Activation of 17195 T effectors by FVIII-2191-2220. (A) Proliferation of 17195TCR-transduced CD4 T cells with FVIII-2191-2220. Prestimulated primary CD4 T cells were transduced with 17195TCR or with a mock vector and maintained for 10 days in culture media supplemented with IL-2. For proliferation assays, cells were labeled with cell proliferation dye (eFluor 450) and then reactivated with irradiated DR1-PBMCs plus peptide FVIII-2191-2220 (0.5 μg/mL), pOVA (0.5 μg/mL), or anti-CD3ε antibody (0.5 μg/mL) for 4 days. Cell proliferation was measured by flow cytometry using a standard dye dilution assay. Representative results are shown for CD4 cells from 2 different donors that were transduced with 17195TCR. (B) Staining of transduced, expanded GFP⁺ 17195 T effectors shown in A using PE-labeled DR1 tetramers loaded with FVIII-2191-2220. (C) Proliferation of GFP⁻ and GFP⁺ T cell effectors stimulated with OVA peptide, FVIII-2191-2220, or anti-CD3ε antibody. Transduced T effectors (2 × 10⁶ per well) were stimulated using irradiated DR1-APCs (Responder:Stimulator = 1:5) as in A. After culturing for 10 days, the cells were harvested, and viable GFP⁻ and GFP⁺ CD4 cells were counted using flow cytometry.