Identifying differential metabolic dependencies in CD4+ T cell subsets using targeted in vivo CRISPR/Cas9-based screening
T cell metabolic programming is dynamic and shifts to support the energetic, biosynthetic, and signaling demands of T cell inflammatory or regulatory functions. Failure to meet these metabolic requirements can cause T cell dysfunction and immunological disease. Conversely, metabolic pathways can be targeted to reestablish homeostasis in an immunometabolic approach to immunotherapy. The Rathmell Lab and others have shown that CD4+ T cell subsets are fueled by distinct metabolic programs, and that each displays differential dependencies on specific pathways and genes. Methods for systematic evaluation of these dependencies, however, have been largely lacking thus far. Here we have endeavored to identify these subset-specific metabolic dependencies. First, we examined amino acid and transporter requirements in CD4+ T cells, identifying cystine uptake, the xCT transporter, and ROS regulation to be critical to effector T cells. This supported the significance of nutrient availability in the microenvironment in shaping T cell metabolism and function and shed light on the advantages of in vivo over in vitro models for immunometabolism studies. Next, we describe the development and application of an unbiased in vivo CRISPR/Cas9-based screening workflow to test targeted metabolic pathways of interest in primary CD4+ T cells in vivo. We demonstrated the efficacy of this approach in a screen using an EAE model with a glutamate metabolism targeted library that identified SNAT1 to be specifically critical in pathogenic Th1 cells but not Th17 cells. We further showed that SNAT1 loss in Th1 cells led to impaired proliferation, which was mediated by decreased intracellular glutamine and dampened mTORC1 signaling with associated shift in the metabolic program from glycolysis to mitochondrial respiration. A second screen using a lung inflammation model with a one carbon metabolism targeted library identified MTHFD2 as a potential anti-inflammatory drug target. Specifically, we showed that MTHFD2 deficiency was associated with decreased proliferation and effector function, Th17 cell transdifferentiation with induction of FoxP3 expression and acquisition of suppressive function, and enhanced Treg cell differentiation. These data suggest that MTHFD2 may function as a metabolic checkpoint in the Th17-Treg cell axis, with MTHFD2 deficiency shifting the balance from pro-inflammatory to anti-inflammatory phenotype. Indeed, inhibition or genetic ablation of MTHFD2 ameliorated disease severity in multiple in vivo models of inflammation and autoimmunity. As demonstrated here, in vivo CRISPR screening allows for the interrogation of metabolic pathways in primary T cells in a systematic and unbiased manner to identify physiologically relevant metabolic dependencies. This may in turn lead to development of more efficacious immunotherapies with fewer adverse effects than those currently available to improve patient care.