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Casein Kinase 1

van de Veen W

van de Veen W., Stanic B., Yaman G., Wawrzyniak M., S?llner S., Akdis D. exert a more diverse range of immune effector and regulatory functions. Distinct functional B cell subsets have been identified on the basis of their cytokine production profiles. Immunosuppressive B regulatory (reg) cells ((encoding IL-8), (score) log2 normalized counts of genes encoding secreted immunomodulatory proteins that are differentially expressed between proangiogenic B and nonangiogenic B cell clones (FDR 0.01, log2 fold change 0.5). The top box indicates genes with known proangiogenic effects, the middle box indicates genes with unknown or pleiotropic effects on angiogenesis, and the bottom box indicates genes with known anti-angiogenic effects. (B and C) Reads per kilobase million (RPKM) expression values from normal goat serum data (top) and real-time qPCR gene expression after prolonged ( 3 weeks) in vitro expansion (bottom) of proangiogenic (= 5) and nonangiogenic (= 5) clones (mean SEM). * 0.05 and Vipadenant (BIIB-014) ** 0.01, Mann-Whitney test. (B) Genes that were up-regulated in proangiogenic clones. (C) Genes that were down-regulated in proangiogenic clones. (D) Representative images of HUVEC tube formation assay to quantify proangiogenic effect of B cell clones (scale bars, 400 m). Negative control, IMDM +2% FCS; positive control, EGM medium with growth factors. (E) Quantitative analysis of rate of HUVEC tube formation induced by supernatants of pro- and nonangiogenic B cell clones (mean SEM). * 0.05 and ** 0.01, Mann-Whitney test. To assess the functional capacity of proangiogenic B cell clones, we tested their potential to promote tube formation of human umbilical vein endothelial cells (HUVECs) ((encoding CD112), (encoding CD73), CD276, (encoding CD49b), (encoding CD121a), and (encoding CD325) showed the most uniform differential expression profile with high expression on proangiogenic clones and low expression on nonangiogenic clones. Consistently up-regulated surface expression of CD49b and CD73 was observed on proangiogenic B cell clones by flow cytometry (Fig. 2B). CD49b and CD73 were also both expressed on a subset of peripheral B Vipadenant (BIIB-014) cells, while peripheral B cells did not express CD112, CD325, and CD276, and all B cells were positive for CD53 (Fig. 2C). On the basis of Vipadenant (BIIB-014) these data, CD49b and CD73 represented potential surface markers for the identification of proangiogenic B cells. Open in a separate window Fig. 2 Proangiogenic B cells are characterized by expression of CD49b and CD73.(A) Heat map showing gene-scaled (score) log2 normalized counts of CD markerCencoding genes that are differentially expressed between proangiogenic B and nonangiogenic B cell clones (FDR 0.01, log2 fold change 0.5). (B) Flow cytometry analysis of CD73 and CD49b surface expression on proangiogenic (black line) (= 5) and nonangiogenic (red line) B cell clones (= 20) (mean SEM). Grey dotted line indicates isotype control. * 0.05 and ** 0.01, Mann-Whitney test. (C) Flow cytometry analysis of surface expression of CD73 and CD49b on freshly isolated peripheral blood B cells. CD73+CD49b+ B cells form a distinct population among circulating B cells Staining of CD49b and CD73 on peripheral B cells from healthy individuals revealed a distinct CD73+CD49b+ RDX population (Fig. 3A). Real-time quantitative PCR (qPCR) mRNA expression analysis of proangiogenic cytokines by B cell populations sorted based on surface expression of CD49b and CD73 showed that the expression of was up-regulated in CD73+CD49b+ B cells compared to CD73?CD49b? B cells (Fig. 3B). Surface expression of CD39 as well as the Vipadenant (BIIB-014) VEGF receptor FLT1 was higher on CD73+CD49b+ B cells (Fig. 3C). The frequency of CD49b+ B cells was significantly increased after 3 days of in vitro stimulation of total B cells with CD40L + IL-21, whereas B cell stimulation with CD40L + IL-21 led to a reduction of CD73+ B cells (Fig. 3D). Open in a separate window Fig. 3 CD49b+CD73+ B cells form a distinct population of B cells and express proangiogenic cytokines.(A) Gating of CD49b+CD73+ B cells in PBMCs of healthy Vipadenant (BIIB-014) donor. (B) mRNA expression of proangiogenic cytokines in B cell populations sorted based on their expression of CD49b and CD73 (= 4). (C) Flow cytometric analysis of CD39 and FLT1 expression on CD49b+CD73+ B cells stained directly ex vivo. (D) Effect of 3-day in vitro stimulation of primary B cells on the expression of CD49b and CD73 (= 4). Proangiogenic B cells show increased frequencies in circulation and are present in esophageal tissue of patients with EoE To.