Effects of extended anti-ICOS treatment in mice after hFVIII plasmid transfer.hFVIII plasmid-treated mice (n = 9) were injected intraperitoneally with anti-ICOS 16 times during a 4-week period. (A) Persistent high levels of FVIII were expressed in mice as shown by hFVIII activity. (B) No inhibitors developed in mice as evaluated by Bethesda assay. (C) Absence of in vitro proliferation of CD4⁺ T cells isolated from tolerized mice in response to stimulation with hFVIII. Eight weeks after hFVIII plasmid transfer and anti-ICOS treatment, spleen CD4⁺ T cells were isolated and stimulated with hFVIII or PHA or cultured without stimulation for 72 hours in the presence of irradiated APCs. A total of 1 μCi of ³H-thymidine per well was added for the last 18 hours of culture. Spleen CD4⁺ T cells from mice treated with anti-ICOS alone, hFVIII plasmid alone, and untreated naive mice were used as controls. Data are presented as the mean counts per minute plus or minus SD by subtracting data obtained from T cells only in the absence of APCs and are representative of 2 separate experiments. *P < .05, **P < .01, compared with groups indicated by arrows.