Naked plasmid transfer of hFVIII plasmids into hemophilia A mice with short-term anti-ICOS treatment either alone or in combination with CTLA4-Ig or antimurine CD40L antibody (MR1). A total of 50 μg of the plasmid in 2 mL of saline solution was injected into the tail vein of hemophilia A mice (n = 4) in 5 to 8 seconds. Immunosuppressive agents were administered intraperitoneally. Anti-ICOS mAb was administrated 8 times in a 2-week period, CTLA4-Ig was injected 2 times at day 0 and 2, and anti-CD40L mAb was administered 5 times over a 2-week period. Circulating hFVIII activity was evaluated in plasma at regular intervals by a modified clotting assay (A,C,E,G) and confirmed by a chromogenic COATEST assay. Inhibitory antibody titers were evaluated by Bethesda assay and expressed as Bethesda units/mL (B,D,F,H). (A,B) No immunosuppression. (C,D) Anti-ICOS mAb alone. (E,F) Anti-ICOS + CTLA4-Ig. (G,H) Anti-ICOS + anti-CD40L mAb. The respective set of figures (A,B, C,D, E,F, and G,H) show data from the same experiment, and an individual mouse was represented by the same symbol in a set.