Fig. 6: Deep characterization of sample heterogeneity in MEL-4.
a, Phylogenetic tree of tumors and cell lines, annotated with mutations in known driver genes (black) and B2M defects (red). b, mIF-derived CD8+ T cell infiltration in GI and P samples. Dots represent CD8 densities in the stroma and tumor in each tile. c, Digital reconstruction and density map from mIF quantification of Sox10+ cells expressing HLA-ABC and HLA-DR in GI and P lesions. d, mIF quantification of HLA-ABC and HLA-DR on cancer cells (Sox10+) GI and P lesions. e, Heat map comparing expression levels of genes encoding for TSAs in healthy tissues (GTEx, 90th percentile expression value; left) and MEL-4 tumors and the HLA-low and HLA-high isogenic cell lines derived from the P lesion with and without IFNγ treatment (right). f, DIA-based quantification of HLA-I and HLA-II tumor-specific peptides identified by MS DDA and DIA across MEL-4 tumors and cell lines with and without IFNγ treatment. Gray boxes reflect the absence of detection. g, FACS quantification of HLA in the primary P lesion cell line. Diagram of HLA-low and HLA-high cell isolation by FACS sorting and histograms of HLA-ABC expression of isolated HLA-low and HLA-high populations before and after IFNγ treatment. h, Expression levels of genes involved in the APPM. Green and orange boxes show their expression levels in healthy sun-exposed skin (GTEx, n = 701 samples) and MEL samples (TCGA, n = 468 samples), respectively. Connected dots represent gene expression levels in HLA-high and HLA-low cell lines treated or not with IFNγ. In box plots, 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× the IQR from the 25th and 75th percentiles, respectively. i, Tumor reactivity of antigen-specific TCR clonotypes from MEL-4. Reactivity was assessed by CD137 upregulation of TCR-transfected primary autologous activated CD8+ T cells following coculture with IFNγ-treated or untreated HLA-low and HLA-high isogenic cell lines. Data are presented as the mean values ± s.d. of technical replicates (n = 2). The positivity threshold is described in the Methods. Irr_Ctrl, irrelevant TCR; mock, transfection with water. Panel g created with BioRender.com.
