CD4⁺CD25⁺ cells are required in vivo and in vitro for anti-CD3–derived tolerance and mediate suppression through a contact-dependent mechanism. (A) Experimental outline for the in vivo depletion of CD4⁺CD25⁺ cells. Anti-CD25 was administered intraperitoneally on days 0 and 5, whereas anti-CD3 was given intravenously on days 3 to 7. On day 10, mice were killed or were immunized with FVIII weekly for 4 weeks. (B) Raw data demonstrating that the spleen was completely depleted of CD4⁺CD25⁺ cells after injection of anti-CD25 (clone PC61, n = 3) or isotype control (IgG1, n = 3) during anti-CD3 treatment (10 μg/day for 5 days). Splenocytes from untreated aged-matched mice are shown for reference (n = 3). (C) Quantification of the effect of anti-CD3/anti-CD25 on CD4⁺ and CD4⁺CD25⁺ populations as shown in panel B. Anti-CD3, as shown in previous experiments, can deplete CD4⁺ cells, and this is clearly evident in the isotype control and anti-CD25 group compared with untreated mice. An increase in CD4⁺CD25⁺ cell levels is also evident in the anti-CD3–treated isotype control mice. However, despite anti-CD3 treatment, which increases CD4⁺CD25⁺ cell levels, anti-CD25 almost completely depleted this population. (D) Evaluation of FVIII inhibitor formation after depletion of CD4⁺CD25⁺ cells. During anti-CD3 treatment (10 μg/day for 5 days), mice were treated with anti-CD25 (n = 6) or isotype control antibody (n = 6), which was followed with 4 FVIII immunizations as previously described. The subsequent immune response to FVIII was analyzed by the Bethesda assay 1 week after the final FVIII immunization. Tolerance to FVIII was completely abrogated in the anti-CD25 mice, but not in the isotype controls. (E) CD4⁺CD25⁺ or (F) CD4⁺CD25⁻ cells isolated from tolerant anti-CD3–treated mice were cocultured, in the presence of FVIII, with total splenocytes from FVIII-immunized, HBSS control animals (no anti-CD3 therapy) in the same well (coculture, CC), or were separated by a transwell membrane (transwell, TW). Data are mean plus or minus SEM.