CSA but not RAPA or MMF interferes with Treg suppressor function in vivo. Expansion of luc⁺ donor T_CONV cells in animals receiving T_CONV cells alone or T_CONV and Treg cells or T_CONV and Treg cells in conjunction with CSA (10 mg/kg), RAPA (0.5 mg/kg), or MMF (90 mg/kg) as shown for 3 representative animals at days 4, 6, 8, and 14 after BMT (A) and as quantified by emitted photons over total body area at serial time points after BMT (B). GVHD was induced as described in “Materials and methods.” Data from 3 independent experiments are combined. (A) Proliferation of luc⁺ T_CONV (first column from left) is reduced by cotransfer of Treg cells (second column from left). Addition of CSA reduces the suppressive effect of Treg cells (third column from left). In contrast RAPA allows for Treg function (fourth column from left) and MMF has minimal impact (fifth column from left). (B) TCD-BM (▴,n = 15), with T_CONV cells (•,n = 15), with T_CONV and Treg cells (▾,n = 10), with T_CONV and Treg cells and CSA (▵,n = 10), with T_CONV and Treg cells and RAPA (□,n = 10), or with T_CONV and Treg cells and MMF (○,n = 10). Signal intensity is significantly higher in animals receiving T_CONV and Treg cells and CSA as compared to RAPA (▵ versus □, P = .001, day 14) and MMF (▵ versus ○, P = .009, day 14). (C) Percentage survival of Balb/c recipients is significantly lower when combining Treg cells with CSA as compared to RAPA (▵ versus □, P = .001) or MMF (▵ versus ○, P = .007). (D) Expansion of luc⁺ T_CONV cells at day 10 after BMT in the GIT. (Di) Background signal in the GIT of animals having received only TCD-BM without any luc⁺ cells. Proliferation of T_CONV cells in mLNs and the GIT (Dii) is reduced by cotransfer of Treg cells (Diii). Addition of CSA reduces the protective effect of Treg cells (Div). RAPA (Dv) and MMF (Dvi) do not abrogate Treg suppression of T_CONV proliferation in vivo.