Generation of enhanced affinity variants of the 3D TCR. (A) Schematic of the 3D TCRα chain CDR3 region showing the location of the mutations that confer higher affinity for WT1 peptide/MHC. (B) 58^−/− T cells (CD8-negative) transduced to express 3D-wt, 3D-NYH, or 3D-PYY TCRs (black line) and the 58^−/− parental line as a control (gray histograms) stained with WT-1/D^b DimerX at 125 nM. (C) WT-1/Db DimerX titration analysis by flow cytometry at 4°C and relative mean fluorescence intensity values were used to generate equilibrium-binding curves. (D) T-cell activation in the presence of WT-1 peptide titrated on T2-D^b cells with 3D-wt, 3D-NYH, and 3D-PYY.