Abstract 5851: The role of IL-9 on NK and T cell infiltration and immune response to promote tumorigenesis in HNSCC

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Head and neck squamous cell carcinomas (HNSCC) are the sixth most common cancer type worldwide and result in 400,000 deaths annually. Interleukin-9 (IL-9) is a pleiotropic cytokine largely produced by CD4+ Th9 cells. IL-9 is known to function in allergic inflammatory processes and is involved in both positive and negative regulation of immune responses. The role of IL-9 in cancer, however, differs depending on the cancer type and the tumor microenvironment, harboring both tumorigenic and anti-tumorigenic effects. The role of IL-9 in HNSCC specifically is poorly understood. We begin here to elucidate the possible functions of IL-9 on the immune system in the context of HNSCC.IL-9 plasma levels were determined by ELISA and cell proliferation was measured using a metabolic absorbance assay. Kaplan-Meier Plotter was used to determine if tumor IL-9 mRNA expression affects survival probability in HNSCC patients. Immune cell infiltration was determined by a novel infiltration assay designed by our laboratory. Briefly, we co-cultured Cal27 spheroids, a 3D model made with the HNSCC cell line Cal27, with peripheral blood mononuclear cells (PBMCs) isolated from HNSCC patients. The spheroids were then dissociated and the percentage of live infiltrating PBMCs was analyzed by flow cytometry. To assess the influence of IL-9 on immune cell killing, NK and CD8+ T cell cytotoxicity was measured by co-culturing CFDA-SE-labeled HNSCC cell lines with HNSCC patient-derived NK or T cells treated with recombinant IL-9 or a neutralizing antibody to IL-9 followed by flow cytometry analysis to determine the percentage of dead target cells.IL-9 plasma levels were found to be significantly increased in HNSCC patients as compared to healthy subjects. Additionally, high tumoral mRNA expression of IL-9 was associated with decreased survival in HNSCC cohorts. IL-9 did not affect tumor cell proliferation in vitro. However, higher concentrations of IL-9 significantly decreased immune cell infiltration into Cal27 spheroids, while a neutralizing antibody to IL-9 reversed these effects. Additionally, while studies are ongoing, IL-9 may decrease NK and CD8+ T cell cytotoxicity against HNSCC cell lines.Since IL-9 did not directly enhance or inhibit HNSCC cell proliferation in vitro, we extended our studies to explore IL-9’s potential effects on the immune system. Importantly, IL-9 significantly decreased immune cell infiltration into HNSCC spheroids, suggesting that IL-9 inhibits tumor immune cell infiltration. IL-9 may also decrease immune cell cytotoxicity against HNSCC cells. Taken together, this data suggests that IL-9 negatively regulates the immune system in HNSCC, allowing for tumor growth. Future studies will focus on the effects of IL-9 on immune cell cytotoxicity and immune infiltration in vivo and will utilize single-cell transcriptional profiling to delineate the multifaceted role of IL-9 in the HNSCC immune microenvironment. Citation Format: Samantha L. Nusbaum, Maria Lehn, Vinita Takiar, Dalia El-Gamal, Trisha Wise-Draper. The role of IL-9 on NK and T cell infiltration and immune response to promote tumorigenesis in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5851.