CD147 is a broadly expressed cell surface glycoprotein of the Ig superfamily whose expression is up-regulated upon T cell activation. In order to elucidate a possible role of CD147 in T cell biology, we established 15 specific mAb. Seven distinct epitopes were defined by the mAb panel. Most of the mAb bound only to phytohemagglutinin (PHA)-activated but not resting T cells. We demonstrate that this was not because of true expression of activation-dependent neoepitopes but rather due to bivalent binding of the relatively low-affinity mAb (affinity constant KA values between 2.25 x 10(8) and 7 x 10(9) M-1) to the more densely expressed and/or more clustered CD147 molecules on the activated T cells. In contrast, the mAb with higher affinity (KA > 7 x 10(9) M-1) could stably bind in a monovalent fashion even to the relatively low dense CD147 molecules on resting T cells. This model might more generally explain the nature of 'activation epitopes' described previously in other leukocyte surface molecules. Finally, we provide evidence that induction of ordered dimerization of CD147 by a mAb directed to a unique epitope results in strong inhibition of CD3-mediated T cell activation.

AimsProprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising therapeutic target for the treatment of hypercholesterolaemia and atherosclerosis. PCSK9 binds to the low density lipoprotein receptor and enhances its degradation, which leads to the reduced clearance of low density lipoprotein cholesterol (LDLc) and a higher risk of atherosclerosis. In this study, the AT04A anti-PCSK9 vaccine was evaluated for its therapeutic potential in ameliorating or even preventing coronary heart disease in the atherogenic APOE*3Leiden.CETP mouse model.Methods and resultsControl and AT04A vaccine-treated mice were fed western-type diet for 18 weeks. Antibody titres, plasma lipids, and inflammatory markers were monitored by ELISA, FPLC, and multiplexed immunoassay, respectively. The progression of atherosclerosis was evaluated by histological analysis of serial cross-sections from the aortic sinus. The AT04A vaccine induced high and persistent antibody levels against PCSK9, causing a significant reduction in plasma total cholesterol (−53%, P < 0.001) and LDLc compared with controls. Plasma inflammatory markers such as serum amyloid A (SAA), macrophage inflammatory protein-1β (MIP-1β/CCL4), macrophage-derived chemokine (MDC/CCL22), cytokine stem cell factor (SCF), and vascular endothelial growth factor A (VEGF-A) were significantly diminished in AT04A-treated mice. As a consequence, treatment with the AT04A vaccine resulted in a decrease in atherosclerotic lesion area (−64%, P = 0.004) and aortic inflammation as well as in more lesion-free aortic segments (+119%, P = 0.026), compared with control.ConclusionsAT04A vaccine induces an effective immune response against PCSK9 in APOE*3Leiden.CETP mice, leading to a significant reduction of plasma lipids, systemic and vascular inflammation, and atherosclerotic lesions in the aorta.

Tamm-Horsfall glycoprotein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in mammalian urine. A critical role for THP in antibacterial host defense and inflammatory disorders of the urogenital tract has been suggested. We demonstrate that THP activates myeloid DCs via Toll-like receptor-4 (TLR4) to acquire a fully mature DC phenotype. THP triggers typical TLR signaling, culminating in activation of NF-kappaB. Bone marrow-derived macrophages from TLR4- and MyD88-deficient mice were nonresponsive to THP in contrast to those from TLR2- and TLR9-deficient mice. In vivo THP-driven TNF-alpha production was evident in WT but not in Tlr4-/- mice. Importantly, generation of THP-specific Abs consistently detectable in urinary tract inflammation was completely blunted in Tlr4-/- mice. These data show that THP is a regulatory factor of innate and adaptive immunity and therefore could have significant impact on host immunity in the urinary tract.

