The function of chimeric Tregs is antigen-specific. (A) CD8⁻/CD4⁺/CD25^bright cells were sorted from the spleens of mixed chimeras (B6 → NOD) or naive B6 mice. Sorted Tregs were mixed with NOD lymph node–responder cells in decreasing ratios (1:1, 1:0.25, and 1:0.125) and stimulated with irradiated B6 or NOD-stimulator splenocytes. T-cell proliferation was measured at 5 days. Results are means ± SEM of 3-4 independent experiments. (B) Sorted chimeric Tregs were mixed with NOD lymph node–responder cells in decreasing ratios (1:1, 1:0.25, and 1:0.125) and stimulated with irradiated B10.BR (third-party) stimulator splenocytes. Irradiated B6 stimulator splenocytes serves as a control (supplemental Figure 3). T-cell proliferation was measured at 5 days. Results are means ± SEM of 4 independent experiments. (C) Experimental design for the evaluation of the function of chimeric Tregs in vivo. (D) 10 000 B6 HSCs + 10 000 B10.BR HSCs with or without 100 000 sorted chimeric Tregs or naive B6 Tregs were transplanted into NOD recipient mice conditioned with 950 cGy of TBI in competitive repopulation assays (n = 4-9). PBL typing was performed by staining with anti–H-2K^b, H-2K^k, and H-2K^d mAbs at 30, 60, and 90 days. Analysis of donor (B6 or B10.BR) origin and recipient (NOD) origin were based on lymphoid gate. The bar represents the mean.