Distinct Bioenergetic Features of Human Invariant Natural Killer T Cells Enable Retained Functions in Nutrient-Deprived States

Invariant natural killer T (iNKT) cells comprise a unique subset of lymphocytes that are primed for activation and possess innate NK-like functional features. Currently, iNKT cell-based immunotherapies remain in early clinical stages, and little is known about the ability of these cells to survive and retain effector functions within the solid tumor microenvironment (TME) long-term. In conventional T cells (T CONV ), cellular metabolism is linked to effector functions and their ability to adapt to the nutrient-poor TME. In contrast, the bioenergetic requirements of iNKT cells – particularly those of human iNKT cells – at baseline and upon stimulation are not well understood; neither is how these requirements affect effector functions such as production of cytokines and cytolytic proteins. We find that unlike T CONV , human iNKT cells are not dependent upon glucose or glutamine for these effector functions upon stimulation with anti-CD3 and anti-CD28. Additionally, transcriptional profiling revealed that stimulated human iNKT cells are less glycolytic than T CONV and display higher expression of fatty acid oxidation (FAO) and adenosine monophosphate-activated protein kinase (AMPK) pathway genes. Furthermore, stimulated iNKT cells displayed higher mitochondrial mass and membrane potential relative to T CONV . Real-time Seahorse metabolic flux analysis revealed that stimulated human iNKT cells utilize fatty acids as substrates for oxidation more than stimulated T CONV. Together, our data suggest that human iNKT cells possess different bioenergetic requirements from T CONV and display a more oxidative metabolic program relative to effector T CONV . Importantly, iNKT cell-based immunotherapeutic strategies could co-opt such unique features of iNKT cells to improve their efficacy and longevity of anti-tumor responses.