Differential Effects of Calcineurin and Mammalian Target of Rapamycin Inhibitors on Alloreactive Th1, Th17, and Regulatory T Cells

Background. Previously, we had reported the role of tacrolimus (TAC) versus sirolimus (SRL) on the generation of regulatory T cells (Tregs) in primary MLR assays with SRL, demonstrating a uniquely supportive effect. However, the mechanisms associated with their actions on alloreactive human T cells are not fully understood. Therefore, we tested whether TAC and SRL differentially affect already alloactivated human CD4(+) T-cell subsets. Methods. Alloreactive CD4(+)CD45RA(-)/CD45RO(+) T cells generated in 9-day MLR were cocultured with anti-CD3 and autologous antigen presenting cells plus interleukin (IL)-2 in presence of TAC, SRL, or both, and the Tregs generated after another 5 to 6 days were phenotypically, molecularly, and functionally characterized. Results. Tacrolimus significantly and SRL modestly inhibited interferon (IFN)-gamma (Th1) and IL-17 (Th17)-producing cells. At clinical therapeutic concentrations, SRL, however, significantly increased forkhead/winged helix transcription factor P3 (FOXP3(+)) Tregs, whereas TAC inhibited this T-cell population dose dependently and significantly. When used in combination, TAC and SRL had additive effects on inhibition of IFN-gamma- and IL-17-producing cells. This was in contrast to the ability of SRL to reverse TAC-mediated inhibition of FOXP3-expressing cells. Proinflammatory cytokines (IL-1 beta, IL-6, and tumor necrosis factor-alpha) added to cultures caused significant decrease in FOXP3(+) Tregs that was again reversed by SRL. Sirolimus-derived Tregs were phenotypically normal, anergic to allostimulation, and suppressed proliferation of allogeneic effector T-cells. Conclusions. Thus, although TAC inhibits all alloreactive T cells, SRL promotes the differentiation and expansion of donor-specific Tregs without secondary reprogramming to IFN-gamma(+)FOXP3(+) and IL-17(+)FOXP3(+) Treg subsets. These results, although performed in an artificial in vitro model, add clinically applicable information on how these agents affect T-cell subpopulations.