Abrogation of GVHD is associated with a decreased expansion of CD4⁺ effector T cells. Irradiated WT and RelB^−/− BMCs were transplanted with BM from Balb/c (CD45.2⁺) donors in combination with T cells from congenic Balb/c (CD45.1⁺) donors, and spleens and thymi were harvested and dissociated 10 days after BMT. (A) Splenocytes were stained with mAb against H2D^b and H2D^d to examine engraftment. (B) Splenocytes and thymocytes were stained with CD3, CD45.1. and CD4; CD3 T cells were gated and examined for CD4 and CD45.1 expression. Splenocytes were stained with CD3, CD45.1, CD4, and Foxp3, and (C) CD45.1 expression by FoxP3⁺ Treg cells was examined and (D) CD4⁺ T effector (CD3⁺CD4⁺FoxP3^neg) and CD4⁺ T_reg cells were enumerated **P < .01; WT (n = 6) versus RelB^−/− BMCs (n = 6). (E) The percentage of CD4⁺FoxP3⁺ and CD4⁻FoxP3⁺ cells in WT and RelB^−/− allograft recipients 10 days after BMT. **P < .01. Data represent mean ± SEM from individual animals. (F) Suppressive function (as described in “Materials and methods”) of day 10 ex vivo sort-purified CD4⁺CD25⁺ T_reg cells from WT or RelB^−/− BMCs recipients. (G) Four-color flow cytometry was used to phenotype and enumerate splenic FoxP3⁺ T cells (number in upper right quadrants represent percentage of CD4 or CD8 populations which were FoxP3⁺).