Treatment with anti-CD3 increases the number of CD4⁺ cells that express FoxP3 and GITR. Splenocytes were pooled and analyzed from anti-CD3–treated, HBSS-treated, or untreated animals (n = 2-3/treatment group) 1 week after the final of 4 FVIII immunizations (1 of 3 and 1 of 2 representative experiments for the BALB/c and C57BL/6 mice, respectively, is shown). There is a significant increase in the number of CD4⁺ cells expressing GITR and FoxP3 from anti-CD3–treated BALB/c (A,B) and C57BL/6 (C,D) mice. In addition, the C57BL/6 anti-CD3–treated mice expressed significantly more CTLA-4 (I) and mTGF-β (J) on CD4⁺CD25⁺ cells than the HBSS-treated and untreated mice, but this did not occur in tolerant BALB/c mice (F,G). Furthermore, there was a significant reduction in CD62L on CD4⁺CD25⁺ cells from both the BALB/c (E) and C57BL/6 (H) mice. Anti-CD3 (tol.) and anti-CD3 (nt) refer to anti-CD3–treated C57BL/6 mice inhibitor negative or positive, respectively, 1 week after FVIII immunizations. ns indicates not significant.