389 Ex vivo expanded ieILC1-like NK cells induce significant tumor cell death and demonstrate metabolic flexibility and macrophage reprogramming capacity under hypoxic conditions

<h3>Background</h3> Natural killer (NK) cells offer unique therapeutic potential given their antigen-independent cytotoxicity. NK cell dysfunction in hypoxic tumor microenvironments remains a barrier for NK cell therapies against solid tumors. Through single-cell RNA sequencing of primary head and neck squamous cell carcinomas, we identified CD103⁺CD49a⁺ NK cells (termed ieILC1-like NK cells) that demonstrate upregulated expression of effector molecules.¹ We developed a novel method for differentiating and expanding these cells <i>ex vivo.</i>¹ Here, we found that ieILC1-like NK cells induce significant tumor cell clearance under 1% oxygen conditions. To determine attributes associated with their enhanced cytotoxicity, we investigated the metabolic fitness and stemness of these cells, and their impact on macrophages under hypoxic conditions. <h3>Methods</h3> Expanded ieILC1-like NK cells were generated by culturing conventional NK (cNK) cells with irradiated PCI-13 cells (1:1) and IL-15 (10ng/mL).¹ ieILC1-like and cNK cells were placed in hypoxia chambers set to 1% oxygen for up to 96 hours (n=3–6/group). Conditioned medium (CM) was collected after hypoxia exposure. Incucyte Live Cell analysis determined <i>ex vivo</i> killing capacity post-exposure (E:T = 1: 2.5). Mitochondrial mass and activity, amino acid transporter expression, and stemness and activation markers were measure by flow cytometry post-exposure. Metabolism genes and NADH/NAD concentrations were measured by quantitative gene expression and enzymatic assays performed in duplicate. Macrophage chemoattraction was assessed by transwell migration of polarized macrophages (1x10⁵ cells/M-type) exposed to collected CM. Macrophage polarization potential was determined by culturing unpolarized macrophages in collected CM, and measuring M-type markers by flow cytometry. All data met normality and equal variance assumptions. One-way ANOVAs or unpaired t-tests determined statistical differences. <h3>Results</h3> ieILC1-like NK cells showed improved killing capacity (p=0.03), increased percentages of CD25⁺ (p=0.012), MitoTracker Red⁺ (p=0.002), MitoTracker Green⁺ (p=0.005), CD43⁺ (p=0.02) and CD38⁺ (p=0.13) cells, and reduced percentages of CD98⁺ (p=0.0003) and CD71⁺ (p=0.03) cells after hypoxia exposure. ieILC1-like NK cells demonstrated upregulated expression of LDHA (p&lt;0.0001) and an 85% increase in NADH:NAD compared to cNK cells post-exposure. CM from ieILC1-like NK cells attracted more anti-tumorigenic M1 (p=0.018) and fewer pro-tumorigenic M2 macrophages (p=0.016), and fewer macrophages were CD206⁺ after exposure to ieILC1-like NK cell CM (p=0.04). <h3>Conclusions</h3> ieILC1-like NK cells uniquely demonstrate enhanced killing capacity that co-occurs with increased mitochondrial activity, stemness, and anti-tumor M1 macrophage attraction in hypoxic settings. This work reveals a novel anti-tumor capacity of ieILC1-like NK cells, and supports clinical investigation of these cells for solid tumor treatment. <h3>Reference</h3> Moreno-Nieves UY, Tay JK, Saumyaa S, Horowitz NB, Shin JH, Mohammad IA, Sunwoo JB. Landscape of innate lymphoid cells in human head and neck cancer reveals divergent NK cell states in the tumor microenvironment. <i>Proceedings of the National Academy of Sciences</i>, 2021;<b>118</b>(28):e2101169118.