1061 The role of interleukin-9 in head and neck squamous cell carcinoma immune response and tumorigenesis

Background Head and neck squamous cell carcinomas (HNSCC) are the sixth most common cancer worldwide and result in 400,000 deaths annually.1 Interleukin-9 (IL-9) is a pleiotropic cytokine largely produced by CD4+ Th9 cells.2 IL-9 is known to function in allergic inflammatory processes and is involved in positive and negative regulation of the immune response. The role of IL-9 in cancer differs depending on the cancer type and the tumor microenvironment, harboring both tumorigenic and anti-tumorigenic effects.3–8 However, the role of IL-9 is poorly understood in HNSCC. Here, we begin to elucidate possible functions of IL-9 in HNSCC. Methods IL-9 plasma and PBMC levels were determined by ELISA and cell proliferation was measured using a metabolic absorbance assay. Immune cell infiltration was determined by co-culturing Cal27 spheroids with HNSCC PBMCs, dissociating the spheroids, and analyzing the percentage of live infiltrating PBMCs by flow cytometry. Results IL-9 plasma levels were found to be significantly increased in HNSCC patients as compared to healthy counterparts, and higher pre-treatment plasma levels of IL-9 in HNSCC patients were associated with tumor recurrence. IL-9 did not affect tumor cell proliferation in vitro. However, higher doses of IL-9 significantly decreased immune cell infiltration into Cal27 spheroids. When natural killer (NK) cells were depleted from peripheral blood mononuclear cells (PBMCs), there was a significant increase in IL-9 in both HNSCC PBMCs and healthy counterpart PBMCs. Conclusions IL-9 did not directly enhance or inhibit HNSCC cell proliferation. Importantly, IL-9 significantly decreased immune cell infiltration into HNSCC spheroids, suggesting that IL-9 inhibits immune cell infiltration into the tumor in vivo. Because IL-9 levels were increased when NK cells were depleted from PBMCs, it is likely that NK cells are inhibiting the production or the release of IL-9. Future studies will focus on the effects of IL-9 on immune cell cytotoxicity, immune infiltration in vivo, and elucidating the inhibitory effects of NK cells on IL-9 production/release. References Levy DA, et al. Programmed death 1 (PD-1) and ligand (PD-L1) inhibitors in head and neck squamous cell carcinoma: A meta-analysis. World J. Otorhinolaryngol. - Head Neck Surg. 2022;8:177–186. Lee JE, et al. The Role of Interleukin-9 in Cancer. Pathol. Oncol. Res. 2020;26:2017–2022. Angkasekwinai P, Dong C. IL-9-producing T cells: potential players in allergy and cancer. Nat. Rev. Immunol. 2021;21:37–48. Zheng N, Lu Y. Targeting the IL-9 pathway in cancer immunotherapy. Hum. Vaccines Immunother. 2020;16:2333–2340. Fang Y, et al. IL-9 inhibits HTB-72 melanoma cell growth through upregulation of p21 and TRAIL. J. Surg. Oncol. 2015;111:969–974. Gruss HJ, Brach MA, Drexler HG, Bross KJ, Herrmann F. Interleukin 9 is expressed by primary and cultured Hodgkin and Reed-Sternberg cells. Cancer Res. 1992;52:1026–1031. Purwar R, et al. Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells. Nat. Med. 2012;18:1248–1253. Heim L, et al. IL-9 Producing Tumor-Infiltrating Lymphocytes and Treg Subsets Drive Immune Escape of Tumor Cells in Non-Small Cell Lung Cancer. Front. Immunol. 2022;13:859738. Ethics Approval Human samples were collected as part of University of Cincinnati IRB approved protocol #2014–4755 after informed consent.