Immune tolerance to cFVIII is associated with a cFVIII-expandable population of CD4⁺CD25⁺Foxp3⁺ T cells. (A) A representative flow cytometric analysis obtained with CD4⁺ T cells, derived from the spleens of hemophilia A mice that received Lenti-HCR/hAAT-cFVIII as neonates (cFIII-Tolerized) or did not receive gene transfer and were not challenged intravenously with cFVIII (Naive) and that were challenged with FVIII. Isolated splenocytes were stained directly (Pre-cFVIII) for CD4⁺CD25⁺Foxp3⁺ or after 72 hours in vitro stimulation with cFVIII (Post-cFVIII). (B) Comparison of the mean percentage of CD4⁺CD25⁺Foxp3⁺ T cells within the CD4⁺ cell population from the tolerized and naive mice before and after in vitro cFVIII stimulation. Error bars represent SEM, and * indicates a significant (P < .01) increase in CD4⁺CD25⁺Foxp3⁺ T cells.