IFN (and mice (macrophage killing assay; and splenocytes were cultured in the presence of ovalbumin peptide257C264 for 6?days, subsequently CD8+ T cells were isolated and co-cultured with ovalbumin peptide257C264-pulsed CFSEhigh labelled bone marrow-derived macrophages and unpulsed CFSElow labelled bone marrow-derived macrophages (CD8+ T cells, incubation with OVA257C264-primed CD8+ T cells significantly reduced the survival of OVA257C264-pulsed BMDMs (Fmice. CD8+ T cell are the main drivers of atherogenesis in mice To further evaluate the contribution of CD8+ T cells to atherosclerosis, or bone marrow was transplanted into lethally irradiated recipient mice. initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques. Open in a separate CNX-2006 window mice, whereas antibody-mediated depletion of CD8+ T cells impedes CNX-2006 the formation of atherosclerotic lesions.3,5,6 Despite the well-described functions of T cell subsets in atherosclerosis, the regulatory mechanisms by which they undergo activation CNX-2006 and polarization during atherogenesis are less extensively studied. The (CBL) E3 ubiquitin ligasescomprising CBL-B, C-CBL, and CBL-Cform one of the protein families that modulate T cell activation and polarization.7promotes T cell tolerance through ubiquitination and degradation of downstream effectors, such as phosphoinositide phospholipase C and phosphoinositide 3-kinase, and thus is a negative regulator of T cell activation.7,8deficiency is linked to enhanced toll-like receptor (TLR)4 signalling and increased macrophage activation and migration in diet-induced obesity11 and lung inflammation models,12 processes that are also relevant for the atherosclerosis. Considering the significant regulatory activity of CBL-B in T cell and macrophage biology, we evaluated the expression pattern of CBL-B in human atherosclerotic lesions and investigated the function of CBL-B in experimental atherosclerosis. Translational perspective In this study, we demonstrate that the E3-ligase (CBL-B) is expressed in human atherosclerotic plaques, and that its expression decreases with plaque progression. Using an atherosclerotic mouse model, we found that CBL-B exerts profound anti-atherogenic effects by regulating CD8+ T cell and macrophage activation. Activation of CBL-B, therefore, represents a promising anti-inflammatory CNX-2006 therapeutic strategy in atherosclerosis. Methods Human studies Coronary artery specimens were obtained from autopsy from the Department of Pathology of the Amsterdam UMC and immediately fixed in 10% formalin and processed for paraffin embedding. All use of tissue was in agreement with the Code for Proper Secondary Use of Human Tissue in CNX-2006 the Netherlands. CBL-B expression was analysed by immunohistochemistry, as described in the Supplementary material online. Gene expression of CBL-B in human atherosclerosis was examined by microarray-based transcriptional profiling of carotid endarterectomy specimens (BiKE dataset13,14). Animal studies Male and mice were bred and housed at the animal facility of the University of Amsterdam and kept on a normal chow diet. All mice were treated according to the study protocol (permit nos. 102601 and 102869) that were approved by the Committee for Animal Welfare of the University of Amsterdam, the Netherlands. Detailed methods are provided in the Supplementary material online. Results Casitas B-cell lymphoma-B co-localizes with macrophages and T cells in human atherosclerotic plaques Human coronary atherosclerotic plaques, histologically classified as intimal xanthomas or pathological intimal thickenings (initial/intermediate atherosclerosis) expressed higher levels of CBL-B+ cells when compared with fibrous cap atheromata (advanced atherosclerosis) (is expressed in human atherosclerotic lesions and co-localizes with macrophages and T cells. (was not differentially expressed between atherosclerotic plaques from symptomatic and asymptomatic patients (data not shown), indicating that CBL-B predominantly affects plaque development and not plaque rupture. Casitas B-cell lymphoma-B deficiency aggravates atherosclerosis in Apoe?/? mice is expressed in CD68+ macrophages and CD3+ T cells in murine atherosclerotic plaques (Supplementary material online, and mice were generated and fed a normal chow diet for 20?weeks. The extent and phenotype of atherosclerosis was determined in the aortic arch and the aortic root (or mice. Open in a separate window Figure 2 deficiency aggravates atherosclerosis in mice. (((and mice (the brachiocephalic trunk is shown; haematoxylin and eosin staining). Scale bar: 50?m. (((and mice. Scale bar: 500?m. (Cmice contained significantly more CD45+ cells (and mice were not only larger (mice contained fewer CD68+ macrophages when compared with mice (HKmice (30.4??2.6% vs. 45.0??3.8% vs. 2.0??0.1% mice, we analysed the effects of CBL-B on monocytes and macrophages. Deficiency of CBL-B increased the expression of the chemokine receptors BBmonocytes and BMDMs exhibited an increased migratory capacity towards CCL2 (Ddeficiency induces an atherogenic phenotype in macrophages. Quantification of mRNA expression of chemokine receptors CCR1, 2, 5, and 7 in monocytes (((mice (and mice towards 10?ng/mL MCP-1 by transwell assay (((and Sox17 mice after 24?h exposure to oxLDL (BMDMs produced significantly more reactive oxygen species (ROS) (FHwas increased in aortic arch lysates of mice, the M2 markers and were not affected (Supplementary material online, and were not affected (Supplementary material online, and mice. Immunohistochemistry demonstrated a trend towards increased CD3+ T cell abundance in the advanced plaques of the aortic roots of mice (8.0??3.2% vs. 12.0??3.2%.