Background Natural killer (NK) cells are innate immune cells that generate inflammatory responses important for anti-tumor responses. NK cell therapies have been increasingly explored and tested as a novel approach to cancer immunotherapy in recent years. In contrast to currently approved CAR-T technologies, NK cell therapy can potentially be administered off-the-shelf with low risk of graft-versus-host disease compared to T cells.¹ Recently, we identified intraepithelial ILC1-like (ieILC1-like) NK cells as the subset of NK cells within the tumor microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC) with the highest cytotoxicity.² Here, we explore how these cells may modulate the TME and interact with other immune cells in the anti-tumor response.

Methods We used various methods to assess the range of cytokine production by ieILC1-like NK cells in the context of cancer. Co-culture of activated conventional NK (cNK) cells (CD56+CD49a+CD103-CD3-CD14-CD19-CD20-) or ieILC1-like NK cells (CD56+CD49a+CD103+CD3-CD14-CD19-CD20-) with K562 chronic myelogenous leukemia (CML) cells was performed and cytokines were analyzed via Luminex immunoassay of cultured supernatants, evaluating a panel of 80 cytokines and chemokines. Validation was performed by intracellular flow cytometry of the cells in this co-culture system.

Results Luminex analysis revealed that the co-culture of ieILC1-like NK cells with K562 cells resulted in elevated levels of CXCL10 in cultured supernatants compared to that of K562 cells alone, ieILC1-like NK cells alone, or cNKs co-cultured with K562. Similarly, cultured supernatants of ieILC1-like NK cells with K562 had elevated levels of IL-13 compared to that of K562 alone, ieILC1-like NK cells alone, or cNKs co-cultured with K562. To determine the source of these cytokines, we performed intracellular flow cytometry. We found that the K562 cells were the primary source of CXCL10 when co-cultured with ieILC1-like NK cells. Unexpectedly, however, IL-13 was uniquely produced by a subset of IFN-gamma-producing ieILC1-like NK cells following stimulation by tumor cells.

Conclusions The ieILC1-like NK cells induce CXCL10 production by K562, most likely via IFN-gamma. CXCL10 is a pro-inflammatory chemokine involved in immune cell recruitment to the TME.³ Unexpectedly, we identified a subset of ieILC1-like NK cells that produce IL-13, a Th2-type cytokine that has the potential to dampen anti-tumor immune responses and possibly promote tumor survival and proliferation.⁴ Thus, the CD49a+CD103+ ieILC1-like NK cell population is heterogeneous, and further studies to understand the functional differences of these distinct subsets are necessary.

Acknowledgements We thank the Stanford Human Immune Monitoring Center for their assistance with the Luminex assays involved in this project.

References

1. Wolf NK, Kissiov DU, Raulet DH. Roles of natural killer cells in immunity to cancer, and applications to immunotherapy. Nat Rev Immunol. 2022;23:90–105.

2. Moreno-Nieves UY, Tay JK, Saumyaa S, et al. Landscape of innate lymphoid cells in human head and neck cancer reveals divergent NK cell states in the tumor microenvironment. Proc Natl Acad Sci U S A. 2021;118(28):e2101169118.

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4. Terabe M, Park JM, Berzofsky JA. Role of IL-13 in regulation of anti-tumor immunity and tumor growth. Cancer Immunol Immunother. 2004;53(2):79–85.