Metabolic Stress Caused by Inadequate Glutamine Uptake in Activated T Cells Can Drive Th2 Cytokine Production in NRF2-Dependent Fashion

Genome-wide association studies have identified members of the CARD11/MALT1/BCL10 (CBM) complex as susceptibility loci for common allergic disorders, while carriers with rare loss-of-function (LOF) variants in MALT1 or CARD11 can develop severe atopy and elevated T cell Th2 cytokine production, in addition to other immunopathology. Loss of CBM complex function impairs lymphocyte receptor–mediated activation of NFκB as well as glutamine uptake—thereby selectively reducing signaling in mTORC1, but not mTORC2. In tumor models, glutamine starvation results in mTORC2-dependent activation of NRF2—a known transcriptional inducer of Th2 cytokines. Because patients with NFkB deficiency do not develop significant Th2 phenotypes, while those with gain-of-function mutations in the PI3K/AKT/mTORC2 pathway can, we therefore investigated how glutamine deprivation and mTORC1/mTORC2 dysregulation might lead to Th2-associated allergic predisposition.CARD11 LOF mutations were introduced or corrected via CRISPR-Cas9 base editing of primary CD4+ T cells or studied in a CARD11 LOF mouse model. Cytokine production, glutamine uptake, reactive oxygen species (ROS), and NRF2 expression were measured by flow cytometry. Transcriptomics were measured via bulk RNA sequencing.We observed reduced glutamine uptake and reduced mTORC1 but not mTORC2 signaling upon TCR stimulation in primary patient CD4+ T cells, base-edited CD4+ T cells expressing patient mutations, or the addition of bioorthogonal glutamine competitors. These conditions, as well as glutamine starvation of dividing WT CD4+ T cells, resulted in increased Th2 phenotypes, while base editing of patient CD4+ T cells to WT or glutamine supplementation decreased Th2 phenotypes. Increased ROS production was detected in glutamine-deprived WT CD4+ T cells and in human and mouse CARD11 LOF CD4+ T cells, which was associated with higher NRF2 protein expression. Finally, small molecule inhibition or genetic knockdown of NRF2 or antioxidant treatment suppressed the elevated Th2 cytokine production in the CARD11 LOF and glutamine-deprived WT CD4+ T cells.Decreased CBM function or glutamine uptake may promote Th2 differentiation and cytokine production by triggering a stress response characterized by ROS production and NRF2 activation, which can drive Th2 phenotypes. Strategies to mitigate these processes may be helpful in treating CBM-associated disease and other forms of Th2-driven disease.