Evaluation of CD4⁺ and CD4⁺CD25⁺ populations in BALB/c and C57BL/6 anti-CD3–treated mice after FVIII immunizations. 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). The number of CD4⁺ cells is significantly reduced in the anti-CD3–treated BALB/c (A) and C57BL/6 (C) mice after FVIII immunizations, whereas the CD4⁺CD25⁺ populations are increased in both the BALB/c (B) and C57BL/6 (D) mice, although to a much greater extent in the latter. 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.