The Vα2 CDR1 αY31 side chain contributes to the antigen specificity of multiple Vα2+ T cells. (A) Soluble J809.B5 αY31A or (B) J809.B5 TCR TCRs were analyzed for equilibrium affinity binding to immobilized IAb-FMRKA via SPR. Listed KD are the average of three independent experiments, sensograms are representative analyses. (C) IL-2 production of T cell hybridomas expressing the J809.B5 (filled squares) or J809.B5 αY31A (open circles) TCR in response to titrating concentrations of FMRKA peptide (ng/mL) presented by IAb expressing fibroblasts. IL-2 production of T cell hybridomas expressing the (D, J) J809.G3, (E, K) J809.H1, (F, L) 13.B1, (G, M) 13.D5, (H, N), 14.A6 or (I, O) 14.C6 TCRs (filled squares) or TCRs carrying alanine substitutions at the CDR1 residue αY31 (open circles), in response to (D–I) titrating concentrations of 3K peptide presented by IAb expressing fibroblasts, or (J–O) titrating concentrations of alternative peptide identified using baculovirus display library presented by IAb expressing fibroblasts. IL-2 secretion is reported as Units/mL. Data are the average of three independent experiments. See (Table S2) for EC50 and SEM values.