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

Abstract

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 complete and durable responses. Investigations into strategies to further enhance the cancer killing capacity of these cells are therefore crucial in the effort to improve immunotherapies. One avenue for manipulation of CTLs is through alterations to cellular metabolic programs. T cells radically alter their metabolism upon activation. Prior studies have shown pan-inhibition of glutaminolysis using the inhibitor 6-Diazo-5-oxo-l-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. Herein, I utilize 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-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) – the enzyme responsible for catalyzing the conversion of glutamate to glutamine – conferred a fitness advantage to CTLs. In future experiments, inhibition and genetic deletion of GS will be utilized to investigate the role of GS in CTL activation, proliferation, effector cytokine production, and memory formation.