Proliferation and cytokine secretion of subject GS1-56A T cells in response to FVIII_2194-2213. Three T-cell clones and 1 line (10 000 cells) were stimulated with FVIII_2194-2213 or irrelevant peptide (FVIII_2178-2197) at concentrations from 0.01 to 100 μM presented by 100 000 irradiated PBMCs from a DRB1*01:01-typed donor. Forty-eight hours later, 50 μL of cell supernatant was removed from each well for cytokine analysis and replaced with 1 μCi [³H]thymidine. Cells were harvested after 18 hours of further incubation, and [³H]thymidine incorporation was measured. Cell supernatants in quadruplicate wells were pooled for measurement of IFN-γ, IL-4, IL-17A, and IL-21 by sandwich ELISAs using purified recombinant cytokines to generate standard curves. (A) Proliferation results are expressed as averages of quadruplicate determinations ± standard deviations. Proliferation in response to the FVIII_2178-2197 peptide was negligible when compared with proliferation in the absence of peptide. Background proliferation levels: 56A-C2, 171.0 ± 69.9 counts per minute (cpm); 56A-C8, 84.2 ± 10.9 cpm; 56A-C15, 99.4 ± 7.2 cpm; and 56A-L2, 209.0 ± 61.4 cpm. (B) IFN-γ secretion levels. (C) IL-4 secretion levels. (D) IFN-γ:IL-4 ratios. IL-17A and IL-21 secretion were not detected.