Dendritic Cell Migration and Antigen Presentation Are Coordinated by the Opposing Functions of the CD82 and CD37 This information is current as of September 26, 2021. Eleanor L. Jones, Janet L. Wee, Maria C. Demaria, Jessica Blakeley, Po Ki Ho, Javier Vega-Ramos, Jose A. Villadangos, Annemiek B. van Spriel, Michael J. Hickey, Günther J. Hämmerling and Mark D. Wright

J Immunol 2016; 196:978-987; Prepublished online 4 Downloaded from January 2016; doi: 10.4049/jimmunol.1500357 http://www.jimmunol.org/content/196/3/978 http://www.jimmunol.org/

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Dendritic Cell Migration and Antigen Presentation Are Coordinated by the Opposing Functions of the Tetraspanins CD82 and CD37

Eleanor L. Jones,* Janet L. Wee,*,† Maria C. Demaria,* Jessica Blakeley,* Po Ki Ho,* Javier Vega-Ramos,‡ Jose A. Villadangos,‡,x Annemiek B. van Spriel,{ Michael J. Hickey,† Gunther€ J. Ha¨mmerling,‖ and Mark D. Wright*

This study supports a new concept where the opposing functions of the tetraspanins CD37 and CD82 may coordinate changes in migration and Ag presentation during dendritic cell (DC) activation. We have previously published that CD37 is downregulated upon monocyte-derived DC activation, promotes migration of both skin and bone marrow–derived dendritic cells (BMDCs), and restrains Ag presentation in splenic and BMDCs. In this article, we show that CD82, the closest phylogenetic relative to CD37, Downloaded from appears to have opposing functions. CD82 is upregulated upon activation of BMDCs and monocyte-derived DCs, restrains migration of skin and BMDCs, supports MHC class II maturation, and promotes stable interactions between T cells and splenic DCs or BMDCs. The underlying mechanism involves the rearrangement of the via a differential activation of small GTPases. Both CD372/2 and CD822/2 BMDCs lack cellular projections, but where CD372/2 BMDCs spread poorly on fibro- nectin, CD822/2 BMDCs are large and spread to a greater extent than wild-type BMDCs. At the molecular level, CD82 is a negative regulator of RhoA, whereas CD37 promotes activation of Rac-1; both tetraspanins negatively regulate Cdc42. Thus, this http://www.jimmunol.org/ study identifies a key aspect of DC biology: an unactivated BMDC is CD37hiCD82lo, resulting in a highly motile cell with a limited ability to activate naive T cells. By contrast, a late activated BMDC is CD37loCD82hi, and thus has modified its migratory, cytoskeletal, and Ag presentation machinery to become a cell superbly adapted to activating naive T cells. The Journal of Immunology, 2016, 196: 978–987.

