Microbiota‐induced expansion of the intraepithelial lymphocyte (IEL) compartment results in an altered bioenergetic profile and reduced IFNγ production in γδ IELs

Intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IEL) are located within the intestinal epithelium and serve as the first line of defense against pathogen invasion. We recently identified a novel hyperproliferative and hypermotile γδ IEL (γδHYP ) phenotype that can be transferred both horizontally and vertically to wildtype (WT) mice via the gut microbiota. Given the close relationship between metabolism and immune cell function, as well as the influence of commensals on T cell metabolism, we hypothesized that the bioenergetic potential of γδHYP IELs is increased to support this enhanced proliferation and motility. Transmission electron microscopy of sorted small intestinal γδ IELs revealed that γδHYP IELs exhibit a 70% increase in the number of mitochondria per cell (p=0.005) accompanied by a 24% increase in mitochondrial area (p=0.04) and 40% increase in aspect ratio (p=0.02) relative to WT. Since elongated mitochondria may be indicative of increased oxidative phosphorylation, we next performed Seahorse mitochondrial stress assays on sorted γδ IELs to assess the bioenergetic capacity of these lymphocytes. We find that γδHYP IELs exhibit a 50% increase in spare respiratory capacity compared to WT (p=0.014). Bulk RNA sequencing was performed on WT and γδHYP IELs and transcriptomic analysis revealed that γδHYP IELs upregulate genes involved in cell cycle (p<0.0001), as well as cholesterol and lipid metabolism (p<0.0001) compared to WT. Given the role of cholesterol and lipid metabolism in T cell activation and proliferation, we next analyzed intracellular cholesterol and lipid content within WT and γδHYP IELs using Filipin III and LipidTOX staining followed by flow cytometry. Interestingly, while there is a 3-fold increase in neutral lipids in γδHYP IELs (p<0.0001), cholesterol content is equivalent between WT and γδHYP IELs. Glucose and mitochondrial metabolism, as well as neutral lipid content, was recently shown to influence cytokine production by γδ T cells. Upon stimulation with phorbol myristate acetate (PMA) and ionomycin, the frequency of IFNγ+ γδ IELs was reduced by 59% with a 44% decrease in IFNγ mean fluorescence intensity (MFI) in γδHYP IELs relative to WT (p<0.0001). Although IFNγ production is reduced in γδHYP IELs, transcriptomic analysis revealed an increase in genes associated with antimicrobial defense including Reg3g (p<0.0001). In support of this, we find that γδHYP mice exhibit reduced bacterial burden in the spleen and liver 6 days post-infection with Salmonella Typhimurium compared to WT. In summary, our data demonstrate that the increased mitochondrial mass and oxidative phosphorylation observed in γδHYP IELs, in addition to increased neutral lipid accumulation, may contribute to this hyperproliferative and hypermotile phenotype and decreased IFNγ production. Further understanding of the metabolic mechanisms regulating γδ IEL homeostasis and effector function may ultimately allow fine tuning of mucosal surveillance to protect against intestinal injury or infection.