ABSTRACT Spontaneous T cell pressure against an evolving tumor is a conserved mechanism of anti-tumor immune control. On the other hand, immune therapies directed to alleviate paralyzing T cell dysfunction within solid tumors only benefits a small proportion of patients with metastatic disease. The field is in desperate need for the development of complementary therapies capable of enhancing sustained T cell-mediated control of primary and metastatic tumor growth. However, myeloid cells within tumors present a substantial barrier towards overcoming the immune dysfunction present in most cancers. Here, we demonstrate that recognition of commensal bacteria through TLR5 signaling results in sustained myeloid dysfunction and impaired response to PD-L1 blockade. TLR5 signaling broadly impairs myeloid function, resulting in infiltration of tumors with poorly functional T cells. In TLR5 KO mice, PD-L1 blockade achieves significantly increased overall survival and leads to durable and long-term remission for mice bearing aggressive ovarian tumors. The effect of TLR5 signaling is recapitulated in non-responsive melanoma and breast tumor models, suggesting that this signaling pathway is a conserved mechanism of immune suppression and failure for PD-L1 blockade. Mechanisms underlying myeloid dysfunction and failure of PD-L1 blockade have largely focused upon interactions between tumors and immune cells. However, our data implicate a mechanism whereby recognition of commensal bacteria by TLR5-expressing immune cells initiates myeloid dysfunction and failure of PD-L1 blockade. Based upon these data, inhibition of TLR5 signaling emerges as a means of restoring anti-tumor T cell function across a broad range of tumor types. However, because TLR5 signaling is canonically associated with activation of adaptive immunity in other settings, it is critical to understand how TLR5 signaling impairs myeloid function within the tumor microenvironment. The overarching goal is to define how commensal microorganisms impair anti-tumor immunity and response to PD-L1 blockade. Here, we will test the hypothesis that chronic encounter with microbiome-derived TLR5 ligands and autocrine amplification of IL-6 polarize TLR5-expressing myeloid cells within the tumor microenvironment (TME). This results in impaired ability of myeloid cells to prime and/or recall tumor-reactive T cells and subsequent failure of PD-L1 blockade. Aim 1 will define how TLR5 signaling on myeloid cells affects tumor growth and response to PD-L1 blockade. Aim 2 will determine how TLR5 signaling impairs myeloid function. Aim 3 will leverage in vivo labelling of commensal microorganisms to establish how encounters between commensal microorganisms and TLR5 expressing immune cells within the tumor microenvironment impair anti- tumor immune function. Mechanistically, very little is known as to how the microbiome negatively impacts host anti-tumor immune function. The studies proposed herein will fill this gap i...