CD46 is a cell surface complement inhibitor widely expressed in human tissues in contrast to mice where expression is limited to the testes. of CD28. We also identify a downregulation of microRNA-150 in CD46-costimulated T cells and Narirutin identify Narirutin the glucose transporter-1 (GLUT1) encoding transcript SLC2A1 as a target of microRNA-150 regulation connecting microRNA-150 with modulation of glucose uptake. We also investigated microRNA expression profiles of Narirutin CD46-induced ‘switched’ IL-10-secreting Th1 T cells and found increased expression of microRNA-150 compared to IFN-γ-secreting Th1 cells. Knockdown of microRNA-150 led to a reduction in IL-10 but not IFN-γ. CD46 therefore controls both Th1 activation and regulation via a miRNA-150-dependent mechanism. and measles virus (14). CD46 is expressed on all human nucleated cells but absent in murine somatic tissues (15) meaning that murine-based immunological studies do not contribute to the understanding of the role of CD46 in human T cell responses. As a complement inhibitor CD46 binds C3b and acts as co-factor for its inactivation by the serine protease factor I. C3b can be generated in an autocrine fashion by activated T-cells and has also been identified as a ligand for the T cell costimulatory function of CD46 (13 16 This activation-associated T cell-derived generation of C3b and dependence of human CD28-costimulated Th1 responses on CD46/C3 leads to a model of autocrine CD46 signaling functioning downstream of CD28 during human Th1 cell activation. The clinical importance of CD46-mediated regulation of Th1 responses is supported by the altered expression of CD46 isoforms in T cells from multiple sclerosis patients (17) and by the failure of T cells from patients with rheumatoid arthritis to develop the full IL-10-secreting regulatory phenotype upon sustained CD46 costimulation compared to healthy controls (10). It is therefore of interest to investigate and potentially therapeutically harness the mechanisms by which this mode of immunoregulation functions. The downstream molecular effector pathways are still incompletely mapped and we have focused on CD46-mediated alterations in microRNA (miRNA) expression. MiRNAs have important roles as regulators of immune cell differentiation and function (18) and more specifically have Rabbit polyclonal to IL11RA. been shown to affect T cell regulation development signaling and metabolism (19-21). Several miRNAs have high specificity of expression in lymphocytes Narirutin and their expression is required for normal lymphocyte function. We found that CD46 signaling in CD4+ T cells leads to a strong reduction in Narirutin miRNA-150 (miR-150) levels and we then identified miR-150 targets which include regulators of T cell metabolism and cytokine secretion. Furthermore miR-150 is required for IL-10 secretion from CD46-stimulated Th1 cells. We therefore highlight the role of miR-150 in CD46-induced Th1 activation and regulation. Materials and methods Purification and activation of T cells All primary cells were purified from fresh peripheral blood collected from healthy volunteers according to the permission of the local ethics committee in Lund and with informed written consent. Blood was taken using EDTA-coated vacuum tubes diluted in PBS EDTA at room temperature and PBMCs purified using Lymphoprep (Axis Shield) according to manufacturer’s instructions. CD4+ T cells were then purified using positive selection magnetic cell sorting (Miltenyi biotech) and purity (above 95%) verified by staining with Allophycocyanin-labeled anti-CD4 (Immunotools). Cells were washed and resuspended in RPMI (Invitrogen) with 10% FCS and 50 U/ml IL-2 (Immunotools) and 3.5×105 plated out per well in 48 well plates coated overnight with 2 μg/ml anti-CD3 (OKT3 BD biosciences) and 2 μg/ml of either anti-CD28 (CD28.1 BD Biosciences) or anti-CD46 (Tra2-10 Sheffield university hybridoma biobank UK). Antibodies and Proteins Anti-CD25-FITC anti-CD69-Allophycocyanin and anti-CD46-Phycoerthyrin (PE) (Immunotools) were used to assess CD antigen expression. Fc-CD46 and Fc-CD55 were expressed in CHO cells and purified on protein A columns Narirutin as described in (22). For flow cytometry of purified T cells viable cells were gated by exclusion of cells stained with fluorophore-labeled AnnexinV (Immunotools). Cytokine detection Cytokine secretion was measured using Miltenyi biotech flow cytometry cytokine capture kits for IL-10 and IFN-γ according to manufacturer’s instructions. Alternatively cytokines in supernatant were measured by ELISA (Peprotech/Mabtech). RNA.