Natural killer (NK) cells are key effectors in cancer immunosurveillance, and they can eliminate tumor cells through the release of cytotoxic granules triggered by interactions with natural ligands or through antibody-dependent cellular cytotoxicity (ADCC). NK cell-based treatment has had therapeutic success for hematologic malignancies, but strategies to treat solid tumors have been more limited. One of the biggest barriers to NK cell immunotherapy is immunosuppression within the tumor microenvironment. An important aspect of this immunosuppression is the highly hypoxic environment created by proliferating tumor cells. To further study the effect of hypoxia on NK cell function, our lab has been collaborating with XCell Biosciences to use their novel incubators that precisely control oxygen and pressure levels. We incubated NK cells isolated from peripheral blood mononuclear cells (PBMCs) at three different oxygen (O2) levels: 12% O2 to mimic the peripheral blood, 5% O2 to mimic the bone marrow, and 1% O2 to mimic the solid tumor microenvironment. All comparisons were made against a standard incubator condition. NK cells were incubated for three different time points—24 hours, 72 hours, and 7 days—and activated with low dose IL-15. We conducted a mass cytometry analysis to assess phenotype of PBMCs and enriched NK cells incubated at the different oxygen conditions. NK cells from the 1% O2 condition express fewer activating receptors (NKG2D, Nkp30, Nkp46, DNAM-1) and less perforin and granzyme than NK cells from the higher oxygen conditions. However, the NK cells from 1% O2 conditions express high levels of the activating receptors CD69 and CD16 (which triggers ADCC). This altered activation state is reflected in cell-killing assays using an IncuCyte machine. Here, fluorescently labeled target cells and NK cells are incubated together and images are taken for 48 hours to measure target cell death. We observed that natural cytotoxicity against K562 targets is severely impaired in the NK cells at the lowest oxygen condition but ADCC responses using the antibody rituximab against Raji targets remain intact. We observe a sharp decrease in proliferation, as measured by CellTrace dilution and Ki-67 expression in the NK cells in the 1% O2 condition. We also detect a decrease in glycolytic and mitochondrial metabolism, as measured by a Seahorse Analyzer, as the oxygen levels decrease. Supernatants collected from NK cells in the lower oxygen conditions (12% O2 and 5% O2) contain more inflammatory cytokines (IFNγ, TNFα, GM-CSF) and chemokines (CCL3, CCL4, CCL5) than NK cells in high oxygen conditions, indicating a shift from cytotoxic to a more immunomodulatory state. These results indicate that hypoxia changes the phenotype and function of NK cells. Overall, the insights gained from these experiments can help overcome hypoxia-induced immune suppression in the tumor microenvironment and enhance NK cell-based immunotherapy for solid tumors.

Citation Format: Upasana Sunil Arvindam, Pippa Kennedy, Bri Ettestad, Peter Hinderlie, Jeffrey S. Miller, Martin Felices. Hypoxia induces phenotypic and functional changes in NK cells which negatively impacts antitumor immunity in the solid tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B108.