Highly enriched Treg cell preparations inhibit lineage-depleted bone marrow from syngeneic wild-type but not MHC class II^−/− mice. (A) CD4⁺CD25⁺ T cells from B6-CD8^−/− spleen and lymph node cells were prepared by initially depleting B cells with anti–mouse IgG and IgM Abs. Remaining cells were labeled with anti–CD4-CYC and anti–CD25-PE. Cells were separated on a FACSAria. Postsort analysis indicated enrichment at 99.7% (A inset). These cells were activated for 3 days as described. Bone marrow populations from B6-wt and B6-MHC class II^−/− mice were depleted of T cells and cocultured with these activated Tregs. B6-wt (P < .05) but not B6-MHC class II^−/− cultures containing Tregs showed diminished CFU-IL3 levels. After 1.5 days of coculture, cells were harvested and plated for CFU-IL3. (B) Tregs obtained from the same highly enriched preparation as in panel A were cocultured with lineage-depleted B6-wt and B6-MHC class II^−/− BMCs as in panel A. To more stringently lineage deplete the marrow populations, the Miltenyi Biotec lineage depletion kit (biotinylated mAb cocktail) was initially used, and the remaining cells were labeled with streptavidin-FITC and anti–c-kit-PE mAb. After isolation by cell sorting (FACSAria), the FITC⁻PE⁺ fraction was > 99.6% for the 2 bone marrow populations (B insets). B6-wt (P < .001) but not B6-MHC class II^−/− cultures with Tregs exhibited decreased CFU-IL3 numbers. CFU-IL3 were performed as described.