CSA administration is associated with reduced numbers of CD4⁺FoxP3⁺cells and has only minor impact on the expansion of donor-derived CD4⁺CD25⁺cells. Expansion of luc⁺ donor Treg cells in Balb/c mice (H-2k^d) receiving T_CONV and Treg cells (both H-2k^q) alone or T_CONV and Treg cells along with CSA (10 mg/kg), RAPA (0.5 mg/kg), or MMF (90 mg/kg) as shown for representative animals on day 7 after BMT (A) and as quantified by emitted photons over total body area at serial time points after BMT (B). (A) Expansion of luc⁺ Treg (first column from left) is slightly reduced by conjunction of CSA (second column from left) and to a lesser extent by MMF (first column from right). Addition of RAPA (third column from left) has no impact on Treg expansion. (B) TCD-BM (^*, n = 10), 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 decreased in animals receiving T_CONV and Treg cells and CSA or MMF as compared to RAPA (▵ versus □, and ○ versus □, NS). (C) Splenic CD4 donor T cells (H-2k^q) were investigated for FoxP3 expression in Balb/c recipients on day 7 after BMT, having received T_CONV and Treg cells alone (first column from left) in conjunction with CSA (second column from left), RAPA (third column from left), or MMF (fourth column from left). The percentage of FoxP3⁺ Treg cells is significantly reduced in the presence of CSA as compared to PBS injection (P = .005) or RAPA (P = .004).