Anti-CD4 mAb is effective in inducing tolerance following sensitization. (A) C57Bl/6 mice were sensitized with hFVIII-alum on days 1 and 14 and sacrificed on day 30. Leukocytes from spleen, pooled lymph nodes, and bone marrow were collected and adoptively transferred intravenously into congenic Rag2^−/− or C57Bl/6 recipients. The recipient mice were then treated with hFVIII-alum plus anti-CD4 mAb (or an isotype control) on the day of cell transfer and 14 days later (consistent with protocol represented in Figure 1A), and all groups of mice were subsequently challenged with 3 × 1 U hFVIII on days 10, 12, and 14 following the last administration of anti-CD4. (B) Quantification of serum hFVIII-specific IgG1 in Rag2^−/− recipient mice. Animals that received primed T and B cells were competent to produce anti-hFVIII IgG1 (n = 5; ***P < .001), but antibody production was abrogated in mice treated with anti-CD4 (n = 5; ***P < .001). (C) Concentration of serum hFVIII-specific IgG1 in C57Bl/6 mice transferred with primed T and B cells. Animals adoptively transferred with primed cells produced higher levels of hFVIII-specific IgG1 than animals that did not received primed cells (n = 5; ***P < .001). Treatment with anti-CD4 prevented production of hFVIII-specific IgG1 by the primed lymphocytes (n = 5; ***P < .001). Data are representative of 2 independent experiments. (D) C57Bl/6 mice were sensitized with hFVIII on days 1, 5, 10, and 15 and treated with hFVIII-alum and anti-CD4 (intraperitoneally or subcutaneously) on days 22 and 36. Control mice received anti-CD4 without hFVIII. One control group had not been previously exposed to hFVIII. All mice received hFVIII intravenously on days 54, 56, 64, 66, 74, and 76. Anti-hFVIII IgG1 was quantified on the indicated days. Mice treated with hFVIII-alum plus anti-CD4 (regardless of the route of administration) were protected from production of anti-hFVIII IgG1 following subsequent exposure to hFVIII (n = 6; ***P < .001). ip, intraperitoneal; N.D., not detected; sc, subcutaneous.