Next Generation Sequencing analysis using splenic CD8 T cells isolated from Wild Type-OT-I (WT) and NIK -CD4Cre-OT-I (KO) mice

Metabolic reprograming towards aerobic glycolysis is a pivotal mechanism that shapes immune responses. While deregulated T cell metabolism is associated with autoimmune diseases, metabolic deficiency contributes to T cell exhaustion in tumor microenvironment. Here we describe a posttranslational mechanism of glycolysis regulation mediated by the NF-kB-inducing kinase (NIK). NIK deficiency impairs glycolysis induction, rendering CD8 effector T cells hypofunctional with features of exhaustion in tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8 T cell metabolism and prevents exhaustion, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. Interestingly, although NIK is known as a kinase mediating activation of noncanonical NF-kB, NIK regulates T cell metabolism via an NF-kB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK deficiency causes autophagic degradation of HK2, at least in part due to aberrant ROS accumulation. NIK phosphorylates, and maintains the activity of, glucose-6-phosphate dehydrogenase (G6PD), an enzyme mediating production of the antioxidant NADPH required for preventing ROS accumulation and oxidative stress. We provide genetic evidence that the G6PD-NADPH redox system has a vital role in regulating HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a posttranslational mechanism of metabolic regulation involving the G6PD-NADPH redox system. Overall design: Splenic naïve CD8 T cells cultured with plate-bould anti-CD3 (1ug/ml) plus solube anti-CD28 (1ug/ml) for 24 hours. mRNA profiles of 6 to 8 week old wild type-OT-I (WT) and NIK-CD4Cre-OT-I (KO) mice were generated by deep sequencing, in triplicate