Supplementary Figure 4: Replicate screening of the HLA-A*2402_EWW-engaging TCR showing read counts and data from an irrelevant sample

Results obtained from the DNA barcode-based analysis of T cells transduced with a TCR recognizing the HLA-A*2402 restricted peptide, EWWRSGGFSF (replicate of the data shown in Fig. 1d-f). The analysis was performed with all possible variations of peptides created by single-position amino acid substitutions. The hierarchy of pMHC interactions expressed as (a) clonality reduced read counts or (b) log₂FC relative to a triplicate baseline sample (see Supplementary Note). A healthy donor PBMC sample (BC-D42) was screened with the same MHC multimer panel in parallel. The plotted order of read counts or log₂FC of each pMHC-associated DNA barcode is determined by the hierarchy obtained from screening the HLA-A*2402_EWW responsive TCR. (c-f) Heat map showing the clonality reduced read counts or log₂FC of (c,d) the HLA-A*2402_EWW responsive TCR or (e,f) the healthy donor BC-D42, respectively. Each row represents a given amino acid and each column a position in the peptide sequence. The amino acids of the original peptide target are marked with black borders. c and e showing read counts, and d and f showing Log₂FC are colored according to the same keys respectively. (g) The replicate recognition pattern of the HLA-A*2402_EWW interacting TCR, here visualized as a sequence logo based on the data from a-d.