Supplementary Figure 9: The HLA-A*2402_EWW-engaging TCRs recognize peptides with multiple amino acid substitutions

(a) Heat map showing the amino acid preferences of the TCR recognizing the HLA-A*2402 restricted peptide, EWWRSGGFSF, expressed as log₂FC of read counts relative to a triplicate baseline sample (see Supplementary Note). The TCR transduced clones were stained with a MHC multimer library composed of peptides with all single amino acid substitutions of the original EWWRSGGFSF peptide, as well as double amino acid substitutions covering 12 naturally occurring amino acids, where positions 4-8 were substituted two amino acids at a time. The heat map includes only double amino acid substitution variants (the effect of single amino acid substitutions can be seen in Fig. 1 and Supplementary Fig. 4). Double substitution variants resulting in peptides with a %Rank>0.5 were excluded from the analysis (total number of double substitution peptide variants, n=776, Supplementary Data 7). The data largely confirm the findings when screening only with the single substitution library, that there is some flexibility of the amino acids at positions 4 and 5, while almost no other double substitutions are tolerated. The barcode-based analysis of double substitutions is representative of duplicate analyses. (b) Dot plots from staining the HLA-A*2402_EWW engaging TCR with fluorescently labeled MHC multimers carrying one of 14 variations of EWWRSGGFSF and an irrelevant peptide (p*, the UV conditional peptide). The variants are comprised of peptides with 3 to 8 amino acid substitutions compared to the original peptide sequence (1). The substitutions were either at positions that had no preference for certain amino acids for TCR recognition (positions 1, 3 or 9, as visualized in the TCR fingerprint, Fig. 1f) or substituted with an amino acid that was prominent in the TCR fingerprint (positions 2, 4, 5, 6, 8 and 10). The MHC anchor at positions 2 were in some cases instead substituted with tyrosine (Y), which is the preferred amino acid for the peptide-MHC interaction (peptides 8, 9, 10, 11, 13, see Supplementary Fig. 6). We saw that the TCR were able to recognize peptide variants with up to 6 amino acids substituted compared to the original sequence (peptide 4 and 6) when the substituted amino acids were represented in the TCR fingerprint. The respective peptide sequences (substitutions in red), %Rank, MFI and percentages out of total CD8 T cells are indicated within the contour plots. The fluorescent-based MHC multimer stainings were performed once.