Dendritic Cell Migration Semaphorin 7A Promotes Chemokine-Driven

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

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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 connectivity 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 polymerization 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 novel regulator of blotting. chemokine-induced migration of activated DCs that is central for MLR initiating adaptive immune responses. A total of 1 3 105 allogeneic PBLs isolated from buffy coats were cultured overnight in the presence of DCs at a 1:5 or 1:25 ratio in 200 ml RPMI + Materials and Methods 10% FCS. PBLs were subsequently stained for CD3 and CD69 to identify Isolation/generation of human and murine DCs activated T cells and analyzed by flow cytometry. Human monocytes, myeloid DCs (myDCs), and plasmacytoid DCs (pDCs) Collagen matrix preparation were isolated from buffy coats of healthy donors, obtained after informed Bovine type I collagen (55.5% [v/v]; stock concentration 3.1 mg/ml; final consent, using Ficoll density gradients. Monocytes were subsequently ob- concentration 1.72 mg/ml [for moDCs] or 1.95 mg/ml [for BMDCs]; tained by plate adhesion and differentiated into moDCs in 6 d in RPMI 1640 Downloaded from PureCol, Advanced BioMatrix) was turned into fibrillar collagen matrices (Life Technologies) containing 10% FCS, glutamine, and antimycotics/ by raising the pH to 7.4 using 3.7% (v/v) 0.75% Na-bicarbonate solution antibiotics supplemented with IL-4 (300 U/ml) and GM-CSF (450 U/ml). (Life Technologies), together with 7.4% (v/v) minimum essential Eagle’s For maturation, moDCs were cultured for an additional 2 d in the above medium (Sigma-Aldrich) and 33.3% (v/v) RPMI 1640 containing 10% medium supplemented with IL-1b plus IL-4 (300 U/ml) and GM-CSF (450 FCS, and polymerizing at 37˚C. U/ml) or, alternatively, with IL-1b (5 ng/ml), IL-6 (15 ng/ml), TNF-a (10 m m ng/ml), and PGE2 (10 g/ml) (cytokine mixture), or LPS (1 g/ml), or a Transwell migration combination of R848 (4 mg/ml; Alexis Biochemicals) and polyinosinic- polycytidylic acid (poly I:C; 20 mg/ml; Sigma-Aldrich), where indicated. For Transwell-migration assays, a detached cytokine mixture of 5 3 104–1 3 http://www.jimmunol.org/ Human myDCs were isolated from PBMCs using a MACS CD1c (BDCA-1) 105 matured moDCs was applied in 100 ml RPMI 1640 containing 10% FCS Dendritic Cell Isolation Kit and human pDCs were isolated from PBMCs to 6.5-mm diameter Transwell inserts that were separated from the lower using a MACS BDCA-4 Plasmacytoid Dendritic Cell Isolation Kit (both chamber by polycarbonate membranes containing 5-mm pores (Costar). The from Miltenyi Biotec). For maturation, myDCs and pDCs were cultured lower compartments were filled with RPMI 1640 containing 10% FCS and, overnight in X-Vivo medium (Lonza) supplemented with 2% human serum when indicated, 1 mg/ml CCL21 (R&D Systems). Inserts were coated with and a combination of poly I:C (20 mg/ml) and R848 (4 mg/ml; myDCs) or 75 ml collagen matrix, as indicated, prior to applying the cells. Cells were with R848 alone (4 mg/ml; pDCs). Purity of isolated myDCs and pDCs was allowed to migrate through the bottom of the chamber for 1–2 h (without determined by staining for a combination of CD11c and CD1c (myDC) or collagen), for 4–5 h (BMDCs with collagen), or overnight (moDCs with for CD123 (pDCs; all Abs used were from BD Biosciences). collagen). The amount of transmigrated cells relative to input was measured

To generate bone marrow–derived DCs (BMDCs), bone marrow cells by flow cytometry for a set time period, while keeping volumes equal be- by guest on September 28, 2021 from mouse femurs were cultured in RPMI 1640 medium supplemented tween conditions. with 10% FCS, glutamine, antimycotics/antibiotics, and 2-ME in the presence of 20 ng/ml GM-CSF (PeproTech) for 8 d to generate GM-CSF Adhesion assays BMDCs or in the presence of 5 ng/ml GM-CSF and 200 ng/ml Flt3L (CellGenix) for 15 d to generate Flt3L BMDCs (23). For plate-adhesion assays, flat-bottom 96-well tissue culture–treated plates + were coated with fibronectin (FN; Roche) or GRGDS peptides (AnaSpec) Primary CD11c DCs were obtained from mouse spleens. Spleens were m m excised and injected with collagenase type II (Worthington) and DNase at 20 g/ml and, when indicated, subsequently with 1 g/ml CCL21 (R&D (Roche) for 45 min at 37˚C. After addition of 10 mM EDTA and resus- Systems). Cells were allowed to attach to the culture plates in RPMI 1640 pending, cells were applied to nylon mesh to remove debris, and DCs were with 10% FCS for 5 min, and nonadherent cells were removed by exten- enriched by a 