In vivo depletion of host CD25⁺ cells enhances CFU-IL3 after transplantation of syngeneic BMCs. (A) Lymph node cells (3-4 mice/group) from control rat IgG– and anti-CD25–treated (PC61 mAb) BALB/c (H2^d) mice (1 mg intraperitoneally at days −4 and −2) were stained on day 0 for CD4 (PerCp) and CD25 (PE, clone 7D4) followed by anti-Foxp3 (FITC) intracellular staining. In comparison with rat IgG–treated control mice (A top panels), the CD4⁺ cells of lymph nodes from anti-CD25 mAb–treated mice (A bottom panels) exhibited few Foxp3⁺ Tregs. Results are representative of 2 independent experiments demonstrating a decrease in Foxp3 levels in CD4⁺CD25⁺ Tregs in anti-CD25–treated mice. (B) BALB/c (H2^d) mice were treated with control rat IgG or anti-CD25 mAb as described in panel A (3-4 mice/group) and were administered lethal TBI on day 0 and escalating doses of syngeneic TCD-BMCs (3-4 mice/group). Splenic CFU-IL3 were assessed on day 7. Triplicate cultures were established and the results presented as the average total CFU-IL3/spleen ± SD (ANOVA), after multiplying CFU frequency by the total number of nucleated splenocytes. Enhanced BM engraftment occurred after the depletion of host CD4⁺CD25⁺ FoxP3⁺ Tregs. One of 2 experiments is presented. (C) Depletion of CD25⁺ cells in recipients undergoing HSCT with donor B6-wt or B6-MHC class II^−/− marrow differentially affects CFU-IL3 progenitor cell levels early after transplantation. B6 mice were prepared and treated with antibodies as in panel A, and then administered lethal TBI on day 0 (9.5 Gy) and escalating doses of B6-wt or B6-MHC class II^−/− TCD-BMCs (3-4 mice/group). Splenic CFU-IL3 were assessed on day 7. Enhanced CFU-IL3 occurred after transplantation of B6-wt but not MHC class II–deficient TCD-BMC inoculum. P values illustrate groups that exhibited statistically significant (P < .05) differences.