LAUSR

Simple Summary Tumors use immunosuppressive signals to evade detection by the immune system. While recurrent and metastatic head and neck squamous cell carcinoma has historically carried a poor prognosis, therapies targeting the immunosuppressive PD1:PDL1 axis have improved survival in certain patients. Defining mechanisms regulating PDL1 in various contexts may inform refinement of immunotherapy protocols. We identified a role for Toll-like Receptor 2 (TLR2) signaling in driving PDL1 expression. In antigen-presenting cells, TLR2 functions to initiate response to pathogens, and it is overexpressed or genetically altered in some tumors. We found that the synthetic TLR2 ligand Pam3CSK4, as well as whole bacteria, induced PDL1 expression in specific HNSCC cell line models, suggesting that TLR2 may contribute to immune evasion in chronically inflamed tissues. Abstract As immunotherapies targeting the PDL1 checkpoint have become a mainstay of treatment for a subset of head and neck squamous cell carcinoma (HNSCC) patients, a detailed understanding of the mechanisms underlying PDL1-mediated immune evasion is needed. To elucidate factors regulating expression of PDL1 in HNSCC cells, a genome-wide CRISPR profiling approach was implemented to identify genes and pathways conferring altered PDL1 expression in an HNSCC cell line model. Our screen nominated several candidate PDL1 drivers, including Toll-like Receptor 2 (TLR2). Depletion of TLR2 blocks interferon-γ-induced PDL1 expression, and stimulation of TLR2 with either Staphylococcus aureus or a bacterial lipopeptide mimetic, Pam3CSK4, enhanced PDL1 expression in multiple models. The data herein demonstrate a role for TLR2 in modulating the expression of PDL1 in HNSCC models and suggest that microbiota may directly modulate immunosuppression in cancer cells. Our study represents a step toward disentangling the diverse pathways and stimuli regulating PDL1 expression in HNSCC and underscores a need for future work to characterize the complex microbiome in HNSCC patients treated with immunotherapy.