15/11.5/0% gradient of iodixanol in HBSS (prepared from sive washing. Adherent cells were subsequently detached by PBS con- 60% iodixanol OptiPrep stock; Sigma-Aldrich). For maturation, GM-CSF taining 2 mM EDTA at room temperature, and the number of adherent BMDCs and CD11c+ splenocytes were cultured overnight with a mixture cells relative to input was measured by flow cytometry for a set time- of TLR ligands containing LPS (0.5 mg/ml; Sigma-Aldrich), poly I:C (10 window while keeping volumes equal between conditions. m g/ml), and CpG 1668 (6.4 ng/ml; Sigma-Aldrich). Flt3L BMDCs were Atomic force microscopy adhesion measurements matured overnight with LPS alone (0.1 mg/ml). Force measurements on living mature DCs were performed in force- Small interfering RNA knockdown of Sema7A on human distance mode using a BioScope Catalyst AFM (Bruker, Santa Barbara, moDCs CA) mounted on an inverted microscope (TCS SP5 II; Leica, Mannheim, 3 6 m Germany). The temperature was kept at 37˚C throughout the experiments Day-6 moDCs were electroporated (5–10 10 cells) with 15 M non- by a petri dish heater (Bruker). Polystyrene microspheres (10 mm diameter; targeting (NT) small interfering RNA (siRNA; Dharmacon) or 15 mM Polysciences) were glued onto atomic force microscopy (AFM) cantilevers SMARTpool of three Sema7A-targeting siRNAs (Stealth RNAi siRNA; (NP-S type D; Bruker) (24). Bead-functionalized cantilevers were cleaned by Life Technologies; H55112366, H55112367, and H55112368), using the immersion in 2% Hellmanex III (Hellma, Mullheim,€ Germany) overnight exponential program at 300 V, resistance at ‘, and capacitance at 150 mF and thoroughly rinsed with Milli-Q water and ethanol, and then (after a final in a 4-mm cuvette in the Bio-Rad Gene Pulser Xcell electroporator, and rinse in Milli-Q water) were allowed to dry. Before experiments, cantilevers subsequently cultured for 2 d in the presence or absence of maturation were incubated at 37˚C in FN (20 mg/ml) in PBS. The spring constant of stimuli, as indicated. each cantilever was calibrated before adhesion measurements using thermal Flow cytometry noise analysis (25). To measure the adhesion of a single DC to the FN-coated bead on the cantilever, siRNA-treated mDCs were immobilized on ConA- Human cells were stained with PBS containing 0.5% BSA with the fol- coated WillCo dishes (WillCo, Amsterdam, The Netherlands) and sub- lowing Abs: CCR7 (clone 150503, R&D Systems), CD83 (Immunotech), merged in culture medium supplemented with 10 mM HEPES containing and Sema7A (clone MEM-150; Exbio). Murine cells were stained with 10% FBS and 0.5% antibiotics/antimycotics (pH 7.4). ConA-coated WillCo PBS containing 0.5% BSA with the following Abs: CD86 (clone Gl-1), dishes were prepared by overnight incubation at 4˚C in ConA (Sigma- CD11c (clone N418), CD4 (clone RM4-5), and F4/80 (clone A3-1; all Aldrich; 0.2 mg/ml) in PBS; before seeding mDCs, dishes were rinsed from BioLegend); CD3 (clone 145-2C11) and B220 (clone RA3-6B2; both with PBS. Adhesion of the FN-coated bead to different cells was measured from BD Biosciences); and CD8 (clone 53-6.7; Exbio). The presence of by pushing the bead into contact with flat parts of the DCs and applying a 2- Sema7A protein on DCs of wild-type (WT) littermates and its absence on nN contact force for 10 s. Subsequently, the cell was retracted at 10 mm/s The Journal of Immunology 461 and allowed to recover for a time period equal to that during which the cell converted into mean square displacement (MSD) using the following was in contact with the substrate before repeating adhesion measurements equation: (26). Five measurements were performed per cell and used for subsequent calculation. Detachment forces were determined after baseline correction of MSDðdtÞ¼ð½xðtÞ 2 xðt þ dtÞ2 þ½yðtÞ 2 yðt þ dtÞ2Þ=dt F-D curves with a macro in Matlab (27). Force-step analysis and cell elasticity were determined from F-D curves using in-house Igor Pro 6 (Wave- where dt is the time interval and x(t)andy(t) are the position in x and y at time t. Metrics) algorithms (28). The cell cortex elasticity was determined using The MSD was calculated as a summation over time, for different time intervals. retraction F-D curves by linear fitting the region when adhesion built up to For confocal analyses, DCs were overlaid with RPMI 1640 medium m the maximum force (force-range 0 nN to Fmax,Fig.7C). containing 10% FCS, with or without 1 g/ml CCL21, and incubated for the indicated times, after which cells were fixed in 4% paraformaldehyde Microscopy for 1 h, washed/permeabilized (PBS 1% BSA and 0.1% saponin), and labeled with Alexa Fluor 488–conjugated phalloidin (Invitrogen). Cells To monitor the appearance of moDCs