Low-Dose Cyclosporin A Does Not Abrogate the Suppressive Function of In Vitro Expanded Human CD4⁺CD25⁺ Regulatory T Cells.

Graft-versus-host disease (GVHD) is a major complication after allogeneic stem cell transplantation caused by donor T cells in the stem cell graft. Prophylactic treatment of GVHD usually comprises immunosuppressive medication including cyclosporin A (CsA) that blocks calcineurin phosphatase activity and thereby inhibits nuclear factor of activated T cells (NFAT)-mediated cytokine production. CD4⁺CD25⁺ natural regulatory T (Treg) cells have been shown to suppress the proliferation and cytokine secretion of conventional CD4⁺ and CD8⁺ T cells in vitro and to protect from GVHD lethality in murine models of allogeneic BMT. As the adoptive transfer of human CD4⁺CD25⁺ Treg cells as a means of GVHD prophylaxis is currently under investigation, we aimed to explore the influence of CsA on survival, cytokine production and suppressive activity of this T cell subpopulation. To this end, in vitro expanded human CD4⁺CD25^high and CD4⁺CD25⁻ T cells were stimulated with anti-CD3 and anti-CD28 antibodies and simultaneously exposed to various concentrations of CsA. While T cell death occurred in both in vitro expanded subpopulations at high CsA concentrations of 1000 ng/ml, with only 10 to 20% viable cells after 72h exposure, CD4⁺CD25^high T cells showed significantly enhanced resistance to lower CsA concentrations of 100 ng/ml with still approx. 60% viable cells, as compared to only 20% viability in expanded CD4⁺CD25⁻ T cells. At this lower concentration, CsA also blocked the proliferation of the two subpopulations in response to anti-CD3/CD28 stimulation, as determined by CFSE-dilution, and inhibited the IL-2 and IFN-γ production of CD4⁺CD25⁻ T cells as well as the IL-10 production by expanded CD4⁺CD25^high Treg cells, as shown by intracellular staining after stimulation with PMA/ionomycin. Addition of exogenous IL-2 protected both subpopulations from CsA-induced cell death and restored their proliferative capacity but was unable to abrogate the block in cytokine production. Surprisingly, the suppressive activity of in vitro expanded CD4⁺CD25^high Treg cells was neither affected by a pre-incubation of the cells with 100 ng/ml CsA, nor by its presence during the suppression assay. These results indicate that in vitro expanded CD4⁺CD25⁺ Treg cells are less sensitive to CsA-induced apoptosis and that their yet unknown mechanism of suppression does not involve the calcineurin pathway.