Education-dependent activation of glycolysis promotes the cytolytic potency of licensed human natural killer cells

Background: The mechanism by which natural killer (NK) cell education results in licensed NK cells with heightened effector function against missing self-targets is not known. Objective: We sought to identify potential mechanisms of enhanced function in licensed human NK cells. Methods: We used expanded human NK cells from killer immunoglobulin-like receptor (KIR)/HLA–genotyped donors sorted for single-KIR⁺ cells to generate pure populations of licensed and unlicensed NK cells. We performed proteomic and gene expression analysis of these cells before and after receptor cross-linking and performed functional and metabolic analysis before and after interference with selected metabolic pathways. We verified key findings using freshly isolated and sorted NK cells from peripheral blood. Results: We confirmed that licensed human NK cells are greater in number in peripheral blood and proliferate more in vitro than unlicensed NK cells. Using high-throughput protein analysis, we found that unstimulated licensed NK cells have increased expression of the glycolytic enzyme pyruvate kinase muscle isozyme M2 and after KIR cross-linking have increased phosphorylation of the metabolic modulators p38-α and 5′ adenosine monophosphate–activated protein kinase α. After cytokine expansion and activation, unlicensed NK cells depended solely on mitochondrial respiration for cytolytic function, whereas licensed NK cells demonstrated metabolic reprogramming toward glycolysis and mitochondrial-dependent glutaminolysis, leading to accumulation of glycolytic metabolites and depletion of glutamate. As such, blocking both glycolysis and mitochondrial-dependent respiration was required to suppress the cytotoxicity of licensed NK cells. Conclusions: Collectively, our data support an arming model of education in which enhanced glycolysis in licensed NK cells supports proliferative and cytotoxic capacity.