after electroporation with siRNA, within the matrices were imaged using an Olympus FV1000 Confocal mature moDC cell cultures were directly imaged on a Leica DM IL inverted Laser Scanning Microscope with a 403 water objective (NA 0.8) and an 3 microscope using a 20 air objective (NA 0.4) and a Leica DC300 camera. Olympus U-CMAD3 camera. Images were acquired using Olympus im- 3 6 For confocal and time-lapse microscopy analysis, DCs (1 10 cells/ml aging software and subsequently analyzed with FIJI software. Cell den- RPMI 1640) were embedded into collagen matrices and monitored by dricity (1 2 circularity) was measured for individual cells after applying a confocal microscopy or time-lapse microscopy experiments 1–2 h thereafter. mask, as shown in Fig. 6A. For circularity measurement, FIJI software For time-lapse microcopy, a suspension of DCs in collagen was loaded in applies the following formula: circularity = 4p(area/perimeter2). wax chambers (generated from cover glasses hermetically sealed with paraffin), which, after collagen polymerization, was overlaid with medium Mice containing 1 mg/ml CCL21 (R&D Systems). Wax chambers were sealed to prevent a decrease in pH due to air exposure and were kept at 37˚C while Sema7A2/2 mice were generated and genotyped previously (16). Sema7A+/+ migration was monitored with a step interval of 2 min by time-lapse littermates (referred to as WT mice) or Sema7A+/2 littermates (referred to as Downloaded from bright-field video microscopy using a 203 air objective (NA 0.3) on a heterozygous [HZ]) animals) were matched for age and gender and used be- bright-field inverted microscope (Leica) and a CCD camera (Sentech). A tween 9 and 14 wk of age. Mice were housed in top-filter cages and fed a 16-channel frame grabber software (Vistek) was used for image acquisi- standard diet with freely available water and food. All in vivo studies complied tion. Cell tracks of randomly selected cells were followed for 3 h, be- with national legislation and were approved by local authorities for the care ginning 30 min after the start of imaging, using the manual tracking and and use of animals with related codes of practice. Animal studies were ap- chemotaxis plug-ins that are incorporated into FIJI software. The speed proved by the Animal Ethics Committee of the Nijmegen Animal Experiments was calculated as the length of each cell path/time, and xy trajectories were Committee (DEC 2013-158). http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Sema7A is expressed on moDCs upon maturation. (A) Flow cytometry of representative samples of immature moDCs and cytokine mixture (IL-1b,IL-6,TNF-a,andPGE2)-matured moDCs stained with Sema7A (ﬁlled graph) or isotype-control Abs (open graph). (B) Expression of Sema7A in day-8 immature moDCs or 48-h cytokine mixture–matured moDCs from multiple donors (eight donors in four independent experiments), expressed as the mean ﬂuorescence intensity (MFI) fold change (6 SEM) over isotype-control labeling) (left panel) and as the mean percentage (6 SEM) of Sema7A+ cells (right panel). (C) Western blot showing the expression of Sema7A protein at the expected size of 80 kDa in 48-h cytokine mixture–matured moDCs. (D) Mean percentage (6 SEM) of Sema7A+ DCs after a 48-h maturation with cytokine mixture or TLR ligands (four donors in two independent experiments). (E) Expression of Sema7A, CCR7, and CD83 on moDCs after the addition of cytokine mixture. Data are mean percentage (6 SEM) of the maximum measured expression at 48 h in moDCs from three donors [t = 48 h used for (B)and (D)]. *p , 0.05, ***p , 0.001, paired t test per donor. 462 SEMAPHORIN 7A GOVERNS DENDRITIC CELL MIGRATION

FITC painting LeicaDMLBmicroscopeusinga103 air objective (NA 0.4) and a Leica DC300 camera. In vivo DC migration was studied by FITC-painting experiments, as described previously (29). Briefly, abdominal skin was shaved prior to ap- Statistical analysis plying a FITC solution. FITC solution was prepared by diluting a 10% (w/v) solution of fluorescein-5-isothiocyanate in DMSO to a 1% final concentration Statistical analyses were performed in GraphPad Prism 5 software using the with a 1:1 mixture of acetone and dibutyl phthalate. Draining (inguinal) and appropriate testing methods, as indicated in the figure legends. nondraining (brachial) LNs were taken after 24 h, and LN cells were isolated by DNase and collagenase digestion. LN cells were stained for FITC, CD11c, Results and CD86 to identify migrated DCs and analyzed by flow cytometry. Sema7A is a general DC maturation marker conserved Ear emigration between mice and humans Ear emigration assays were performed as described previously (29). Briefly, Consistent with