Hypoxia profoundly alters Natural Killer cell phenotype and function: implications for immunotherapy within the solid tumor microenvironment

Abstract

Abstract For Natural Killer (NK) cell-based immunotherapy to succeed against solid tumors, NK cells need to enter a highly immunosuppressive tumor microenvironment (TME) and retain functionality. The objective of this study is to evaluate how hypoxia exerts an immunosuppressive effect on NK cells. We used the novel AVATAR™ 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). NK cells incubated at 1% O2 have decreased proliferation and cytotoxicity compared to NK cells incubated at higher oxygen conditions. To assess what contributes to these changes, we conducted a mass cytometry (CyTOF) analysis, real time imaging assays, metabolic assays and gene expression (RNA-seq and ATAC-seq) analysis. We observed oxygen dependent changes in expression of activating receptors, Ki-67, perforin and granzyme. Hypoxia impacts aggregation of perforin and granzyme granules at the immune synapse. Under hypoxic conditions, NK cell metabolism resembles cancer cell metabolism with increased glycolysis, amino acid synthesis and central carbon metabolism. These changes are accompanied by mitochondrial defects. Gene expression analysis revealed that changes in NK cell metabolism and function are mediated at the epigenetic level by histone demethylases. Treatment with histone demethylase inhibitors rescued NK cell cytotoxicity as measured using an IncuCyte machine. These results indicate that NK cells who enter the TME are fundamentally different than those in the bone marrow or blood stream. The insights gained from this study will be leveraged to overcome hypoxia induced immune suppression in the TME to enhance NK cell-based immunotherapy for solid tumors.