We used our adhesion frequency assay19,20 (Fig. 1a), which analyzes cross-junctional interactions upon initial brief cell-cell encounters, to quantify the 2D binding between HLA-DR1 presenting a melanoma antigenic peptide derived from the glycolytic enzyme triosephosphate isomerase (TPI:HLA-DR1, referred hereafter as pMHC, unless specified)21 and its cognate E8 TCR, wild-type (WT) CD4, or a high-affinity mutant (MT) CD4 generated via directed evolution7. Here, TPI1 is linked to melanoma.