Treg cells limit cytotoxic alloreactive immune responses in the setting of MC. (A) Irradiated host HLA-DR⁺ APCs were cocultured with responder T cells isolated posttransplant. Cells were cocultured at a ratio of 1:1 for 5 days and then the number of residual live cells was determined by propidium iodide and Accu-Chek counting beads. An index of in vitro cytotoxicity was then defined as the percentage of remaining stimulator host HLA-DR⁺⁺ cells divided by the percentage of remaining responder T cells. (B) Ratio of host HLA-DR⁺ cells: post-HSCT T cells, 1.3: 1 in the context of FC compared with 5:1 in the context of MC (*P = .011), indicating that T cells from patients with MC are less able to eliminate HLA-DR⁺⁺ APCs. The mean and standard error are shown, and analysis was undertaken with a 2-tailed Mann-Whitney U test (FC, n = 11; MC, n = 12). (C) Coculture assays were reestablished from patients with MC following depletion of CD25⁺ cells (Treg cells) before in vitro culture. The ratio of host HLA-DR⁺⁺ cells to T cells was significantly decreased, indicating that Treg cells act directly to suppress donor alloreactive cytotoxicity in the context of MC (*P = .0312). The mean and standard error are shown; analysis was undertaken with a Wilcoxon matched pairs signed rank test (n = 7).