our previous proteome analysis (22), we found that dorsal and ventral skin sheets were placed in medium containing 1 mg/ml Sema7A was strongly expressed on mature, but not resting, moDCs LPS alone or LPS and 1 mg/ml CCL21 and cultured for 48 h. Emigrated using both flow cytometry and Western blotting (Fig. 1A–C). Ex- DCs were quantified using flow cytometry. pression could be triggered by a mixture of IL-1b, IL-6, TNF-a, Immunohistochemistry and PGE2, as well as by various TLR ligands (R848, poly I:C, LPS), indicating that Sema7A is a general maturation marker of human 2/2 Mouse lymphoid organs were taken from Sema7A mice and WT moDCs (Fig. 1D). Sema7A expression was induced relatively late Sema7A+/+ littermates and embedded in OCT. Frozen sections (5 mm) were fixed in acetone for 10 min at 220˚C. Sections were blocked with 5% goat during maturation and similar in timing to the chemokine receptor

serum and stained with anti-CD3 (clone CD3-12; Serotec), anti-B220 (clone CCR7 (Fig. 1E) (30). Downloaded from RA3-6B2; BD Biosciences), anti-CD11c (clone N418; eBioscience), or Next, we studied whether Sema7A was also upregulated during CD68 (clone MCA1957; Serotec), followed by biotin-conjugated secondary the maturation of primary DCs from humans and mice. Indeed, all Abs (Invitrogen). Staining was revealed using an SA-alkaline phosphatase labeling kit (Vector Laboratories) with Fast Red substrate (Sigma-Aldrich), DC subsets tested increased Sema7A expression upon maturation, and slides were counterstained with Mayer’s hematoxylin and em- albeit to a variable extent (Fig. 2). Both human myDCs and pDCs bedded in Kaiser’s glycerol gelatin (Merck). Slides were imaged on a isolated from the blood of healthy donors expressed Sema7A when http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Sema7A is a general DC maturation marker conserved between mice and humans. (A) Line graphs showing the expression of Sema7A (filled) versus isotype-control labeling (open) on myDCs and pDCs from one representative experiment. myDCs were matured with a combination of poly I:C and R848, and pDCs were matured with R848 only. (B) Percentage (6 SEM) of immature and mature Sema7A+ myDCs (six donors in four independent experiments) and pDCs (three donors in three independent experiments). (C) Representative line graphs showing the expression of Sema7A on murine immature or overnight-matured CD11c+ splenocytes (Spl). (D) Percentage (6 SEM) of Sema7A+ cells after overnight maturation of conventional GM-CSF BMDCs, Flt3L BMDCs, and CD11c+ splenocytes (all from three mice). GM-CSF BMDCs and CD11c+ splenocytes were matured with a mixture of TLR ligands (poly I:C, LPS, R848, CpG), and Flt3L BMDCs were matured with LPS alone. ***p , 0.001, paired t test per donor. ns, nonsignificant. The Journal of Immunology 463 matured with TLR ligands (Fig. 2A, 2B). In mice, most mature experiments in Sema7A2/2 mice, as well as in WT and HZ primary CD11c+ DCs derived from spleen, as well as the majority (Sema7A+/2) controls. Skin DCs in WT and HZ littermates readily of Flt3L BMDCs and some (10–20%) GM-CSF BMDCs, expressed migrated to the LNs and represented ∼50% of CD11c+ DCs in the Sema7A (Fig. 2C, 2D; for gating see Supplemental Fig. 1A). Taken inguinal LNs after 24 h (Fig. 3B; for gating see Supplemental Fig. together, we show that, similar to CCR7, Sema7A expression is a 1B). In Sema7A2/2 mice, DC migration to the LNs was impaired, general feature of maturation in murine and human DCs. because only 40% of CD11c+ DCs in the LNs originated from the skin after 24 h. This reduction was not significant because of the Sema7A modulates DC migration in vivo high experimental variation, especially in Sema7A2/2 mice. This Because Sema7A was demonstrated to regulate cell adhesion/ high variation was likely caused by the complexity of the migration migration in the neuronal system and in skin tumors (16, 17), process in vivo, which is composed of several sequential steps (e.g., we hypothesized that Sema7A on DCs may be involved in the peripheral detachment, migration through the lymphatics, LN in- regulation of adhesion and/or migration during DC maturation. vasion), each of which may be affected differentially by the absence To this end, we investigated the functional consequences for DC of Sema7A. To further elucidate how Sema7A affects DC migra- migration in vivo using Sema7A-knockout (Sema7A2/2) mice tion, we exploited the ear emigration model in which separated skin DCs. We first analyzed the composition and architecture of sec- sheets of WT or Sema7A2/2 mice were exposed to LPS, with or ondary lymphoid organs in these mice. The architecture of the without CCL21, to induce skin DC migration. DCs from both WT LNs (Fig. 3A) and spleen (data not shown) appeared unaltered and Sema7A2/2 mice readily migrated out of the skin when ex- with respect to B cell follicles, T cell zones, and localization of posed to LPS alone, demonstrating that maturation, basal migration,

