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Distinct B cell signatures and tertiary lymphoid structures are driven by two etiologies in head and neck cancer


Background: Current FDA-approved immunotherapies aim to reinvigorate CD8+ T cells, but the contribution of the humoral arm of the immune response in human cancer remains poorly understood [1,2]. B cells can mediate anti-tumor immunity by presenting antigen , producing tumor-specific antibodies and immunomodulatory cytokines [3-5]. Head and neck squamous cell carcinoma (HNSCC) can be induced by human papillomavirus (HPV+) and carcinogens (HPV-), and the immune infiltrate in the two etiologies has been reported to be distinct, in particular, more B cells in HPV+ HNSCC patients [6]. Further, the presence of B cells in HNSCC correlates with increased patient survival [6]. Our study seeks to differentiate B cell phenotype and function in HPV+ and HPV- HNSCC and identify putative immunotherapeutic targets.

Methods: We utilized a multi-level approach to categorize B cells in HNSCC patients. Single cell RNA sequencing (scRNAseq) was performed on CD45+ tumor infiltrating lymphocytes (TIL) and peripheral blood leukocytes (PBL) from HPV+ and HPV- HNSCC patients. Data was analyzed using the Seurat R toolkit in R studio. HNSCC TIL and PBL were stained via multiparameter flow cytometry panel using the Cytek Aurora to allow for unbiased analysis of B cell subsets via computational spectral unmixing. Paraffin embedded slides from HNSCC primary tumors were utilized for single parameter immunohistochemistry of CD20 (n=25 each for HPV + and HPV - disease), and multispectral immunofluorescence was performed on matched tumors from scRNAseq for CD20, CD4, CD8, CD68, PanCK, DAPI and Foxp3.

Results: We demonstrated distinct trajectories for B cells in HPV+ and HPVdisease. B cell signatures in HPV- HNSCC patients were predominantly memory B cells and plasma cells, while the signatures in HPV+ HNSCC were naïve and germinal center B cells. We quantified B cells and CD4 T cells in tertiary lymphoid structures (TLS), and the presence of germinal center-rich TLS was associated with HPV+ disease. GC-rich TLS within the tumor bed of HPV+ patients correlated with increased overall survival. Independent of HPV status, we have identified an atypical B cell signature increased in the HNSCC TME compared to healthy and inflamed tonsil tissue with no cancer. This atypical B cell subpopulation is also elevated in the periphery of patients with advanced, metastatic disease.

Conclusions: Characterization of B cell phenotype and function in HNSCC is important for devising new therapeutic options for patients. Table 1 (abstract P546). See text for description Fig. 1 (abstract P546). See text for description Fig. 2 (abstract P546). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):283 Page 26 of 237 Development of therapeutics to enhance B cell responses in the TME should be prioritized as a compliment to T-cell mediated therapies.

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