TCR affinity ranges and human Jurkat cells and primary T cells transduced with the wild-type and affinity-matured TCR constructs. (A) Schematic diagram showing the natural TCR-to-peptide–MHC affinity range, as well as the affinities of the Tax TCR and affinity-matured TCRs to either the pTax peptide or the pHuD peptide and the positions they occupy in the affinity range. Indicated are the dissociation constant K_D of the TCR-HLA/peptide binding and the half-life t_1/2 (in seconds) of the interaction. Jurkat cells (B) or activated primary T cells (C) were transduced with pMP71 vectors encoding the indicated TCR. Jurkat cells were stained with anti-CD3 and Vβ13 antibodies (B) and mock transduced primary T cells or TCR transduced primary T cells were stained with anti-CD3 and CD8 antibodies and either Vβ13 antibodies or tetramer (C) 72 hours after transduction. Primary T cells were gated on the CD3⁺ T-cell population. The Jurkat cell transduction shown is a representative of at least 6 independent experiments that demonstrated similar results. The primary T-cell transduction shown is a representative of at least 20 independent experiments that demonstrated similar results.