Donor CD4⁺CD25⁺ T cells increase long-term multilineage chimerism levels. B6-GFP TCD BM (1 × 10⁶) was transplanted alone or with 1 × 10⁶ CD4⁺CD25⁺ T cells into 7.0-Gy irradiated BALB/c recipients. Peripheral blood was harvested and flow cytometry was performed to determine chimerism 1, 2, and 6 months after BMT by expression of GFP and MHC class I (n = 7-16 recipients; A). □ indicates BM only; ○, BM + CD4⁺CD25⁺. Data presented are pooled from 6 experiments. Horizontal bars indicate the average percent of positive staining of donor MHC class I for each group. MHC class I staining was also used to determine chimerism 1 month after BMT in bone marrow, lymph nodes, and spleens of transplant recipients (n = 3/group from one of the individual experiments included in panel A) (B). Light gray bars indicate BM only; dark gray bars, BM + CD4⁺CD25⁺. Multilineage donor chimerism was assessed 6 months after BMT in recipients who received transplants of B6-GFP BM and B6 CD4⁺CD25⁺ T cells by splenic expression of GFP, CD8, and CD4 and by expression of MHC class I, B220, and Mac-1 in peripheral blood (C). ▦ indicates spleen; ▪, blood. Data represent pooled results from individual mice (n = 3-9/group) obtained from 2 of the experiments included in panel A. Error bars are mean % chimerism and SEM from individual mice (B,C).