The liver X receptor (LXR) is a key transcriptional regulator of cholesterol, fatty acid, and phospholipid metabolism. Dynamic remodeling of immunometabolic pathways, including lipid metabolism, is a crucial step in T cell activation. Here, we explored the role of LXR-regulated metabolic processes in primary human CD4+ T cells and their role in controlling plasma membrane lipids (glycosphingolipids and cholesterol), which strongly influence T cell immune signaling and function. Crucially, we identified the glycosphingolipid biosynthesis enzyme glucosylceramide synthase as a direct transcriptional LXR target. LXR activation by agonist GW3965 or endogenous oxysterol ligands significantly altered the glycosphingolipid:cholesterol balance in the plasma membrane by increasing glycosphingolipid levels and reducing cholesterol. Consequently, LXR activation lowered plasma membrane lipid order (stability), and an LXR antagonist could block this effect. LXR stimulation also reduced lipid order at the immune synapse and accelerated activation of proximal T cell signaling molecules. Ultimately, LXR activation dampened proinflammatory T cell function. Finally, compared with responder T cells, regulatory T cells had a distinct pattern of LXR target gene expression corresponding to reduced lipid order. This suggests LXR-driven lipid metabolism could contribute to functional specialization of these T cell subsets. Overall, we report a mode of action for LXR in T cells involving the regulation of glycosphingolipid and cholesterol metabolism and demonstrate its relevance in modulating T cell function.

Liver X Receptors (LXRs) are important transcriptional regulators of intracellular cholesterol, fatty acid, and phospholipid levels. LXRs can dampen inflammation in macrophages by promoting cholesterol efflux and altering plasma membrane lipid rafts; microdomains enriched for cholesterol and glycosphingolipids that regulate immune-cell signalling. However, little is known about the impact of LXR activators on T cell plasma membrane lipid rafts, which are critical for T-cell antigen receptor (TCR) signalling. Here we show that the LXR synthetic agonist GW3965 significantly increases glycosphingolipid levels and reduces cholesterol and lipid order (stability) in the plasma membrane of human CD4 + T cells. Moreover, the GSL biosynthesis enzyme UGCG was identified as a novel direct target of LXR activation. Importantly, LXR-mediated changes in T cell lipid composition were associated with altered spatiotemporal distribution of lipids and signalling molecules at the immune synapse. Crucially, LXR activation altered the kinetics of proximal TCR-signalling events in activated T cells, reduced T cell proliferation and enhanced the production of IL-2 and IL-4. Thus, LXR activation influences T cell function via alteration of the plasma membrane lipid profile. Finally, the expression of UGCG and other LXR-regulated genes and lipids was significantly dysregulated in T-cells isolated from people with multiple sclerosis. Overall, a novel action of LXR has been characterised that involves modulation of lipid raft-associated cholesterol and glycosphingolipids in CD4 + T-cells, which could be of therapeutic relevance in multiple sclerosis. Keywords liver X receptor -CD4 + T cell -lipid metabolismcholesterolglycosphingolipid-multiple sclerosis 3