Atherosclerosis and its complications, such as myocardial infarction, stroke, and peripheral artery disease, are the leading cause of morbidity and mortality in the U.S. Therefore, novel therapies are urgently needed to inhibit the progression of atherosclerosis. CD4+Foxp3+ regulatory T cells (Treg) suppress vascular inflammation and atherosclerosis in both humans and mice. However, the mechanisms underlying Treg suppression of atherosclerosis remain poorly defined. Interleukin-35 (IL-35) is a newly characterized anti-inflammatory cytokine, mainly secreted from Treg, which inhibits inflammation resulting from various conditions, including autoimmune diabetes and arthritis, etc. Compared with the other well-characterized anti-inflammatory cytokines, IL-10 and TGF-β, IL-35 is more powerful, and IL-35 significantly converts effector T cells and B cells to inducible Treg and regulatory B cells. IL-35 is a heterodimer composed of p35 and Epstein-Barr virus induced gene 3 (EBI3) subunits. Both IL-35 subunits are strongly expressed in endothelial cells (EC) and macrophages (M) in patients' atherosclerotic plaques, suggesting that IL-35, but not IL-27, is upregulated in atherosclerotic lesions. Compared with healthy controls, IL-35 is decreased in the plasma of patients with coronary artery disease (CAD) while related cytokines IL-12 and IL-27 are increased in patients with CAD. However, the roles of IL- 35 in suppressing EC activation and atherogenesis have not been studied. The goal o is to determine the roles and mechanisms underlying IL-35 suppression of EC activation and atherosclerosis. We have a long publication record of studying EC activation, EC dysfunction, monocyte (MC) recruitment, atherosclerosis, and decreased Treg in acceleration of vascular inflammation. We have obtained strong preliminary data and published two papers (J.B.C.; PLOS ONE) showing that 1) IL-35 is a responsive cytokine, which is not constitutively expressed in most cell types and is significantly induced in lipopolysaccharide (LPS)-induced inflammation and in atherogenic apolipoprotein E (ApoE-/-) mice; 2) IL-35 shares EBI3 subunit with its relative cytokine, IL-27. In contrast to IL-27, IL-35 inhibits EC activation induced by proatherogenic Toll-like receptor 4 (TLR4) ligand, LPS, by suppressing the expression of vascular cell adhesion molecule 1 (VCAM-1) via mitogen-activated protein kinase (MAPK)- AP-1 dependent pathway in human aortic EC; 3) IL-35 inhibits lysophosphatidylcholine (lysoPC, an oxLDL- derived proatherogenic lipid stimulus)-induced EC activation; 4) Mechanistically, IL-35 inhibits lysoPC- induced generation of mitochondrial reactive oxygen species (mtROS) in human aortic EC; 5) We established an IL-35 therapy model in ApoE-/- mice and found that IL-35 therapy inhibits atherosclerosis; and finally, 6) we have generated EBI3-/-/ApoE-/- double knock-out (KO) mice and are also in the process of generating two additional double KO mice, including IL-12Rβ2 (a...