In Vivo Effects of Cyclosporine A, Mycophenolate Mofetile and Rapamycin on Adoptively Transferred CD4⁺CD25⁺ Regulatory T Cells in Prevention of Experimental Acute Graft-Versus-Host Disease.

CD4⁺CD25⁺ regulatory T (Treg) cells reduce GVHD without abrogating GVT effects in murine models and hold promise for clinical application. However, in contrast to most clinical situations, Treg have been investigated in models without immunosuppressive drugs. Therefore, we aimed to assess the impact of Cyclosporine A (CSA), Mycophenolate mofetile (MMF) and Rapamycin (RAPA) on Treg function both in vitro and in a lethal acute GVHD model. Gfp⁺ Treg reisolated from mixed leucocyte reactions (MLR) containing CSA but not RAPA showed significantly reduced ability to suppress CD4⁺CD25⁻ T-cell proliferation in secondary MLRs (p=0.025) in a CFSE based assay. The CSA effect could be reversed to 85±3.4% by the addition of IL-2 (50 IU/ml) to the primary culture. In vivo bioluminescence imaging after major mismatch bone marrow transplantation demonstrated reduced early proliferation of donor derived luciferase-labeled conventional T-cells (Tc^luc+) in animals treated with Treg. The addition of RAPA and MMF did not interfere with this suppressive effect. Conversely, combining Treg with CSA led to a significantly increased Tc^luc+ proliferation as measured in photons/second/mouse (p=0.002). Treatment with Treg/CSA was associated with increased GVHD risk and lowest survival of the Treg/drug combinations. Immunofluorescence microscopy of secondary lymphoid organs revealed less donor derived Treg (CD4⁺FOXP3⁺H-2q⁺) per high power field in animals receiving CSA as compared to RAPA or MMF. Our data indicate that CSA, but not RAPA or MMF interferes with Treg function possibly through an IL-2 dependent mechanism. In vivo, the combination of Treg with RAPA or MMF allowed for adequate GVHD suppression and may therefore provide guidance for clinical trials.