Requirements for Prolongation of Allograft Survival with Regulatory T Cell Infusion in Lymphosufficient Hosts

Background For the clinical applicability of regulatory T cells (Tregs) in transplantation, it is critical to determine if donor antigen specificity is required for their immunosuppressive function. We developed an allospecific CD4 + T cell receptor transgenic (TCR-tg) mouse as a source for large numbers of Tregs with defined allospecificity and tested whether they are more effective than polyclonal Tregs at suppressing allograft rejection. Materials and Methods CD4 + CD25 + CD62L hi T cells were sorted from the spleen and peripheral lymph nodes of wild-type (WT-Tregs) and TCR-tg (Allo-Tregs) mice, and expanded using IL-2 and anti-CD3/anti-CD28 conjugated magnetic beads. Tregs were tested for their ability to suppress the proliferation and cytokine production of alloreactive CD4 + CD25 − T cells in mixed leukocyte assays. Syngeneic WT hosts were adoptively transferred 5 × 10 6 Tregs and transplanted with allogeneic hearts. Results Using anti-CD3/anti-CD28 conjugated beads, Tregs were expanded in vitro 100-fold and maintained their suppressor phenotype and function. Allo-Tregs were 6–8 times more potent on a cell-for-cell basis than WT-Tregs in suppressing allospecific proliferation in vitro . Allo-Tregs were unable to suppress in the absence of allo-antigen. Adoptive transfer of expanded Allo-Tregs into WT recipients prolonged the graft survival in a F1 heart transplant model compared with WT-Treg or no treatment [20.0 ± 4.4 d ( n = 6) versus 10.4 ± 1.2 ( n = 8) and 9.7 ± 1.6 d ( n = 6)]. Conclusions Unlike polyclonal Tregs, allospecific Tregs are able to prolong allograft survival. However, large numbers of Allo-Tregs were unable to induce tolerance, suggesting that Treg therapy in immunocompetent recipients will require conditioning and/or additional immunomodulation for the induction of tolerance.