Cytokine secretion profile and relative proliferation of splenocytes from cFVIII-tolerized mice after in vitro cFVIII stimulation. (A) Within 48 hours of the last cFVIII challenge, splenocytes were isolated from mice that received temporal vein infusion of either Lenti-HCR/hAAT-cFVIII (n = 3) or Lenti-CMV-cFVIII (n = 3) as neonates, and the cells were cultured in vitro with or without cFVIII. Culture supernatants were evaluated for the presence of IL-6, IL-10, and IFN-γ by ELISA. The bar graphs represent the levels of assayed cytokines. Hemophilia A mice that received Lenti-HCR/hAAT-cFVIII are labeled cFVIII-tolerized, and those that did not receive gene transfer and were not challenged with intravenous infusions of recombinant cFVIII are labeled naive. Mice that were treated with Lenti-CMV-cFVIII and developed inhibitors are labeled inhibitor. Spleens isolated from 3 mice from each group were tested in triplicate with 4 different amounts of cFVIII (0, 0.1, 1.0, and 10 IUs). Results are the mean ± SEM. Significant differences in levels of the various cytokines relative to no FVIII stimulation are incidated: *P < .01. (B) In vitro proliferation analysis of isolated splenocytes cultured in various amounts of cFVIII. Spleens isolated from 3 mice from each group were tested in triplicate with 4 amounts of cFVIII (0, 0.1, 1, and 10 IUs). Results are the mean ± SEM. Significant differences in T-cell proliferation relative to the control are indicated: *P < .01.