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RNA-seq analysis of NDP and F4L5.13 treated bEnd.3 endothelial cell line

ABSTRACT: RNA-seq analysis of NDP and F4L5.13 treated bEnd.3 endothelial cell line

PROVIDER: PRJNA701921 | ENA |

REPOSITORIES: ENA

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Project description:Sphingosine 1-phosphate (S1P) influences T cell migration into and out of secondary lymphoid organs; however, its mechanism of action remains uncertain. Our previous research shows that agonism of the S1P receptor S1P1 inhibits the egress of T lymphocytes from the peripheral tissues into afferent lymphatics. To better define the mechanism of inhibition, we developed an in vitro model to characterize T cell transendothelial migration across lymphatics. Two commercially available endothelial cell lines (MS-1 and SVEC4-10) were characterized by flow cytometry, real time RT-PCR, and Affymetrix Gene Array. These cell lines were grown to confluent monolayers in transwell systems, on either the upper or lower surface of the transwell insert. T cells were isolated from the spleens of (C57BL/6 x C3H/HeJ)F1, S1P1 KO, or S1P1 KO littermate controls, and either treated with the S1P receptor modulator FTY720 or left untreated. Cells were migrated to chemokines (CCL19 or CCL21) for 4 hours, and migration quantified. Flow cytometry, RT-PCR, and array results identified MS-1 as a blood vascular endothelial cell line, expressing high levels of CD31, CD34, and ICAM-1 as well as other endothelial cell markers; while SVEC4-10 closely resemble a lymphatic phenotype, expressing LYVE-1, VEGFR-3, and podoplanin. T cells efficiently migrate across MS-1, whether grown on the upper or lower surface; whereas migration across SVEC4-10 only occurs when cells are grown on the lower surface of the transwell (iSVEC), recapitulating basal (abluminal) to apical (luminal) migration that occurs in vivo. FTY720 inhibits T cell migration across iSVEC, but not across MS-1. Inhibition is due to drug effects only on T cells but not endothelial cells. S1P1 KO T cells treated with FTY720 are not inhibited in their migration across the iSVEC line, showing that S1P1 stimulation is required for migration inhibition. The in vitro model developed here is the first to use endothelial cell lines to analyze the regulation of T cell migration across lymphatic endothelium. The results show there is directional control of T cell migration across lymphatic cells, such that T cells only migrate from a basal to apical direction. Agonism of S1P1 specifically inhibits migration, while absence of the receptor does not. These findings have important implications for the use of S1P1 agonists in transplantation, as inhibition of cell entry into afferent lymphatics and lymph nodes could impede the development of graft rejection. We used microarrays to detail the global gene expression of these two cell lines in order to beter determine their phenotype as blood vascular or lymphatic endothelial cell line for use in our newly-developed in vitro model. Keywords: Cell type comparison

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RNA-seq analysis of NDP and F4L5.13 treated bEnd.3 endothelial cell line

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