Markedly reduced proliferation of CD4⁺T-cell clones and polyclonal lines to BDD-FVIII-F2196K and BDD-FVIII-M2199A. FVIII_2194-2213-specific T-cell clones 32A-18 (A), 17A-19WK-11 (B), 17A-21MO-5 (C), and polyclonal lines 17A-5YR-L1 (D) and 56A-L2 (E) were stimulated with purified recombinant WT-BDD-FVIII, BDD-FVIII-F2196K, and BDD-FVIII-M2199A presented by iMo-DCs. The iMo-DCs were differentiated from CD14⁺ cells isolated from an DRB1*01:01 donor and pulsed with FVIII proteins at final concentrations of 1.6, 3.1, 6.2, 12.5, 25, and 50 nM. CD4⁺ T-cell proliferation was measured by [³H]thymidine incorporation. Stimulation indices were calculated by dividing the cpm of [³H]thymidine incorporated into protein-stimulated cells by the cpm of [³H]thymidine incorporated into unstimulated cells. Results are also plotted as proliferation (cpm) in supplemental Figure 10.