Kinetics of Alloantigen-Specific Regulatory CD4 T Cell Development and Tissue Distribution After Donor-Specific Transfusion and Costimulatory Blockade

Background. The influence of donor-side regulation toward recipient antigens on graft outcome is poorly understood. Methods. Because this influence might be due in part to the accumulation of tissue-resident memory T cells in the donor organ, we used a standard murine tolerization model (donor-specific transfusion plus CD40L blockade) to determine the kinetics of development and peripheralization of allospecific regulatory T cell in lymphoid tissues and liver, a secondary lymphoid organ used in transplantation. Results. We found that donor-specific transfusion and CD40L blockade leads to a progressive and sustained T regulatory allospecific response. The cytokines IL10, TGF beta, and IL35 all contributed to the regulatory phenomenon as determined by trans vivo delayed hypersensitivity assay. Unexpectedly, an early and transient self-specific regulatory response was found as well. Using double reporter mice (forkhead box p 3 [Foxp3]-yellow fluorescent protein, Epstein-Barr virus-induced gene 3 [Ebi3]-TdTomRed), we found an increase in Foxp3+CD25+ regulatory T (Treg) cells paralleling the regulatory response. The Ebi3+ CD4 T cells (IL35-producing) were mainly classic Treg cells (Foxp3+CD25+), whereas TGF beta+ CD4 T cells are mostly Foxp3-negative, suggesting 2 different CD4 Treg cell subsets. Liver-resident TGF beta+ CD4 Tcells appeared more rapidly than Ebi3-producing Tcells, whereas at later timepoints, the Ebi3 response predominated both in lymphoid tissues and liver. Conclusions. The timing of appearance of donor organ resident Treg cell subsets should be considered in experiments testing the role of bidirectional regulation in transplant tolerance.