CD4⁺T-cell clones did not proliferate when stimulated with rFVIII-C2-F2196A. T-cell clones 32A-18 (A), 17A-19WK-11 (B), 17A-21MO-5 (C), and 17A-21MO-11 (D) were stimulated with purified recombinant rWT-FVIII-C2 and rFVIII-C2-F2196A presented by irradiated PBMCs from an DRB1*01:01 donor. Proteins were added at final concentrations of 1, 10, 50, 100, 500, and 1000 nM. T-cell proliferation was measured by [³H]thymidine incorporation. Results are averages of triplicate determinations ± standard deviation (SD). Stimulation indices were calculated by dividing the mean cpm of [³H]thymidine incorporated into protein-stimulated cells by the mean cpm of [³H]thymidine incorporated into unstimulated cells. Results are also plotted as proliferation (cpm) in supplemental Figure 9.