Supplementary Figure 5: The effect of peptide–MHC interaction on analyzing the recognition pattern of the HLA-B*0702_APN-engaging TCR

(a) Sequence logo showing the amino acid preferences for peptide binding to HLA-B*0702 constructed with NetMHCpan-4.0⁴⁹ by finding the binding cores of the top 1% strongest predicted binders among 100,000 natural random 10-mer peptides and visualizing these using R with the Shannon method in Seq2Logo⁴⁸. (b) Scatter plot of the experimentally determined peptide binding to HLA-B*0702 (MHC ELISA, Supplementary Note 3), given as the relative quantity of a pMHC variant (only alanine substitutions) after UV-exchange, compared to that of the original peptide, in correlation to the experimentally obtained recognition properties of the given pHLA-B*0702 interacting with the TCR. The MHC ELISA data is average of 5 experiments (n=2). Each dot in b represents one peptide-MHC variation. The coloring indicates the position of a given amino acid substitution and the asterisks indicates the original peptide. (c) Dot plots from staining the HLA-B*0702_APN engaging TCR with fluorescently labeled MHC multimers carrying one of six variations of APNCYGNIPL and an irrelevant peptide (p*, the UV conditional peptide). 1-3 are all examples of peptides predicted as strong binders to HLA-B*0702 (%Rank<0.5), here only 1 and 2 are recognized by the TCR. 4-6 are examples of peptides that are predicted as poor binders to MHC (%Rank>2), but the TCR is still able to recognize 4 and 5. 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 multimer stainings were performed twice.