675 Oxygen concentration alters natural killer cell phenotype and function in the solid tumor microenvironment

Background Natural Killer (NK) cells can eliminate cancer cells through the release of cytotoxic granules triggered by interactions with natural ligands or through antibody-dependent cellular cytotoxicity (ADCC).1–3 NK cell-based treatments have had therapeutic success for hematological malignancies but strategies to treat solid tumors have been limited due to immunosuppression within the tumor microenvironment (TME).4–6 An important and understudied aspect of NK cell immunosuppression is the low oxygen (hypoxia) environment created by proliferating tumor cells. We used the novel AVATAR incubator system to model oxygen levels of three key tissues that NK cells inhabit in vivo: the peripheral blood (12% O2), the bone marrow (5% O2 ) and the TME (1% O2). Methods NK cells were incubated in the AVATAR incubators for 24 hours, 72 hours and 7 days. We conducted a mass cytometry (CyTOF) analysis to assess phenotype, flow cytometry-based assays to assess proliferation and an IncuCyte machine and immunofluorescent imaging to measure cytotoxicity of NK cells incubated at different oxygen conditions. We evaluated NK cell metabolism using Seahorse assays, gene expression using RNA-Seq and are in the process of evaluating epigenetic regulation using ATAC-Seq. Results NK cells from the 1% O2 condition express fewer activating receptors (CD16, NKG2D, Nkp30, Nkp46, DNAM-1) and less perforin and granzyme than NK cells from the higher oxygen conditions (figure 1). NK cells in the 1% O2 condition also have decreased aggregation of perforin and granzyme granules at the immune synapse. This translates to reduced natural cytotoxicity and ADCC responses against tumor targets (figure 2). We also observe a sharp decrease in proliferation in the NK cells at 1% O2 (figure 3). This is partly due to an increase in CISH gene expression that makes the cells less responsive to cytokine stimulation.7 The RNA-Seq analysis revealed that NK cell metabolism closely resembles cancer cell metabolism under hypoxic conditions, specifically an increased expression of genes related to glycolysis, amino acid synthesis and central carbon metabolism. This change in metabolism was confirmed using Seahorse assays. We also observed changes in genes related to epigenetic regulation specifically, increases in histone demethylases and decreases in DNA methyltransferases (figure 4). Conclusions These results indicate that NK cells who enter the solid TME are fundamentally different than those in the bone marrow or the blood stream. Overall, the insights gained from these experiments can help overcome hypoxia induced immune suppression in the tumor microenvironment and improve NK cell-based immunotherapy for solid tumors. Acknowledgements We thank XCell biosciences for providing us with the AVATAR incubators used for these experiments Trial Registration N/A Ethics Approval N/A Consent N/A References Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol 2008;9(5):503–10. Waldhauer I, Steinle A. NK cells and cancer immunosurveillance. Oncogene. 2008;27(45):5932–43. Voskoboinik I, Smyth MJ, Trapani JA. Perforin-mediated target-cell death and immune homeostasis. Nat Rev Immunol 2006;6(12):940–52. Miller JS, Soignier Y, Panoskaltsis-mortari A, Mcnearney SA, Yun GH, Fautsch SK, et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005;105(8):3051–8. Romee R, Cooley S, Berrien-Elliott MM, Westervelt P, Verneris MR, Wagner JE, et al. First-in-human phase 1 clinical study of the IL-15 superagonist complex ALT-803 to treat relapse after transplantation. Blood 2018;131(23):2515–2527. Bjorklund AT, Carlsten M, Sohlberg E, Liu LL, Clancy T, Karimi M, et al. Complete remission with reduction of high-risk clones following haploidentical NK-Cell therapy against MDS and AML. Clin Cancer Res 2018;24(8):1834–1844. Delconte RB, Kolesnik TB, Dagley LF, Rautela J, Shi W, Putz EM, et al. CIS is a potent checkpoint in NK cell–mediated tumor immunity. Nat Immunol 2016;17(7):816–24.