UVB-induced tumor heterogeneity directs immune response in melanoma

Clonal neoantigen burden is associated with improved response to immune-therapy, however, the underlying functional basis for this relationship in melanoma remains unclear. Here we study this question in a novel, controlled experimental UVB mouse melanoma model that enables one to study the effects of intra-tumor heterogeneity on tumor aggressiveness and immune response, independently of tumor mutational burden (TMB). The induction of UVB-derived mutations in parental melanoma cell-lines gives rise to high TMB tumors that are highly aggressive accompanied by decreased anti-tumor activity of tumor infiltrating lymphocytes (TILs). However, strikingly, UVB single-cell derived melanoma clones with high TMB levels but reduced Intratumor heterogeneity (ITH) are swiftly rejected. Their rejection is accompanied by increased TILs reactivity, increased CD8+ T cell core infiltration and a less suppressive microenvironment. Using phylogenetic tree analyses and mixing experiments of 20 single cell UVB clones that lie along the phylogenetic tree, we further systematically tease apart two main characteristics of tumor ITH: we show that tumor rejection is inversely associated with the number of clones forming the tumor and their diversity. Notably, these results are recapitulated and reinforced in the analysis of melanoma patient data, both in terms of their survival with or without immune check point therapy. Taken together, our results highlight the central importance of clonal mutations in robust immune surveillance and the need to quantify the heterogeneity of patient tumors, a central determinant of their survival and response to checkpoint blockade.