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Background Immunotherapy (IO) has revolutionized treatment of Head and Neck Squamous Cell Carcinoma (HNSCC), but not all patients benefit. Despite advances in understanding the transcriptional states of tumor, stromal, and immune cells via single-cell RNAseq (scRNAseq), the spatial organization of the tumor microenvironment (TME) remains poorly characterized. We hypothesize that a cellular neighborhood present at the tumor margin contributes to immune exclusion and limits responses to IO in HNSCC.Methods To address this hypothesis, we performed spatial transcriptomics (ST) on a tumor microarray (TMA) of 20 treatment-naïve HNSCC patients using Brüker’s CosMx platform. A previously generated scRNAseq atlas 1–3 was integrated with the ST data to enable robust identification of segmented cells from ST. Cell neighborhoods were identified by performing k-means clustering on a matrix containing the proportion of cell types within 300 micrometers of a given cell. We employed 3D spheroids consisting of HNSCC tumor cells, fibroblasts, endothelial cells, and donor immune cells to recapitulate immune invasion into the TME.Results We identified a total of 6 spatially organized cellular neighborhoods in the TME of HNSCC. Neighborhood 5 (N5) represented the tumor core and contained exhausted CD8+ T cells co-expressing multiple inhibitory receptors. Interestingly, neighborhood 3 (N3) was present at the interface between tumor beds and adjacent normal tissue and exhibited signatures of immunosuppression (e.g. TGFβ signaling) and extracellular matrix remodeling (e.g. integrin and collagen pathways). These signatures and its spatial location suggested that N3 is a driver of immune exclusion into the tumor core. Kaplan Meier analysis utilizing the N3 gene signature also showed that HNSCC patients with higher N3 expression displayed worse survival outcomes after IO, demonstrating that immune exclusion drives resistance to IO. N3 also contained more effector-like CD8+ T cells versus N5, suggesting that CD8+ T cells need entry into the tumor core to become exhausted, express inhibitory receptors, and subsequently respond to IO. Our in vitro spheroid model is being leveraged to identify cellular states present in N3 that prevent immune infiltration and represents an experimental platform to therapeutically enhance immune cell infiltration.Conclusions In conclusion, ST enabled the discovery of a cellular neighborhood (N3) that drives immune exclusion and limits response to IO. By mapping immune exclusion mechanisms and modeling them in vitro, this work sets the foundation for interventions that can enhance immunotherapy responsiveness in HNSCC and potentially other solid tumors exhibiting similar immune-exclusionary characteristics.Acknowledgements We thank all members of the Cillo and Bruno labs for feedback and discussion regarding this project. We also thank patients and their families for participating in this study. Finally, we thank UPMC Hillman Cancer Center for startup funds for this project.References Cillo AR, et al. Immune landscape of viral- and carcinogen-driven head and neck cancer. Immunity. 2020;52(1):183–199.e9.Ruffin AT, Cillo AR, et al. B cell signatures and tertiary lymphoid structures contribute to outcome in head and neck squamous cell carcinoma. Nat Comm. 2021;12:3349.Kurten CHL, et al. Investigating immune and non-immune cell interactions in head and neck tumors by single-cell RNA sequencing. Nat Comm. 2021;12:7338.Ethics Approval Specimens were collected with informed consent under the Institutional Review Board approved protocol 99-069.