Abstract DNA mismatch repair deficiency (MMRd) in human cancer is associated with high tumor mutational burden (TMB), frameshift mutation-derived neoantigens, increased T cell infiltration, and remarkable responsiveness to immune checkpoint blockade (ICB) therapy. Nevertheless, about half of MMRd tumors do not respond to ICB for unclear reasons. While tumor cell line transplant models of MMRd have reinforced the importance of TMB in immune response, critical questions remain regarding the role of immunosurveillance in the evolution of MMRd tumors induced in vivo . Here, we developed autochthonous mouse models of lung and colon cancer with highly efficient ablation of MMR genes via in vivo CRISPR/Cas9 targeting. Surprisingly, MMRd in these models did not result in increased immunogenicity or response to ICB. Mechanistically, we showed this lack of immunogenicity to be driven by profound intratumoral heterogeneity (ITH). Studies in animals depleted of T cells further demonstrated that immunosurveillance in MMRd tumors has no impact on TMB but shapes the clonal architecture of neoantigens by exacerbating ITH. These results provide important context for understanding immune evasion in cancers with high TMB and have major implications for therapies aimed at increasing TMB.

Mismatch repair deficiency is not sufficient to increase tumor immunogenicity