CD11c+ cells (DCs) in the T cell areas, as well as the presence of and peripheral detachment were not impaired by Sema7A deﬁ- Downloaded from CD68+ cells (macrophages) throughout the LN. Importantly, there ciency (Fig. 3C; for gating see Supplemental Fig. 1C). Emigrating were no major differences between the total number (data not DCs also expressed high levels of Sema7A (Supplemental Fig. 1D). shown) and relative amounts of lymphoid and myeloid cells be- In the presence of CCL21, DC emigration from WT skin was tween Sema7A2/2 mice and their WT (Sema7A+/+) littermates clearly enhanced (Fig. 3C). In contrast, DC emigration from (Supplemental Fig. 2). Sema7A2/2 mice was severely hampered upon CCL21 addition,

To test whether migration of DCs to the LN in vivo was affected demonstrating that Sema7A acts speciﬁcally on chemokine-induced http://www.jimmunol.org/ by the absence of Sema7A, we performed FITC skin-painting DC migration ex vivo. by guest on September 28, 2021

FIGURE 3. Sema7A is required for DC migration ex vivo. (A) LNs of WT or Sema7A2/2 mice stained for the T cell marker CD3, B cell marker B220, DC marker CD11c, and macrophage marker CD68. Scale bars, 100 mm. (B) Percentage of migrated mature skin DCs (FITC+/CD11c+/CD86+) in skin- draining LNs relative to total mature DCs (CD11c+/CD86+) present in LNs 24 h after applying FITC to the abdominal wall of Sema7A2/2 mice (n = 12) and WT (Sema7A+/+)orHZ(Sema7A+/2) littermates (n = 9). (C) Number of mature skin DCs (FITC+/CD11c+/CD86+) emigrating in 48 h per mg ear tissue from Sema7A2/2 (n = 17) or Sema7A+/+ (n = 15) ear explants exposed to LPS alone or LPS and CCL21. Results in (B) and (C) are pooled from three independent experiments (mean 6 SEM). *p , 0.05, **p , 0.01, unpaired t test. ns, nonsigniﬁcant. 464 SEMAPHORIN 7A GOVERNS DENDRITIC CELL MIGRATION

Sema7A is required for dendrite formation and DC migration control and KD DCs (Fig. 5A); however, migration through col- in response to CCL21 lagen was significantly impaired in DCs lacking Sema7A 2/2 To investigate Sema7A function on human DCs, we used a mixture (Fig. 5B). Likewise, Flt3L BMDCs generated from Sema7A of three siRNAs to knockdown (KD) Sema7A expression in moDCs. mice migrated less well through a collagen matrix (Supplemental Expression of Sema7A was almost completely inhibited by this Fig. 4A, 4B). Thus, in both human and murine DCs, the presence approach (Fig. 4). Importantly, KD DCs were normal with respect of Sema7A appeared most important for DC migration in a more to upregulation of maturation markers (CCR7, CD83), their mor- complex environment where DCs encounter an abundance of phology in culture, and their ability to stimulate allogeneic T cells integrin ligands, as well as in confined spaces. The migration (Fig. 4). Although the siRNA electroporation procedure itself re- defect could not be attributed to aberrant CCR7 expression (Fig. duced the number of matured DCs from 90% to ∼60%, this effect 4C) or a decreased ability of KD DCs to enter the collagen matrix was not different between the NT siRNA–treated DCs and the because microscopic inspection demonstrated that KD DCs Sema7A siRNA–treated DCs (Supplemental Fig. 3). readily entered the matrix (data not shown). The migration defect Having established efficient Sema7A KD, we investigated the was cell intrinsic (rather than mediated via soluble Sema7A or effect of Sema7A KD on CCL21-induced migration of mature DCs cell–cell contact), because the addition of an excess of Sema7A- using the Transwell system in the absence or presence of a collagen expressing WT cells to the experiment could not rescue the mi- matrix. CCL21 triggered DC migration through membranes in gration defect of Sema7A-KD cells (data not shown). Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. siRNA Sema7A KD does not impair DC maturation and Ag presentation. (A) Western blot showing Sema7A protein at 80 kDa in untreated and NT siRNA–treated mature moDCs and its absence in Sema7A siRNA-treated moDCs. Actin (42 kDa) shows protein loading. (B) Expression of Sema7A, CD83, and CCR7 on immature and mature siRNA-treated moDCs. Surface expression of the indicated proteins is depicted in representative line graphs (shaded areas for speciﬁc staining on NT siRNA–treated moDCs; open areas for Sema7A siRNA-treated moDCs; light gray lines for isotype control staining (left panels). Bar graphs show the percentage of positive cells for each maturation marker (right panels) (results of .15 donors from more than eight independent experiments for all). (C) Representative light microscopy images of NT siRNA– and Sema7A siRNA–treated mature moDCs. Scale bar, 25 mm. (D) Activation (e.g., upregulation of CD69) of allogeneic CD3+ T cells by siRNA-treated DCs (ratios refer to DCs/T cell; results of four donors in three independent experiments). ***p , 0.001, paired t test. ns, nonsigniﬁcant. The Journal of Immunology 465

