Residual Treg cells affect in vitro and in vivo regulatory function. (A) B6-wt mice were conditioned with 9.0 Gy TBI and 1 day later transplanted with TCD-BMC from B6-CD4^−/− donors. Approximately 1 month (day 32) posttransplant, CD4⁺CD25⁺ T cells were purified from the spleens and lymph nodes of 2 recipients, pooled (0.5 × 10⁶ Tregs/mouse), and assessed for regulatory activity. CD4⁺CD25⁺ T cells were also purified from normal, nontransplanted B6-wt mice for comparison. Various numbers of the CD4⁺CD25⁺ T-cell populations were cocultured in triplicate with syngeneic B6 CD4⁺CD25⁻ responder T cells plus accessory cells together with anti-CD3 mAb. Cultures were pulsed with ³Tdr for the final 6 hours of incubation and harvested after 72 hours. Data are presented as the percentage inhibition based on the cpm of cultures with Treg cells versus cpm of cultures composed of responder cells, accessory cells, and anti-CD3 mAb without Treg cells. (B) B6-wt and B6-Rag2^−/−γc^−/− (H2^b) recipient mice were conditioned with 9.0 Gy on day −1 and 1 day later (day 0) transplanted with 3 × 10⁶ TCD-BM from B6 IL2Rβ^−/− donors. Spleens were harvested from recipient animals 2 months post-BMT and analyzed for CD4 and CD8 T cells. In addition, CD4 T cells were analyzed for CD62L and CD44 expression. Elevated CD4/CD8 ratios and an activated CD4 T-cell phenotype are present in recipients expressing clinical signs of autoimmune disease. Data represent analysis of 3 individual B6-Rag2^−/−γc^−/− mice and a pool of 3 normal B6-wt recipients.