Metabolic programs drive function of therapeutic natural killer (NK) cells in low oxygen tumor microenvironments

Limited oxygen in solid tumors poses a challenge to successful immunotherapy with NK cells. NK cells have impaired cytotoxicity when cultured in hypoxia (1% oxygen), but not physiologic or atmospheric oxygen (>5%). We sought to analyze the impact of oxygen concentrations on human NK cell biology to determine what factors were driving loss of cytotoxicity and where there were opportunities to overcome hypoxia-induced dysfunction in NK cells. Human NK cells were enriched from healthy blood by magnetic beads selection and then incubated in advanced incubators that were humidified and maintained at 37 degrees Celsius, 5% CO2 with different oxygen (O2) and pressure conditions. Incubators were set to model oxygen and pressure levels found (1) in arterial blood (12% O2, + 2 psi), (2) in bone marrow (5% O2, + 0.6 psi), or (3-4) within the tumor microenvironment (1% O2) at low or high pressure (+ 0.3 psi or + 2 psi). NK cells incubated in a conventional humidified, 5% CO2 incubator (20% O2) served as a control (5). NK cells were extracted after 1, 3 or 7 days in culture. 4-5 biological replicates per condition were obtained from 6 independent donors