TGEM of CD4 T cells from subject GS1-56A using DRB1*01:01 tetramers loaded with pooled FVIII peptides. CD4 T cells were stimulated with pools of peptides having overlapping sequences spanning the FVIII-A2, C1, and C2 domains and then incubated with DRB1*01:01 tetramers loaded with the corresponding peptide pools on day 14. As a positive control, cells from the same subject were stimulated with peptides corresponding to known DRB1*01:01-restricted TT epitopes³⁷ : TT_585-605, TT_666-685, and TT_674-693. Plots show binding of CD3⁺ lymphocytes to APC-anti-CD4 IgG and PE-tetramer. The name of the peptide pool used for staining is indicated above each plot. The cutoff used for selecting pools for staining using individual peptides (decoding) was 1.0% CD4⁺Tetramer⁺ cells. These populations are circled. Pseudocolor plots with percentages of cells in each quadrant were created with FlowJo v10.0.7.