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. Overall design: 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