Extended Data Fig. 5: (Related to Fig. 2). Analysis of the LUAD immunopeptidome throughout tumor evolution.

a) UMAP embedding of clusters used for signature expression analysis in Fig. 2a. b) Gene expression profiles of cell type marker genes indicating robust clustering of known cell types in the healthy lung. c) Representative H&E stains for healthy lung (AT2), Early-, Mid- and Late-stage tumor samples. d) Peptide motifs of 8- and 9-mer peptides identified from healthy AT2 cells. e) Length and affinity characteristics of peptides identified in AT2, Early-, Mid-, and Late-stage tumors. f) Venn diagram comparison of peptides identified in bulk, healthy lung versus those identified specifically presented by normal AT2 cells. Pathways enriched by gene ontology depicted on bottom. g) Comparative analysis of gene signatures derived from peptides detected on AT2 cells versus bulk lung applied to the Tabula Muris data. Volcano plot shows the strong enrichment for an AT2 phenotype in the AT2 immunopeptidome versus bulk lung immunopeptidome. h) Comparison of AT2 and Early-, Mid-, and Late-Stage immunopeptidomes. i) Quantification of percent overlap from h). j) UMAP embedding of clusters from reanalyzing scRNAseq data from Marjonovic et. al. and used for analysis of Fig. 2j. k) Loess regression analysis across all cells scored for AT2, Early, Mid, and Late peptide signatures versus the AT2, GI-Epi, and Mixed transcriptional modules. l) Signature distribution for AT2-, Early-, Mid-, and Late-Stage peptide signatures across all KP scRNAseq clusters. m) Comparison of the immunopeptidome in control tumors (gray), tumors chronically depleted of CD8 T-cells (light teal), and tumors acutely depleted of CD8 T-cells (dark teal). n) Comparison of the immunopeptidome from control tumors (gray) or tumors treated with agonistic-CD40 antibody and Flt3-L (orange). CD8 depletion experiments and CD40/Flt3L experiments were carried out independently and analyzed on different MS runs separated by ~2.5 months.