PROJECT ABSTRACT Cancer immunotherapy, when effective, is lifesaving. Unfortunately, 15-50% of individuals will not respond to therapy; therefore, efforts to ‘flip the switch’ and turn these ‘non-responders’ into ‘responders’ are critical and urgent. Encouragingly, phenomenological studies in humans and preclinical studies in mice have demonstrated that the gut microbiome is associated with cancer immunotherapy response. However, the exact mechanisms that drive microbiome-based changes in immunotherapy response are not known. This project will elucidate how microbial microproteins modulate macrophage function, a critical coordinator of tumor immune response. The central hypothesis of this proposal is that specific gut microbes express microproteins that directly modulate macrophages, thus leading to immunomodulation of cancer. Previous work seeking to deconvolute the signaling interface between microbes and immune cells has done so by: (i) carrying out limited- throughput, arrayed screens, (ii) focusing on microbial small molecule metabolites and (iii) focusing on T-cells. Here, we propose to address many gaps left by these approaches. Namely, we will (i) carry out two orthogonal high-throughput screens (peptide-display polarization assay, Perturb-seq) of 1,000s-10,000s of microbial macromolecules in pooled screens (technical innovation), (ii) focus on proteins, especially an understudied class of microproteins we recently discovered and have now annotated in microbial genomes (conceptual innovation), and (iii) moving one step up in the immunological ‘cascade’ by focusing on macrophages (conceptual innovation). Our strong preliminary data have identified two novel macrophage-modulating microbial proteins that we will mechanistically study in Aim 1, and we have also developed and validated a powerful peptide display system that will be expanded in Aim 2. Building upon these preliminary data, we will: (Aim 1) determine the detailed molecular mechanisms, including targ