FIGURE 5. Sema7A KD impairs chemokine- induced migration. (A) Percentage of human moDCs that migrated relative to input to the lower compartments of a 5-mm-pore Transwell system in the presence or absence of chemokine CCL21. (B)Asin(A), but using a Transwell overlaid with collagen matrix. Each symbol represents DCs from one donor. For the condi- tion with CCL21, NT (triangles), and Sema7A (closed circles), siRNA-treated moDCs from the same donor are connected by a line. DCs from one or two donors were compared per independent experiment. ***p , 0.001, paired t test. ns, nonsigniﬁcant.

To further investigate the mechanism underlying the migra- applying a mechanical force on extracellular matrix–binding ad- tion defect of Sema7A-KD cells, we ﬁrst studied cell mor- hesion receptors using an FN-coated bead. The bead was attached Downloaded from phology and the actin cytoskeleton (F-actin labeling) during to the AFM cantilever that was subsequently brought into contact CCL21-induced migration in a three-dimensional environ- with a ﬂat area of the DC surface and then retracted from the DC ment. Within the collagen matrix, Sema7A-KD DCs appeared surface to break the adhesive bonds one by one (Fig. 7B). Analysis less dendritic (Fig. 6A, 6B). Upon addition of CCL21, NT DCs of obtained AFM-derived force-distance curves allowed us to greatly increased the number of dendrites (Fig. 6A, 6B). In distinguish effects on avidity (i.e., the binding strength exerted by

contrast, Sema7A-KD cells remained impaired with regard to the FN-binding receptors together), affinity (i.e., the binding http://www.jimmunol.org/ dendrite formation compared with NT DCs, indicating that DCs strength/adhesion molecule), and cellular membrane elasticity require Sema7A to efficiently assemble actin-based protru- (i.e., the amount of cytoskeletal support) (Fig. 7C–F) (28). Using sions. Experiments with Sema7A2/2 murine Flt3L BMDCs AFM, we observed that DCs lacking Sema7A were indeed more showed a similar defect in dendrite formation and response to adhesive, in line with the plate-adhesion experiments (Fig. 7F: CCL21, although the responses were rather heterogeneous adhesion strength). This was not caused by an increased affinity of (Supplemental Fig. 4C, 4D). single receptors for their ligands, because the distribution of Because actin polymerization regulates three-dimensional mi- rupture forces required to break single molecule bonds was not gration speed in DCs (7), we next used live cell microscopy to altered (Fig. 7E). Interestingly, the linear elasticity (i.e., the force characterize the movement of individual DCs through collagen required to pull out part of the membrane adhered to the FN- by guest on September 28, 2021 (Supplemental Videos 1, 2). DCs were incorporated into the col- coated bead) of Sema7A-KD DCs was significantly lower com- lagen prior to matrix formation to abrogate any two-dimensional– pared with control DCs, indicating a reduced connection of dependent DC migration effects. In the absence of CCL21, mi- membrane tethers to the cortical actin cytoskeleton in Sema7A- gration speed and MSD of Sema7A-KD DCs were similar to those KD DCs (Fig. 7G). These results indicate that Sema7A-KD DCs of NT DCs (Fig. 6C–E). However, in the presence of CCL21, KD are more adhesive and show an impairment of the actin cyto- DCs clearly migrated less far, and the MSD was 2.4-fold lower skeleton, similar to what we observed for intracollagen protrusion (p , 0.05) over the course of 3 h (Fig. 6C, 6D). The migration formation. Together, our data demonstrate that Sema7A promotes speed of NT DCs clearly increased upon addition of CCL21 in DC migration by increasing the connectivity of adhesion receptors contrast to Sema7A-KD cells, which did not migrate faster in to the actin cytoskeleton to support actin-based protrusion as- response to CCL21 (Fig. 6E). Sema7A-KD DCs did not appear to sembly. stop more frequently or to remain completely immobile. Instead they moved at a lower overall speed throughout the tracking time Discussion (data not shown). Taken together, these data demonstrate that, in Activated DCs are equipped with a complex molecular machinery the absence of Sema7A, DCs are