CD4⁺ and CD4⁺CD25⁺ T lymphocytes from hFVIII plasmid plus extended anti-ICOS–treated mice exert dominant tolerance after adoptive transfer. (A,B) A total of 5 × 10⁶ CD4⁺ cells, 1 × 10⁶ CD4⁺CD25⁻ cells, or 1 × 10⁶ CD4⁺CD25⁺ cells from tolerized mice 4 weeks after gene transfer were adoptively transferred into naive hemophilia A mice. The recipient mice were subsequently challenged with hFVIII plasmid 1 day after adoptive transfer. (A) hFVIII activity and (B) anti-hFVIII antibodies were examined in recipient mice at day 14 after adoptive transfer of indicated cell populations isolated from tolerized mice. (C,D) 5 × 10⁶ CD4⁺ cells were obtained from donor mice 8 weeks after gene transfer and adoptively transferred into naive hemophilia A mice. The recipient mice were then challenged with hFVIII plasmid 1 day after adoptive transfer. (C) hFVIII activity and (D) anti-hFVIII antibodies were measured in recipient mice at day 7 and day 14 after adoptive transfer of total CD4⁺ T cells from mice treated as indicated.