CD3⁺ cells isolated from tolerant (TOL), acutely rejected (AR), and naive nonhuman primates (NHPs) were labeled with CFSE and were cultured with irradiated self, donor cells, or anti-CD2/CD3/CD28 mAbs for 5 days. Naive T cells were cultured with irradiated MHC-mismatched peripheral blood lymphocytes (PBLs). Cultured cells were then stained for CD4 and FOXP3. Representative flow cytometric data (A, anti-donor; C, anti-CD2/CD3/CD28) and mean of % proliferation relative to the response to the self (B, anti-donor; D, anti-CD2/CD3/CD28) are shown. Among these proliferated CD4⁺ cells after donor antigen stimulation, a significant proportion of proliferating CD4⁺ cells was FOXP3⁺ in TOL, while such FOXP3⁺ cell proliferation was minimal in the AR and naive monkeys (A and B). This Treg expansion appeared alloantigen specific as Treg expansion was not observed by polyclonal stimulation with anti-CD2/CD3/CD28 mAbs (C and D). To examine whether Treg expansion is a donor-specific response, CFSE-labeled recipient PBLs from the TOL recipients (n = 7) were cultured with self, donor, and multiple third-party cells for 5 days, after which cultured cells were stained with FOXP3. Significantly greater FOXP3⁺ cell proliferation was observed after MLC with the donor PBLs than following stimulation with third-party PBLs (E). Proliferated cells (%) = proliferated cells (%) with donor, third-party antigens or anti-CD2/CD3/CD28 mAbs – proliferated cells (%) with the self. These assays were performed at 520 ± 86 days in TOL recipients and 175 ± 37 days in AR recipients after bone marrow transplantation. Data are presented as the mean ± SEM. *P < 0.05, ANOVA and the Bonferroni multiple-comparison method was used to test for significant differences among 3 groups; n = 8 (anti-donor) and n = 4 (anti-CD2/CD3/CD28 mAbs) per group. **P < 0.001, n = 7. For analysis of the donor-antigen specificity of Treg expansion, we conducted a 2-way ANOVA with factors of animal (experimental unit) and source of cell (donor or third parties).