Polyunsaturated fatty acids (PUFAs) suppress immune responses and inhibit T cell activation through largely unknown mechanisms. The displacement of signaling proteins from membrane lipid rafts has recently been suggested as underlying PUFA-mediated T cell inhibition. We show here that PUFA treatment specifically interferes with T cell signal transduction by blocking tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase C␥1. A significant fraction of LAT was displaced from rafts by PUFA treatment along with other signaling proteins. However, retaining LAT alone in lipid rafts effectively restored phospholipase C␥1/calcium signaling in PUFA-treated T cells. These data reveal LAT displacement from lipid rafts as a molecular mechanism by which PUFAs inhibit T cell signaling and underline the predominant importance of LAT localization in rafts for efficient T cell activation. Polyunsaturated fatty acids (PUFAs)1 suppress immune responses (1) and are clinically applied as adjuvant immunosuppressive agents in the treatment of inflammatory disorders (e.g. rheumatoid arthritis and inflammatory bowel disease) (2, 3) and following organ transplantation (4). PUFAs of the n-3 series have been shown to be particularly effective and are known to interfere with proinflammatory eicosanoid (prostaglandin/leukotriene) synthesis (1). However, PUFA-mediated inhibition of T lymphocyte activation and function has repeatedly been shown to be independent of eicosanoid synthesis (5-8). The molecular mechanism by which PUFAs inhibit T cell signal transduction has remained largely unknown. Because PUFA treatment changes the lipid composition of lymphocyte membranes (9) and functionally important membrane lipid rafts (10), alterations of rafts have been suggested as underlying the PUFA-mediated inhibition of T cell signaling (11).Membrane lipid rafts or microdomains are specialized regions within the plane of the plasma membrane and have been proposed as playing an essential role in T cell signal transduction (12-16). Lipid rafts are characterized by a high concentration of cholesterol and sphingolipids such as sphingomyelin and glycolipids, and their polar lipids contain predominantly saturated fatty acyl residues (17, 18). Such lipids spontaneously form liquid-ordered membrane regions that are insoluble in non-ionic detergents, facilitating raft isolation as detergentresistant membrane domains (18,19). Cytoplasmic and transmembrane proteins are targeted to lipid rafts mostly by acylation with saturated fatty acyl moieties (palmitoyl and myristoyl) which link these proteins to the cytoplasmic lipid leaflet of microdomains (20). Several proteins involved in T cell signaling such as Src family protein-tyrosine kinases (PTKs) and LAT (linker for activation of T cells) are concentrated in rafts because of post-translational palmitoylation, emphasizing the role of lipid rafts in T cell signaling (14, 21).The activation of T cells requires stimulation of the T cell antigen receptor (TCR)/CD3 complex. Triggering CD3...

BackgroundLow Density Lipoprotein (LDL) hypercholesterolemia, and its associated cardiovascular diseases, are some of the leading causes of death worldwide. The ability of proprotein convertase subtilisin/kexin 9 (PCSK9) to modulate circulating LDL cholesterol (LDLc) concentrations made it a very attractive target for LDLc management. To date, the most advanced approaches for PCSK9 inhibition are monoclonal antibody (mAb) therapies. Although shown to lower LDLc significantly, mAbs face functional limitations because of their relatively short in vivo half-lives necessitating frequent administration. Here, we evaluated the long-term efficacy and safety of PCSK9-specific active vaccines in different preclinical models.Methods and FindingPCSK9 peptide-based vaccines were successfully selected by our proprietary technology. To test their efficacy, wild-type (wt) mice, Ldlr +/− mice, and rats were immunized with highly immunogenic vaccine candidates. Vaccines induced generation of high-affine PCSK9-specific antibodies in all species. Group mean total cholesterol (TC) concentration was reduced by up to 30%, and LDLc up to 50% in treated animals. Moreover, the PCSK9 vaccine-induced humoral immune response persisted for up to one year in mice, and reduced cholesterol levels significantly throughout the study. Finally, the vaccines were well tolerated in all species tested.ConclusionsPeptide-based anti-PCSK9 vaccines induce the generation of antibodies that are persistent, high-affine, and functional for up to one year. They are powerful and safe tools for long-term LDLc management, and thus may represent a novel therapeutic approach for the prevention and/or treatment of LDL hypercholesterolemia-related cardiovascular diseases in humans.