Improving anti-tumor CD8+ T cell function through manipulating glutamine metabolism

Abstract Immunotherapies that bolster the anti-tumor effects of cytotoxic CD8 T lymphocytes (CTLs) have improved outcomes for many, yet most patients fail to achieve durable responses. Investigations into strategies to further enhance the cancer killing capacity of these cells are crucial in the effort to improve immunotherapies. T cells radically alter their metabolism upon activation, and prior studies have shown pan-inhibition of glutamine metabolism (glutaminolysis) using the inhibitor 6-Diazo-5-oxo-norleucine (DON) enhances the function of CTLs in the tumor microenvironment. However, DON’s toxicity combined with this drug’s lack of specificity demonstrate a need for a more directed glutaminolysis targeting approach. I utilized a targeted glutaminolysis CRISPR library to investigate the role of individual enzymes targeted by DON. To interrogate the importance of these enzymes specifically in anti-tumor CTLs, I developed a model in which CRISPR-library edited antigen-specific CTLs were adoptively transferred into tumor-bearing mice. After 7 days of in vivo selection, next generation sequencing was performed on isolated CTLs to evaluate the survival advantage or disadvantage of these gene knockouts. While multiple DON targets resulted in decreased CTL fitness, deletion of the gene encoding for glutamine synthetase (GS) – which converts glutamate to glutamine – conferred an advantage to CTLs. Upon inhibition of GS, cells may increase cellular levels of glutamate, and preliminary data suggest this results in a decrease in glycolysis and increase in mitochondrial respiration capacity. Lastly, in vitro data demonstrate increased inflammatory cytokine production of CTLs upon genetic knockout or pharmacologic inhibition of GS. Supported by grants from NIH (T32 GM007347, R01 CA217987)