Extended Data Fig. 5: TCRseq clustering of EGFR neopeptide stimulated MANAFEST cultures.

To identify groups of TCRs that shared similar sequence structure to the clones that were significantly expanded in PBMC samples from the MANAFEST assay, we clustered together the top 3000 CDR3B sequences from each timepoint (months 30, 40, 45) from each condition (Cytokine, CEF, ex19del, T790M), using the Gliph2 clustering algorithm. a, Gliph2 network plot, coloured by ‘total.score’ metric, herein referred to as cluster importance score. More significant clusters are located towards the middle of the plot. b, Overview of clusters which contain expanded sequences, as defined by the MANA webtool. As all samples were clustered together, the proportion of each condition’s contribution to the cluster is displayed in the bar. Cluster size refers to the number of unique TCR sequences within each cluster. We observed that clusters containing an expanded sequence determined by the MANA webtool were composed of sequences predominantly of the same condition. c, Clusters were segregated into conditions using a proportion cut-off of 50%. All clusters from Gliph2 output which had a sequence count above the threshold are displayed. Significance determined by two-tailed Wilcoxon test. There was no difference in cluster importance scores between the ex19del culture and the positive control (CEF), whilst the T790M condition showed the highest cluster importance score. Taken together, these data suggest that the expanded clones in their respective cultures were driven by peptide-specific stimulation, as we see more significant clustering in the conditions treated with peptide compared to cytokine alone. The box plots represent the upper and lower quartiles (box limits), the median (centre line) and the vertical bars span the 5th to 95th percentiles.