Chimeric Tregs potently enhance HSC engraftment. CD8⁻/CD4⁺/CD25^bright Tregs were sorted from the spleens of mixed chimeras (B6 → NOD). Sorted 23 000 to 50 000 chimeric Tregs + 10 000 B6 HSCs were transplanted into ablatively conditioned NOD recipients. The chimeric Tregs were harvested 2-5 weeks after transplantation. (A) Percent engraftment in NOD recipients of 10 000 B6 HSCs + chimeric Tregs at 1 month. (B) Percentage of donor chimerism at 1 month in transplanted NOD mice. (C) Facilitative ability of chimeric Tregs administered to NOD mice. CD4⁺/CD25⁺ Tregs were sorted at selected time points: 2-week chimeric Tregs (♦) + 3-week (□), 4-week (▲), or 5-week (●) chimeric Tregs. (D) Representative analysis of FoxP3 expression in CD4⁺/CD25⁺ Tregs in chimeric spleen, thymus, bone marrow, and peripheral blood at 2 or 5 weeks after transplantation. Overlay histogram plots display FoxP3 mean fluorescence intensity (MFI) of the CD4⁺/CD25⁺ cells (black line) in comparison with the isotype control stain (shaded histogram). (E) The percentage of total CD4⁺/CD25^bright cells with FoxP3 expression in 2-week (n = 4) or 5-week (n = 4) chimeric Tregs of spleen, thymus, peripheral blood, and bone marrow. (F) Levels of CD152, CD103, and GITR expression on Tregs. Overlay histogram plots display CD103, GITR, and CD152 MFI of B6 CD4⁺/CD25⁺ cells (black line) in comparison with the isotype control stain (shaded histogram). (G) Overlay histogram plots display CD103, GITR, and CD152 MFI of 5-week chimeric CD4⁺/CD25⁺ cells. (H) Percentage of CD103, GITR, and CD152 expression on B6 CD4⁺CD25⁺ cells or chimeric CD4⁺/CD25⁺ cells. (I) Multilineage PBL typing of NOD recipients of B6 HSCs + 5-week chimeric Tregs. The data are from one representative recipient 3 months after transplantation and were analyzed based on the lymphoid and myeloid gate. Results are means ± SEM.