title: EphB and Ephrin-B interactions mediate human mesenchymal stem cell suppression of activated T-cells.
abstract: Mesenchymal stromal/stem cells (MSC) express the contact-dependent erythropoietin-producing hepatocellular (Eph) receptor tyrosine kinase family and their cognate ephrin ligands, which are known to regulate thymocyte maturation and selection, T-cell transendothelial migration, activation, co-stimulation, and proliferation. However, the contribution of Eph/ephrin molecules in mediating human MSC suppression of activated T-cells remains to be determined. In the present study, we showed that EphB2 and ephrin-B2 are expressed by ex vivo expanded MSC, while the corresponding ligands, ephrin-B1 and EphB4, respectively, are highly expressed by T-cells. Initial studies demonstrated that EphB2-Fc and ephrin-B2-Fc molecules suppressed T-cell proliferation in allogeneic mixed lymphocyte reaction (MLR) assays compared with human IgG-treated controls. While the addition of a third-party MSC population demonstrated dramatic suppression of T-cell proliferation responses in the MLR, blocking the function of EphB2 or EphB4 receptors using inhibitor binding peptides significantly increased T-cell proliferation. Consistent with these observations, shRNA EphB2 or ephrin-B2 knockdown expression in MSC reduced their ability to inhibit T-cell proliferation. Importantly, the expression of immunosuppressive factors, indoleamine 2, 3-dioxygenase, transforming growth factor-β1, and inducible nitric oxide synthase expressed by MSC, was up-regulated after stimulation with EphB4 and ephrin-B1 in the presence of interferon (IFN)-γ, compared with untreated controls. Conversely, key factors involved in T-cell activation and proliferation, such as interleukin (IL)-2, IFN-γ, tumor necrosis factor-α, and IL-17, were down-regulated by T-cells treated with EphB2 or ephrin-B2 compared with untreated controls. Studies utilizing signaling inhibitors revealed that inhibition of T-cell proliferation is partly mediated through EphB2-induced ephrin-B1 reverse signaling or ephrin-B2-mediated EphB4 forward signaling by activating Src, PI3Kinase, Abl, and JNK kinase pathways, activated by tyrosine phosphorylation. Taken together, these observations suggest that EphB/ephrin-B interactions play an important role in mediating human MSC inhibition of activated T cells.
authors.affiliation: 1 Mesenchymal Stem Cell Laboratory, School of Medical Sciences, Faculty of Health Sciences, University of Adelaide , Adelaide, SA, Australia .
authors.forename: Thao M
authors.initials: TM
authors.lastname: Nguyen
authors.forename: Agnes
authors.initials: A
authors.lastname: Arthur
authors.forename: John D
authors.initials: JD
authors.lastname: Hayball
authors.forename: Stan
authors.initials: S
authors.lastname: Gronthos
mesh_terms: D049109:Cell Proliferation; D018920:Coculture Techniques; D036388:Ephrin-B2 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000235:genetics / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism / Q000494:pharmacology; D020381:Interleukin-17 / Q000235:genetics / Q000378:metabolism; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D008213:Lymphocyte Activation; D007959:Lymphocyte Culture Test, Mixed; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D061251:Primary Cell Culture; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D036183:Receptor, EphB2 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D036224:Receptor, EphB4 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism
chemical_list: D036388:Ephrin-B2; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D020381:Interleukin-17; D007376:Interleukin-2; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; D007371:Interferon-gamma; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; C586310:EPHB2 protein, human; D036183:Receptor, EphB2; D036224:Receptor, EphB4