PROJECT SUMMARY Obesity has become major problem in western society, affecting ~40% of adults and 20% of children in the US alone, according to the Center for Disease Control and Prevention (CDC). It is well known that obesity can lead to insulin resistance, type II diabetes, and cardiovascular disease, termed “metabolic syndrome”, and that obesity is a major risk factor for leading causes of preventable death such as heart attack and stroke. A key factor driving metabolic disease in obesity is chronic low-grade inflammation in the visceral adipose tissue (VAT). A population of regulatory T cells (Tregs), with a unique transcriptional signature and clonally expanded TCR repertoire, is highly enriched in the VAT and plays an important role in controlling tissue inflammation. However, this population is significantly reduced in obesity, which directly promotes VAT inflammation and symptoms of metabolic disease. Multiple lines of evidence suggest that disruptions in cholesterol homeostasis (CH) is one mechanism promoting a reduction in VAT Tregs during obesity. First, previously published data shows that transcripts associated with CH are reduced in VAT Tregs following long-term high fat diet (HFD) feeding. Second, preliminary data shows that Treg-specific loss of SREBP2 (Srebf2), the master regulator of CH, significantly reduces Tregs in the VAT, but not in other tissues, at steady state and increases HFD-induced VAT inflammation and insulin resistance. Finally, multiple subsets of VAT Tregs have been previously identified which differ in their extent of T cell receptor (TCR) clonal expansion, and preliminary data also shows that Treg-specific loss of SREBP2 alters the subset composition of the VAT Treg compartment, based on key surface markers, and reduces antigen-induced expansion of VAT Tregs. Thus, this proposal hypothesizes that disrupting Treg- intrinsic CH reduces VAT Treg proliferation by impairing TCR signaling and altering subset composition and TCR clonal expansion. To test this, a unique TCR-transgenic mouse model containing a VAT-Treg derived TCR, where Tregs preferentially accumulate in the VAT, will be utilized. Combining this model with ex vivo CRISPR- Cas9 gene ablation and adoptive transfer, allows for the unique advantage of efficiently assessing the impact of ablating genes of interest on VAT Treg accumulation. This model in conjunction with germline knockout models, in vivo and in vitro assays, and single cell -omics approaches will allow for assessing the importance of cellular CH in VAT Tregs. AIM 1 of this proposal will determine the mechanism by which disrupting Treg-intrinsic CH impacts VAT Treg accumulation by assessing Srebf2-sufficient and -deficient VAT Treg proliferation, cell death, and cell recruitment to the tissue, and by identifying the relative importance of cholesterol biosynthesis and uptake for VAT Treg accumulation. AIM 2 will assess the impact of disrupting Treg-intrinsic CH on VAT Treg TCR signaling and o...