Dendritic Cells With TGF-β1 and IL-2 Differentiate Naive CD4+T Cells Into Alloantigen-Specific and Allograft Protective Foxp3+Regulatory T Cells

Background. Naturally occurring, thymic-derived Foxp3+CD25+CD4+ regulatory T cells (nTregs) are pivotal for the maintenance of self-tolerance. nTregs, however, are sparse and lack alloantigen specificity, and these properties pose challenges for their use in clinical transplantation. Methods. We established mixed leukocyte reaction (MLR) with dendritic cells (DCs) as stimulators and CD4+ T cells as responders and supplemented the MLR with IL-2 and TGF-beta 1 and investigated whether DCs + IL-2 + TGF-beta 1 differentiate the polyclonal CD4+ cells into alloantigen-specific and allograft protective Tregs. Results. We found a greater than a 10-fold increase in Foxp3+CD25+ subpopulation (P < 0.01) following stimulation of BALB/c CD4+ cells with C57BL/6 (B6) CD11c+ DCs+IL-2+TGF-beta 1 in the MLR. Levels of TGF-beta 1 messenger RNA (mRNA) (P = 0.01) and the ratios of TGF-beta 1 mRNA to granzyme B mRNA (P = 0.0003) and Foxp3 mRNA to granzyme B mRNA (P < 0.01) were higher in alloantigen-induced Tregs (alloTregs) compared with nTregs. alloTregs suppressed MLR at a 16: 1 responder to suppressor ratio, whereas nTregs suppressed at 4:1. Suppression by alloTregs was alloantigen specific and was observed at the level of responder cells and at the level of stimulator cells. In a fully H-2-mismatched, nonlymphopenic, immunocompetent mouse islet transplantation model, alloTregs but not nTregs prolonged survival of islet allografts without any other immunosuppressive therapy (P = 0.0003), and the protection was alloantigen specific. Conclusions. A combination of CD11c+ DCs, IL-2, and TGF-beta 1 may help differentiate naive, high abundant CD4+ T into alloantigen-specific and allograft protective Foxp3+ Tregs.