Decoding of GS1-56A pooled-peptide tetramer-positive staining results. CD4 T cells stimulated with FVIII-C2-pool 1 peptides (A) or with positive control DRB1*01:01-restricted TT peptides (B) were stained on day 18 using tetramers loaded with the corresponding peptide pool and with the individual peptides comprising the pool. Plots show binding of CD3⁺ lymphocytes to APC-anti-CD4 IgG and PE-tetramer. The name of the peptide pool or individual peptide is indicated above each plot. Pseudocolor plots with percentages of cells in each quadrant were created with FlowJo v10.0.7. Decoding identified strong HLA-DRB1*01:01-restricted T-cell responses to FVIII_2194-2213 and to TT_586-605. Decoding of A2-pool 4 and C1-pool 3 tetramer-positive staining results (Figure 1) indicated both were because of nonspecific binding (supplemental Data).