501 TIM-3 upregulation by tumor-infiltrating NK cells may be associated with NK cell dysfunction in head and neck squamous carcinoma

<h3>Background</h3> Despite significant advancements in the field, the responsiveness of head and neck squamous cell carcinoma (HNSCC) patients to the current generation of immune checkpoint inhibitor (ICI) is modest, with ~15% of patients benefiting from anti-PD-1 monotherapy. This has led to a search for novel druggable immune checkpoint receptors (ICR). Among these, T-cell immunoglobulin mucin 3 (TIM-3) has captured considerable attention due to its intricate immunomodulating mechanisms orchestrated by four distinct ligands. TIM-3 is present on various immune cells, including natural killer (NK) cells, which are innate effector lymphocytes that play a pivotal role in anti-tumor immunity. The impact of TIM-3 on NK cell-mediated antitumor immunity remains ambiguous with both co stimulatory and co inhibitory properties. <h3>Methods</h3> To understand the impact of TIM-3 on NK cell anti-tumor immune responses, we leverage our neoadjuvant clinical trial designed to enhance anti-tumor immunity in subjects with resectable locally advanced HNSCC by combining anti-PD-1 with anti-LAG3 or with anti-CTLA-4 therapies. We generated and profiled single-cell RNA sequencing libraries for CD45⁺CD3^- peripheral blood (PBL) and tumor-infiltrating leukocytes (TIL), including circulating and tumor-infiltrating NK cells isolated from HNSCC patients. Additionally, utilizing an <i>in vivo</i> mice model to specifically delete TIM-3 on NK cells. <h3>Results</h3> Bioinformatic analyses revealed distinct signatures associated with NK cell phenotypes and highlighted the possible correlation between type I interferon and NK cell cytotoxic signatures with patient responsiveness. Notably, we observed an increase in TIM-3 gene (HAVCR2) expression in NK cells, both among TIL and PBL, in comparison to other lymphocytic populations. Furthermore, we observed an increase in <i>HAVCR2</i> but not <i>TIGIT</i> or <i>PDCD1</i> expression by tumor-infiltrating NK cells in non-responding patients. Our data from the murine model planted with murine tonsil epithelium MEER cell line indicates an increase in TIM-3 expression in tumor infiltrating NK cells in comparison with NK cell from spleen compartment. <h3>Conclusions</h3> The role of TIM-3 on tumor infiltrating NK cells remains elusive. Our data stress the importance of TIM-3 expression in the context of tumor infiltrating NK cells. Future mechanistic studies will provide better understanding of the role TIM-3 plays in ICI resistance. Furthermore, targeting TIM-3 and in combination with other ICI may improve NK cell mediated anti-tumor immunity.