Semaphorin 7A Promotes Chemokine-Driven Dendritic Cell Migration Anoek van Rijn, Leonie Paulis, Joost te Riet, Angela Vasaturo, Inge Reinieren-Beeren, Alie van der Schaaf, This information is current as Arthur J. Kuipers, Luuk P. Schulte, Bart C. Jongbloets, R. of September 28, 2021. Jeroen Pasterkamp, Carl G. Figdor, Annemiek B. van Spriel and Sonja I. Buschow J Immunol 2016; 196:459-468; Prepublished online 23 November 2015; Downloaded from doi: 10.4049/jimmunol.1403096 http://www.jimmunol.org/content/196/1/459 Supplementary http://www.jimmunol.org/content/suppl/2015/11/21/jimmunol.140309 http://www.jimmunol.org/ Material 6.DCSupplemental References This article cites 41 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/196/1/459.full#ref-list-1 Why The JI? Submit online. by guest on September 28, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Semaphorin 7A Promotes Chemokine-Driven Dendritic Cell Migration Anoek van Rijn,* Leonie Paulis,* Joost te Riet,* Angela Vasaturo,* Inge Reinieren-Beeren,* Alie van der Schaaf,* Arthur J. Kuipers,† Luuk P. Schulte,* Bart C. Jongbloets,‡ R. Jeroen Pasterkamp,‡ Carl G. Figdor,*,1 Annemiek B. van Spriel,*,1 and Sonja I. Buschow* Dendritic cell (DC) migration is essential for efficient host defense against pathogens and cancer, as well as for the efficacy of DC- based immunotherapies. However, the molecules that induce the migratory phenotype of DCs are poorly defined. Based on a large- scale proteome analysis of maturing DCs, we identified the GPI-anchored protein semaphorin 7A (Sema7A) as being highly expressed on activated primary myeloid and plasmacytoid DCs in human and mouse. We demonstrate that Sema7A deficiency Downloaded from results in impaired chemokine CCL21-driven DC migration in vivo. Impaired formation of actin-based protrusions, resulting in slower three-dimensional migration, was identified as the mechanism underlying the DC migration defect. Furthermore, we show, by atomic force microscopy, that Sema7A decreases adhesion strength to extracellular matrix while increasing the connec- tivity of adhesion receptors to the actin cytoskeleton. This study demonstrates that Sema7A controls the assembly of actin-based protrusions that drive DC migration in response to CCL21. The Journal of Immunology, 2016, 196: 459–468. http://www.jimmunol.org/ endritic cells (DCs) have major potential in new vacci- or other danger signals, DCs mature, resulting in altered adhesive nation strategies for cancer and infectious disease be- and migration capacity, as well as in the upregulation of chemo- D cause of their potent ability to initiate adaptive immune kine receptor CCR7 that recognizes CCL21 and CCL19 present in responses. DCs are dependent on their capacity to migrate to T cell lymphatic vessels and T cell zones of lymph nodes (LNs) (3–6). areas in secondary lymphoid organs to induce effective immune DCs can switch between two modes of migration, adhesion- responses (reviewed in Refs. 1, 2). Upon activation by pathogens independent and adhesion-dependent migration; actin polymeri- zation is the driving force in both cases (7). In the absence of adhesion, DCs can increase retrograde actin flow to maintain *Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, migration speed, and they may use confinement-based pushing by guest on September 28, 2021 Radboud University Medical Center, 6525 GA Nijmegen, the Netherlands; † forces for locomotion in three-dimensional structures (7–9). When Department of Pediatric Oncology, Radboud University Medical Center, 6525 GA Nijmegen, the Netherlands; and ‡Department of Translational Neuroscience, Brain integrin ligands are available or on a two-dimensional substrate, Center Rudolf Magnus Institute, University Medical Center Utrecht, 3584 CX DCs can make their migration more energy efficient by using Utrecht, the Netherlands adhesion receptors as an adhesive clutch (7). The molecular 1 C.G.F. and A.B.v.S. contributed equally to this work. pathways modulating the adhesion and motility of activated DCs, ORCIDs: 0000-0002-9093-010X (J.t.R.); 0000-0001-8030-1572 (A.J.K.); 0000-0003- allowing their effective chemokine-driven migration toward the 2678-6024 (L.P.S.); 0000-0003-4799-333X (B.C.J.); 0000-0003-1631-6440 (R.J.P.). LNs in vivo, have not yet been fully resolved. Received for publication