSBT-Tregs) posttransplant, thereby shedding new light in the mechanisms of the transfusion effect. Compartments and timing of expansion, requirements, and phenotype of DSBT-Tregs are unknown. It is generally assumed that some time is necessary before Tregs develop. However, we show-by adoptive transfer from DSBT-tolerant into naive recipients: 1) the presence of DSBT-Tregs at 5 days posttransplant in spleen and lymph nodes; 2) their gradual expansion in these compartments; and 3) their presence in the graft 14 of 30 days posttransplant. DSBT-Tregs are donor specific and do not protect third-party allografts. Splenocytes from DSBT-treated nontransplanted recipients or from transplanted DSBT-untreated (rejecting) recipients do not transfer tolerance, indicating that both DSBT and graft are required for sufficient numbers of DSBT-Tregs to develop. Thymectomy (or splenectomy) before DSBT (not at transplantation) abrogate DSBT-Tregs generation and tolerance, showing that thymus (and spleen) are required for DSBT-Tregs generation (not for expansion/maintenance). In contrast with other Tregs models, DSBT-Tregs activity is not restricted to CD4(+)CD25(+) but to CD4(+)CD45RC(-) cells, whereas CD4(+)CD45RC(+) cells act as effector cells and accelerate rejection. In conclusion, DSBT alone induces-rapidly posttransplant-the development of alloantigen-specific Tregs in lymphoid tissues and in the graft. DSBT, graft, thymus, and spleen are required for DSBT-Tregs generation. DSBT-Tregs in this model are CD4(+)CD45RC(-) (identical to Tregs protecting from autoimmunity in rats).

Early Presence of Regulatory Cells in Transplanted Rats Rendered Tolerant by Donor-Specific Blood Transfusion