Proliferation and cytokine secretion of T-cell clones isolated from subject 17A 19 weeks and 21 months after inhibitor development and from subject 32A at one time point. Resting T-cell clones were stimulated with FVIII_2194-2213 (0.1, 1.0, and 10 μM) presented on irradiated PBMCs from an unrelated DRB1*0101 donor. Proliferation (n = 3) was measured by addition of [³H]thymidine at 48 hours, and cells were harvested 18 hours later (A). Cell supernatants were collected after 48 hours to measure IFN-γ (B), IL-4 (C), and IL-17 (D) by ELISA. The proliferation and cytokine levels of cells stimulated with buffer as a negative control were subtracted. Results are presented as stacked bar graphs. (E-F) The total amounts of cytokines secreted after stimulation with FVIII_2194-2213 at the 3 concentrations indicated in panel A were summed, and ratios of these total levels were calculated. In all panels, clones are grouped according to their cytokine secretion profiles.