Background 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.

Methods 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 in vivo mice model to specifically delete TIM-3 on NK cells.

Results 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 HAVCR2 but not TIGIT or PDCD1 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.

Conclusions 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.