TCL responses to heat-stressed but not to nonstressed HX575, EPO epitopes, and skewed TCR Vβ usage. (A) TCLs reactive to heat-stressed HX575 were generated from 2 HX575-naïve healthy HLA-DRB1*09:01-negative or -positive donors (donor 2 and donor 8, respectively). Three HX575-reactive TCLs were derived from each of these 2 donors (triangle: donor 2; rhombus: donor 8), which showed pronounced proliferative responses of the TCLs to heat-stressed but not to nonstressed HX575. (B-C) Identification of HX575 T-cell epitopes in donors 2 and 8, by testing TCL reactivity to 31 synthetic EPO peptides (15- or 16-mers) with 10-residue overlaps (Table 2); ctrl = heat-stressed HX575; a specific T-cell response shown as SI was calculated by a ratio of cell proliferation with individual stimuli divided by the cell proliferation with vehicle control; data presented as mean ± SEM; average responses of 3 heat-stressed HX575-specific TCLs from HLA-DRB1*09:01-negative donor 2 (B); average responses of 3 heat-stressed HX575-specific TCLs from HLA-DRB1*09:01-positive donor 8 (C). (D-G) Representative flow cytometric plots showing gating of CD4 T cells, and showing staining for 24 TCR-Vβ families; (D) TCL YS21 derived from HLA-DRB1*09:01-negative donor 2, showing single TCR Vβ22⁺ usage (to peptide 27); (E) TCL YS34 derived from HLA-DRB1*09:01-negative donor 2, showing single TCR Vβ8⁺ usage (to peptide 27); (F) TCL TH38 derived from HLA-DRB1*09:01-positive donor 8, showing single TCR Vβ2⁺ usage (to peptide 7); (G) TCL TH39 derived from HLA-DRB1*09:01-positive donor 8, showing single TCR Vβ13.2⁺ usage (to peptide 10). FSC, forward scatter; SEM, standard error of the mean; SSC, side scatter.