Anti-CD3 treatment in vivo increases regulatory CD4⁺CD25⁺ T cells and prevents FVIII inhibitor formation. (A) The spleens from hemophilia A BALB/c mice (n = 5/time point) were analyzed 1, 8, and 15 days after treatment with anti-CD3 (50 μg/day for 5 days) for expression of CD4, CD8, and CD25 (gated on CD4⁺ cells). Untreated and age-matched hemophilia A BALB/c mice (n = 6) were also studied to determine basel T-cell levels. The transient nature of this response may be due to the very short half-life of anti-CD3 F(ab)′₂ fragments (< 1 day).²²  Data are shown as the mean plus or minus SEM. (B) Dose optimization of anti-CD3 therapy in hemophilia A BALB/c mice. The mice (n= 5-8/group) were treated with 10, 25, 50, 100, or 200 μg/day of anti-CD3 for 5 days. Three days after the final anti-CD3 treatment, mice were immunized with 0.2 μg human FVIII weekly for 4 consecutive weeks. The control mice (n = 8) received HBSS, in place of anti-CD3, followed by 4 FVIII immunizations. One week after the final FVIII immunization, plasma was collected and the immune response to FVIII was assessed by the Bethesda assay. Each point in the figure represents the inhibitor titer in an individual animal and the horizontal bars indicate the mean inhibitor titer in each group.