less protrusive and migrate to govern migration from peripheral tissues to the LNs. We identify more slowly in a three-dimensional environment in response to Sema7A as an essential membrane receptor for chemokine-induced CCL21. DC migration in vitro and ex vivo. The function of semaphorin proteins is mostly known from the neuronal system, where they Sema7A controls the connection of adhesion receptors to the regulate growth and branching of neuronal processes (11). In the cytoskeleton immune system, Sema7A is implicated in the activation of T cells, Although DCs do not require adhesion for migration, especially in monocytes, and macrophages, as well as in neutrophil migration three-dimensional matrices (7), alterations in their adhesion ca- (18, 20, 21, 31), although only in the case of T cells was the effect pacity may still affect their migration speed when integrin ligands immune cell intrinsic (i.e., Sema7A is functional on the T cells are present (e.g., by impairing cell detachment). Therefore, we themselves). We now report that Sema7A expressed on activated investigated whether Sema7A KD affected the adhesive properties human and murine DCs controls adhesion and migration in re- of DCs. sponse to CCL21. Sema7A was not required for DC maturation or Initial plate-adhesion experiments suggested that Sema7A-KD induction of allogeneic T cell responses. Importantly, in mice DCs were slightly more adherent to extracellular matrix compo- lacking Sema7A, the migration of skin DCs to the LN was re- nents than were NT DCs (Fig. 7A). However, experimental vari- duced, and CCL21-induced DC emigration from ear explants was ation was high; therefore, we exploited AFM to decipher the effect severely impaired. The finding that Sema7A deficiency specif- of Sema7A on the individual factors governing DC adhesion by ically affected CCL21-driven migration implies that Sema7A has 466 SEMAPHORIN 7A GOVERNS DENDRITIC CELL MIGRATION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Human DCs lacking Sema7A form fewer dendrites in three-dimensional matrix, and their migration is impaired in response to CCL21. (A) Merged z-stacks of phalloidin-stained F-actin for NT and Sema7A-KD DCs embedded in a three-dimensional collagen matrix and exposed to CCL21 for 1h(upper panels). Masks derived from phalloidin images used to analyze cell dendricity (lower panels). Scale bars, 50 mm. (B) Dendricity index (1 2 roundness) of DCs inside collagen matrices prior to and after stimulation with CCL21 for the indicated times. Perfectly round cells were scored as 0, whereas highly dendritic cells were scored as 1. Results are from four donors in three independent experiments. (C) Trajectories of individual NT or KD DCs from one representative experiment of three (also see Supplemental Videos 1 and 2, respectively). (D) Average MSD curves (6 SD) of NT and Sema7A KD cells at different time points after embedding in collagen matrix with CCL21. At least 15 DCs from three donors (each in independent experiments) were analyzed to calculate MSD curves. At t = 180 min, the average MSD for NT siRNA DCs differed from that of Sema7A-KD DCs (p , 0.05, paired t test). (E) Average speed of single cells migrating through collagen in the absence and presence of CCL21 [same cells used in (D)]. **p , 0.01, ***p , 0.001, unpaired t test. ns, nonsigniﬁcant. an important role in guiding DCs from peripheral tissues into the to the LN upon FITC painting was reduced in plexin C1–knockout lymphatic vessels and in intranodal DC mobility, processes that mice to the same extent as in Sema7A-knockout mice, suggesting are known to be controlled by CCL21 (6, 32). that these two molecules could act in concert (36). Soluble Sema3A produced by the lymphatics was described to support recombinant Sema7A-Fc was unable to bind to Sema7A-KD DCs DC migration by acting on the cytoskeleton upon binding to plexin (data not shown), indicating that an interaction partner for soluble A1 expressed by DCs (33). Furthermore, the secreted viral Sema7A-Fc is not readily available, irrespective of the presence of semaphorins from pox and herpes viruses, which harbor some de- endogenous Sema7A that may occupy potential binding sites. gree of homology to Sema7A, were demonstrated to affect DC Concordantly, Sema7A-Fc could not rescue the defective adhesion, migration, and phagocytosis by acting on the cytoskeleton migration of KD DCs. Thus, it is possible that, on DCs, Sema7A through plexin C1 (34, 35). binds its interaction partner only in its GPI-anchored form in Our experiments revealed that Sema7A acts cell autonomously cis but not in a soluble form. Such in cis interactions with on DC adhesion and protrusion formation. Because