PROJECT SUMMARY Immune checkpoint inhibitors (ICIs) targeting the PD-1 pathway have transformed the management of many advanced cancers, including renal cell carcinoma (RCC), but most patients do not receive durable benefit from these treatments. Whereas many efforts to understand ICI response and resistance in other tumor types have often focused on investigating the role of total mutation burden (and consequent neoantigen load) or total T cell infiltration in the tumor microenvironment (i.e. “hot” vs. “cold” tumors), RCC is biologically distinct from other immunogenic solid tumors. In contrast to other solid tumors, the total mutation burden does not correlate with response to ICI in RCC. Further, despite its relatively modest tumor mutational load, RCC stands out as one of the most highly CD8+ T cell-infiltrated solid tumors at baseline, but the degree of CD8+ T cell infiltration into the tumor-immune microenvironment (TME) does not associate with ICI response. These observations highlight the gaps in our knowledge of the somatic alterations and infiltrating immune cell composition, phenotypic states, and cellular interactions that mediate an effective immune response against RCC in the context of ICI. Thus, there is a critical need to better understand the disease-specific mechanisms of response and resistance to current ICI- based therapies in RCC, which may also uncover the general principles of how a modest mutation burden tumor like RCC can be immunogenic. Prior smaller-scale analyses in RCC identified tumor-intrinsic somatic alterations that impact ICI response in RCC, and co-occurring and interacting immune populations (terminally exhausted CD8+ T cells and immunosuppressive macrophages) that are enriched in advanced disease. We therefore hypothesize that somatic alterations and the TME interact to explain the unique immune landscape of RCC and modulate response to ICI. By leveraging our expertise in immunogenomic analysis and our close collaborations with academic and industry partners, we are now uniquely poised to address this hypothesis. In Aim 1, we seek to systematically define the recurrent somatic alterations that impact immune infiltration and therapeutic response through the interrogation of large-scale genomic data (whole exome sequencing and bulk RNA- sequencing) from over 2,800 RCC tumors (including over 1,500 RCC tumors treated with ICI). In Aim 2, we strive to uncover the cell composition and cellular interactions within the TME that mediate response to ICI through large-scale single-cell transcriptomic analysis of 96 pre-treatment RCC tumor specimens (including 75 tumors subsequently treated with ICI). We aim to validate inferred interactions using advanced spatial phenotyping methods and through functional interrogation using an ex vivo patient-derived tumor fragment model. Overall, this work will identify genetic and immune determinants of effective ICI-mediated anti-tumor immunity in RCC, and will nominate specific th...