Tetramer staining of T-cell clones and lines isolated from severe HA subject GS1-56A and mild HA subject GS1-17A. (A) Staining of T-cell clones from subject GS1-56A with the DRB1*01:01-FVIII_2194-2213 tetramer, DRB1*01:01-FVIII_2186-2205 tetramer, and DRB1*01:01-FVIII_2178-2197 tetramer. The clones shown are representative of those with high-, medium-, and low-avidity binding to the DRB1*01:01-FVIII_2194-2213 tetramer. (B) Staining of 2 T-cell lines from subject GS1-56A with the same tetramers in panel A. (C) Staining of 4 T-cell lines from subject GS1-17A with the DRB1*01:01-FVIII_2194-2213 tetramer. Polyclonal lines from both subjects were isolated by sorting of 250 cells labeled with the PE-DRB1*01:01-FVIII_2194-2213 tetramer. All plots show binding of CD3⁺ lymphocytes to anti-CD4 IgG (FITC label panels A and B, APC label panel C) and PE-tetramer. Pseudocolor plots with percentages of cells in each quadrant were created with FlowJo v10.0.7.