December 12, 2014. Accepted for publication October 28, 2015. Semaphorins represent a family of membrane-associated and secreted proteins that is characterized by an evolutionarily con- This work was supported by Grant 822.02.017 from the Netherlands Organization for Scientific Research (NWO), European Research Council Advanced Grant PATH- served “Sema” domain. More than 25 semaphorins have been FINDER Project 269019, and Grant KWF2009-4402 from the Dutch Cancer Society. identified that are broadly expressed in different organ systems, A.B.v.S. is supported by NWO Innovational Research Incentives Scheme Vidi Grant 864.11.006 and by the Dutch Cancer Society (KUN 2014-6845). J.t.R. is supported including the nervous, cardiovascular, and immune systems (reviewed by NWO Veni Grant 680-47-421 and NWO Medium-Sized Investment Grant in Refs. 10–12). The pleiotropic functions of semaphorin proteins ZonMW Project 91110007 for the atomic force microscopy used. A.v.R. is supported range from axon outgrowth, angiogenesis, bone differentiation, and by an NWO–Radboud Institute for Molecular Life Sciences Graduate Ph.D. Grant. R.J.P. and B.C.J. are supported by the National Epilepsy Fund. immune regulation to tumor metastasis. Semaphorin7A (Sema7A; Address correspondence and reprint requests to Dr. Carl G. Figdor, Department of also known as CD108 or SemaK1) is the only GPI-anchored Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud Uni- semaphoring; thus, it lacks a cytoplasmic domain (13, 14). The ex- versity Medical Center, Geert-Grooteplein 26, 6525 GA Nijmegen, the Netherlands tracellular domain of Sema7A contains a conserved Sema domain or Dr. Sonja I. Buschow at the current address: Department of Gastroenterology and Hepatology, Erasmus MC–University Medical Center, ‘s Gravendijkwal 230, 3015 bearing an RGD motif, a “plexins semaphorins integrins” domain, CE Rotterdam, the Netherlands (S.I.B.). E-mail addresses: carl.figdor@radboudumc. and an Ig domain. The two known receptors for Sema7A are plexin nl (C.G.F.) or [email protected] (S.I.B.) C1 and b1 integrin (a1 and av heterodimers). Binding of Sema7A to The online version of this article contains supplemental material. plexin C1 in melanocytes leads to inhibition of the actin-binding Abbreviations used in this article: AFM, atomic force microscopy; BMDC, bone protein cofilin and reduced cell spreading (15). In contrast, Sema7A marrow–derived DC; DC, dendritic cell; FN, fibronectin; HZ, heterozygous; KD, knockdown; LN, lymph node; moDC, monocyte-derived DC; MSD, mean square binding to integrins in neurons or melanocytes activates FAK and displacement; myDC, myeloid DC; NT, nontargeting; pDC, plasmacytoid DC; poly MAPK pathways, leading to remodeling of the actin cytoskeleton I:C, polyinosinic-polycytidylic acid; Sema7A, semaphorin 7A; siRNA, small inter- and increased cell spreading (16, 17). Thus, Sema7A regulates two fering RNA; WT, wild-type. signaling pathways that have counteracting effects on the actin Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$30.00 cytoskeleton and cell adhesion. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403096 460 SEMAPHORIN 7A GOVERNS DENDRITIC CELL MIGRATION In the immune system, Sema7A expression was reported on DCs from Sema7A2/2 mice were assessed using anti-murine Semaphor- activated T cells, B cells, macrophages, and DCs, where it is in7A (AF1835; R&D Systems). Cells were analyzed on a FACSCalibur implicated in the regulation of immune cell activation (18–21). Our (Becton Dickinson) using FlowJo software. recent proteome study of human monocyte-derived DCs (moDCs) Western blotting uncovered that Sema7A is one of the most highly upregulated Proteins were separated by SDS-PAGE, transferred to Immobilon-P mem- proteins upon DC maturation (22). However, the biological function branes (Millipore, Bedford, MA), and immunolabeled according to standard of Sema7A on DCs remains elusive. Based on the reported actions Western blotting procedures. Western blots were scanned using an Odyssey of Sema7A on melanocyte adhesion and axonal guidance, we hy- imager (LI-COR Biosciences). Goat anti-human Sema7A and rabbit anti- pothesized that Sema7A may be important for activation-induced actin (clone 20-33; both from R&D Systems) Abs were used for Western DC migration. This study identifies Sema7A as a