Extended Data Fig. 6: Extended characterization of samples heterogeneity in MEL-4.

a) T-cell inflammation heatmap of the GI and P tumor samples, in the context of all skin cutaneous melanoma patients in TCGA (n = 468). The top bar shows the inferred inflammation status. The rows represent expression values of immune related genes, and the color indicates their expression level. MEL-4 samples are highlighted at the top and annotated with B2M deficiencies. b) Expression levels of genes involved in the APPM. Green and Orange boxes show healthy sun-exposed skin (GTEx, n = 701 samples) and melanoma (TCGA, n = 468 samples) expression levels of the genes, respectively, and connected dots represent gene expression in MEL-4 tissues. In boxplots, the center line represents the median. The bounds of the box represent the 25th and 75th percentiles, indicating the IQR. The whiskers extend to the smallest and largest values within 1.5 times the IQR from the 25th and 75th percentiles, respectively. c) mIF staining of the GI and P lesions (n = 1 slide). d) Number of peptides identified by MS DDA and DIA in all MEL-4 samples. Binders are defined by a predicted binding rank ≤ 2. The percentage over the blue bar indicates the percentage of binders over the total, written next to the bar. e) Gene expression, represented as color intensity, and DIA-based quantification (as the sum of HLA-I and HLA-II intensities; shown as the width of the circles) of genes encoding for MS-identified tumor-specific peptides across MEL-4 tumors (top) and cell-lines treated or not with IFNγ (bottom). f) Peptide (n = 106) intensities in the tissues (left) and in the cell lines (right) separated between samples without (blue) and with (red) B2M p.Ser31fs. Paired one-sided t-test was applied to assess differences in the distributions. g) Antigen reactivity validation of ELOVL1(P149S)-specific TCR clonotypes from patient MEL-4. Reactivity assessed by luminescence of TCR-transfected Jurkat cells following co-culture with autologous CD4 blasts loaded with neoepitope HSVLSWSWW. Data are presented as mean values +/− SD of technical replicates (n = 2). (CD4 blasts alone: unloaded, Irr_Ctrl: irrelevant TCR, Mock transfection with water, RLU: Relative Light Unit).