Effect of systematic alanine substitutions in FVIII_2194-2205on T-cell proliferation of clones. WT and Ala-substituted FVIII_2194-2205 peptides were added to 5 T-cell clones isolated from 3 HA subjects, incubated with irradiated PBMCs from a donor with an DRB1*01:01 allele as antigen-presenting cells. For clones 32A-18 (A), 17A-19WK-11 (B), 17A-21MO-5 (C), and 17A-21MO-11 (D) each peptide was added at final concentrations of 0.01, 0.1, 1, 5, 10, 50, and 100 μM, cpm of [³H] thymidine incorporated was measured, and EC₅₀ values were calculated. Clone 56A-C2 (E) was stimulated with peptides at 0.05, 0.1, 0.5, 1, and 10 μM final concentrations. The curves that resulted from these data were not sufficient to calculate accurate EC₅₀ values, therefore, cpm in response to 10 μM peptide was plotted. Results are expressed as relative T-cell proliferation (EC₅₀ or cpm of FVIII_2194-2205/EC₅₀ or cpm of the indicated Ala-substituted peptide) ± SE. The EC₅₀ values for clonal responses to FVIII_2194-2205 were 5.7 ± 0.9 μM (A), 48.2 ± 2.1 μM (B), 10.5 ± 0.9 μM (C), and 52.5 ± 3.1 μM (D). Note that the WT peptide already contains an Ala at position 2201. These 5 clones had distinct TCR sequences and were previously shown to bind tetramers with high avidity and to proliferate in response to FVIII_2194-2213 (Table 2). Although the binding affinities of FVIII_2194-2213 and FVIII_2194-2205 to rDRB1*01:01 protein were almost identical, the T-cell clones proliferated more robustly in response to FVIII_2194-2213 compared with FVIII_2194-2205 (supplemental Figure 8). Cytokine analysis of supernatants from stimulated clones is in supplemental Figure 1.