Sema7A lacks binding partners were reported for other semaphorin family a cytoplasmic tail, we anticipate that it alters the intracellular members (37). signaling cascades responsible for regulating CCL21-driven adhe- We observed that DCs lacking Sema7A are more adhesive, lack sion and migration through transmembrane-binding partners. Both cytoskeletal support of adhesion receptors, form fewer actin-rich of the previously described interaction partners of Sema7A, plexin protrusions, and are impaired in CCL21-induced migration. Thus, C1 and b1 integrins, are expressed on mature DCs and, thus, can Sema7A on DCs acts by controlling the balance between adhesion/ potentially bind Sema7A in cis (3, 36). Interestingly, DC migration deadhesion, similar to Sema7A function on tumor cells and The Journal of Immunology 467 Downloaded from http://www.jimmunol.org/

FIGURE 7. Sema7A acts on DC adhesion and the actin cytoskeleton. (A) Plate adhesion of NT siRNA or KD mature moDCs. Plates were coated with by guest on September 28, 2021 FN or RGD peptide. Cells were allowed to adhere for 5 min, and the fraction of adherent cells was quantified by flow cytometry after EDTA detachment. Shown are the mean (6 SEM) number of cells relative to input that adhered to the plate. Results from three donors in two independent experiments. (B) AFM single-cell adhesion measurement setup showing an overview of the FN-coated bead on the AFM cantilever in contact with the DC and the laser used to measure the change in cantilever deflection (left panel). Enlargement of the bead–DC contact site showing the molecules involved (i.e., integrin ab heterodimers and underlying actin cortex connected by adaptor proteins) (right panel). (C) Adhesion parameters that can be derived from the indicated specific sections of AFM-based force-distance curves. (D) Averaged force-distance curves (n . 40 from three cells) obtained with NT siRNA and Sema7A-KD DCs probed with an FN-coated bead attached to the AFM cantilever. (E) Affinity of adhesion receptors depicted as the distribution of rupture forces required to break single molecule bonds in NT siRNA versus KD cells. (F) Adhesive strength (avidity) plotted as the maximal force required to detach the bead from NT siRNA or KD cells. (G) Linear membrane elasticity calculated by linear fitting the part of the force-distance curves indicated in (C) (i.e., the force needed to pull out a part of the membrane and the underlying actin cortex by the FN-coated bead/mm). Each data point in (F) and (G) represents the average of five measurements at one location on a flat part of the DC. Three to five DCs from three donors were analyzed in independent experiments. *p , 0.05, **p , 0.01, unpaired t test. melanocytes (15, 17, 38). A prominent role for the actin-binding DC membrane proteins and the cytoskeleton facilitates both protein cofilin was shown in these studies. Cofilin regulates cell confinement and adhesion-driven migration (7, 8). In conclusion, migration downstream of plexin C1, integrins, and CCR7 (15, our data demonstrate that Sema7A promotes DC migration by 39), making it a possible candidate for controlling Sema7A- enhancing actin-based protrusion formation. In this study, we dependent DC migration. Experiments to unravel a role for identified an important novel receptor in the highly complex cofilin in our studies were hampered by the low number of DCs process of DC migration to the LNs that governs the initiation of available for biochemical experiments, as well as by the uncon- adaptive immune responses. ventional mechanisms regulating cofilin activity (40). CCL21 was shown to facilitate adhesive DC migration by acting Acknowledgments on adhesion- and actin-driven migration speed (6, 41, 42). We now We thank Geert van den Bogaart for discussion and assistance with the anal- demonstrate that Sema7A facilitates CCL21-dependent migration ysis of live microscopy data, Youri Adolfs for help with animal experiments, in a three-dimensional environment. Sema7A-KD DCs proved to the Radboud Institute for Molecular Life Sciences Department of Cell Bi- be defective in actin protrusion formation and displayed an in- ology for use of live cell imaging facilities, and Alessandra Cambi for crit- creased adhesion strength, which, especially in the absence of ical reading of the manuscript. cytoskeletal support of adhesion receptors, slows migration as a result of a lack of force coupling. Importantly, irrespective of li- Disclosures gand engagement of adhesion receptors, the connection between The authors have no financial conflicts of interest. 468 SEMAPHORIN 7A GOVERNS DENDRITIC CELL MIGRATION

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