{
  "abstract": "Background Hypoxia has detrimental effects on lymphocytes in the tumor microenvironment, including reducing tumor invasion, decreasing cytotoxicity, and limiting tumor clearance. It can also dampen the cytotoxicity of freshly isolated NK cells [1] by decreasing STAT3 and ERK signaling. 1 2 However, some studies have shown that hypoxia may increase STAT33 and ERK4 signaling in NK cells expanded in the presence of IL-2 or IL-15, which can enhance NK cell cytotoxicity.1 4 Here, studies by our lab show that hypoxia may increase the cytotoxicity of NK cells expanded ex vivo with IL-2 and IL-15, and these changes may be mediated through increased glucose and HIF-1a signaling.Methods NK cells were isolated from PBMCs and expanded ex vivo under normoxic conditions with IL-15 and IL-2 in NK MACS medium. NK cell cultures were then subjected to 1% oxygen or cultured in normoxia. The phenotype, cytotoxicity, and metabolism of the NK cells was assessed using flow cytometry, overnight cytotoxicity, bulk RNA sequencing, long-term serial killing, and Seahorse analysis. Hypoxic NK cells were then returned to normoxia and transcriptional changes were evaluated after recovery.Results Compared to normoxia, transient hypoxia exposure decreased oxidative phosphorylation and slightly increased basal glycolysis of NK cells ex vivo expanded with IL-2 and IL-15. Hypoxia also increased CD69 expression but only minimally decreased NKG2D and NKp44 expression, while HIF-1a protein expression was not affected. However, hypoxia increased NK cell cytotoxicity 25% on average compared to NK cells cultured in normoxia against K562, U87, U251, MOLT4, and Rs;4-11. Hypoxia also increased transcript levels of glucose and HIF-1a signaling pathway intermediates despite not changing HIF-1a protein levels. After returning NK cells to normoxia for a few days, RNA transcripts were very similar between the normoxic and hypoxia-exposed NK cells, indicating that while this hypoxia-induced increase in cytotoxicity is consistent, alterations of the transcriptional signature may not be imprinted.Conclusions Exposure to hypoxia can increase the cytotoxicity of ex vivo expanded NK cells against multiple tumor cell lines in vitro. However, the duration of this enhanced effect, as well as its durability in an in vivo model, will need to be tested further before this effect can be used clinically to enhance NK cell therapies.References Kennedy PR, Arvindam US, Phung SK, Ettestad B, Feng X, Li Y , et al. Metabolic programs drive function of therapeutic NK cells in hypoxic tumor microenvironments. Sci Adv. 2024;10.Teng R, Wang Y, Lv N, Zhang D, Williamson RA, Lei L, et al. Hypoxia impairs NK cell cytotoxivity through SHP-1-mediated attenuation of STAT3 and ERK signaling pathways. J Immunol Res. 2020;2020.Coulibaly A, Velaszquez SY, Kassner N, Schulte J, Barbarossa MV, and Lindner HA. STAT3 governs the HIF-1a response in IL-15 primed human NK cells. Sci Rep. 2021;11:7023.Lim SA, Moon Y, Shin MH, Kim T, Chae S, Yee C, et al. Hypoxia-driven HIF-1a activation reprograms pre-activated NK cells toward highly potent effector phenotypes via ERK/STAT3 pathways. Cancers. 2021;13:1904.",
  "authors": [
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec company, Gaithersburg, MD, USA"
      ],
      "name": "Nicole Bonan"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec company, Gaithersburg, MD, USA"
      ],
      "name": "Nancy D Marin"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec company, Gaithersburg, MD, USA"
      ],
      "name": "Shahabuddin Alam"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec company, Gaithersburg, MD, USA"
      ],
      "name": "Pulak R Nath"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec company, Gaithersburg, MD, USA"
      ],
      "name": "Pradyot Dash"
    }
  ],
  "title": "390 Hypoxia enhances the cytotoxicity of ex vivo expanded NK cells against multiple tumor cell lines",
  "uid": "c305c773-ab1d-5244-8fd9-52f195c78174"
}