endritic cells (DC) are the most potent of the APCs through pattern recognition receptors, they reduce MHC turnover because they have the unique ability to activate naive Ag- and upregulate expression of MHC/peptide complexes, costimu- D specific T cells (1). However, DC function varies with latory molecules such as CD80 and CD86, and proinflammatory by guest on September 26, 2021 activation state; the classical example being migratory DCs. cytokines, all of which increase their capacity to stimulate T cells Unactivated migratory DCs in the periphery efficiently patrol and direct adaptive cellular immunity (2). Concurrently, DCs through tissues and are specialized for Ag uptake. These highly undergo morphological changes involving the extension of den- endocytic cells are poor stimulators of T cells, because of both drites thought to promote efficient interactions with T cells, a a moderate surface expression and a high turnover of MHC decrease in Ag uptake and processing, and a modification in cell molecules. However, once DCs receive danger signals transduced migration. Thus, rather than migrating randomly through tissue, they now migrate directionally, via the lymphatics to the draining *Department of Immunology and Pathology, Monash University, Melbourne, Victoria lymph nodes (2–4). The mechanisms of migration used by DCs 3004, Australia; †Department of Medicine, Centre for Inflammatory Diseases, Monash have been reported to be both dependent on adhesion molecules University, Clayton, Victoria 3168, Australia; ‡Department of Microbiology and Immu- x (5–7) and adhesion-independent ameboid migration, driven chiefly nology, University of Melbourne, Melbourne 3010, Australia; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University by cytoskeletal protrusion and contractile forces (8). Nonetheless, of Melbourne, Melbourne 3010, Australia; {Radboud Institute for Molecular Life the activated DC that has migrated to the draining lymph node has Sciences, Radboud University Medical Center, G525 GA Nijmegen, the Netherlands; and ‖ now metamorphosed to become a cell specialized at initiating Molecular Immunology, German Cancer Research Center, 69120 Heidelberg, Germany adaptive immunity by presenting Ags to naive T cells and inducing ORCIDs: 0000-0001-6015-5651 (M.C.D.); 0000-0001-6771-8891 (J.A.V.); 0000- 0002-3590-2368 (A.B.v.S.); 0000-0003-2354-357X (M.J.H.); 0000-0002-2177-5214 their activation (2). These major functional changes require ex- (M.D.W.). quisite coordination between the Ag processing and presentation Received for publication February 12, 2015. Accepted for publication December 1, machinery, and the myriad of adhesion, signaling, and cytoskeletal 2015. that regulate DC morphology and migration. This work was supported by grants from the Australian National Health and Medical One type of known to regulate both Ag presentation and Research Council and the Netherlands Organization for Scientific Research (NWO- Vidi Grant 864.11.006 to A.B.v.S.). cell motility is the tetraspanins, a superfamily of four-transmembrane molecules, highly conserved in evolution and expressed in all mam- Address correspondence and reprint requests to Dr. Mark D. Wright, Monash University, Alfred Medical Research and Education Precinct, Commercial Road, malian cells. The best-defined role of tetraspanins in biology is their Melbourne, VIC 4004, Australia. E-mail address: [email protected] molecular organization of cell membranes. Tetraspanins directly The online version of this article contains supplemental material. interact with their molecular partners and organize them into signal- Abbreviations used in this article: BMDC, bone marrow–derived DC; DC, dendritic transducing microdomains (9, 10). Components of - cell; MHC II, MHC class II; MoDC, monocyte-derived DC; TEM, tetraspanin-enriched enriched microdomains (TEM) include membrane proteins such as microdomain; WT, wild-type. integrins, proteases that regulate cell-surface molecule expression Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 posttranslationally, and signaling molecules including kinases and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500357 The Journal of Immunology 979 phosphatases. Tetraspanins regulate the spatiotemporal molecular following primers: forward: 59-AGGTGTTTGCCCTTCTCCTT-39 and 2/2 interactions of their partner proteins and thereby influence complex reverse: 59-CCACCTGTGACAACCAAGTG-39. CD82 PCR was per- cellular events such as activation, adhesion, and migration. An formed with the following primers: forward: 59-TCCTTAAGCCTCAA- GAAAACC-39 and reverse: 59-TGTGAGGGCTCCAGTCTCC-39. PCR emerging role for TEMs is in communication between the cell program was as follows: 95˚C for 15 min, cycle: 95˚C 30 s, 50˚C 30 s, surface and the cytoskeleton. Tetraspanins regulate cytoskeletal- 72˚C 45 s, 35 cycles, 72˚C for 7 min (final annealing), and hold at 4˚C. 2 2 dependent processes such as outside-in integrin signaling and CD82 / mice were shown to lack CD82 mRNA, and levels of CD82 in WT BMDCs were measured via TaqMan real-time PCR for mouse CD82 adhesion strengthening, actin polymerization, cellular polarity, 2 2 (CD82 / expression of CD82 not shown) (Catalog Mm00492061_m1; and spreading (9–11). On the molecular level the tetraspanin Life Technologies). Levels of CD37 in WT BMDCs were measured via CD81 has been shown to interact with three important regulators TaqMan real-time PCR for mouse CD37 (Catalog Mm00514240_m1; Life of cytoskeleton polymerization: the Rho GTPase Rac, Ezrin, and Technologies). Levels of 18S were measured by TaqMan real-time PCR Moesin. Ezrin and Moesin are peripheral membrane proteins that for mouse 18S (Catalog Mm_03928990_g1; Life Technologies). Real-time link the cytoskeleton with the plasma membrane (11). PCR was performed according to the manufacturer’s guidelines. In APCs, tetraspanins have been implicated in many facets of Cell isolation biology including pattern recognition (12), Ag presentation (13), DCs were isolated as previously described (14). In brief, bone marrow was and cell migration (14). A role for tetraspanins in Ag processing is cultured for 7–9 d with 10 ng/ml GM-CSF and IL-4 (R&D Systems) to suggested both by their presence in MHC class II compartments, obtain BMDCs, and activated with 1 mg/ml LPS. Spleens were treated with where they associate with MHC class II and the peptide editors enzymatic digestion and density-gradient centrifugation before magnetic HLA-DM and HLA-DO (15–17), and by their association with bead depletion to obtain splenic DCs. T cells were isolated as previously described (31). In brief, spleens and lymph nodes were prepared into a Downloaded from MHC I and II at the cell surface (15, 18). However, the conse- single-cell suspension before being subjected to magnetic bead depletion. quences of these interactions on Ag presentation are incompletely understood, with studies variously pointing to roles in promoting Expression of CD82 and CD37 on human DCs (19) and restraining Ag presentation (20, 21). Human PBMCs were isolated from buffy coats and monocytes were Recently, we have demonstrated that the tetraspanin CD37 plays enriched by plastic adherence. Monocytes were cultured with 450 U/ml an important role in linking Ag processing and presentation with GM-CSF (Strathmann) and 300 U/ml IL-4 (Strathmann) in complete RPMI 1640 (Invitrogen Life Technologies)/10% FCS for 6 d to generate cell migration during DC activation. CD37 molecularly interacts http://www.jimmunol.org/ immature DCs. DC maturation was induced by addition of 200 ng/ml LPS with MHC (22), is downregulated upon DC activation (23), re- for 24 h. DCs were fixed in 2% paraformaldehyde, mounted on poly-L- strains MHC-mediated Ag presentation (24), but is also required lysine–coated coverslips, and stained with anti-CD37 (WR17) and anti- for optimal DC migration to draining lymph nodes (14). The CD82 (B-L2; Serotec) followed by goat anti-mouse Alexa 488 secondary closest phylogenetic relative to CD37 in the is Abs. Samples were analyzed by flow cytometry or confocal laser scanning CD82 (25), a tetraspanin best characterized as a metastasis sup- microscopy. pressor that influences cellular adhesion and is a prognostic in- In vivo responses dicator in various nonimmune human cancers (26, 27). In APCs, In vivo responses were performed as previously described (14). In brief, CD82 also molecularly associates with MHC, both at the cell IFN-g ELISPOT was performed on restimulated splenocytes 2 wk after surface and in intracellular vesicles (16, 17, 28). We therefore injection of gamma-irradiated B16-OVA. To measure Ag presentation and by guest on September 26, 2021 hypothesize that CD82 is also an excellent candidate to regulate T cell priming in vivo, we injected 3 3 106 CFSE-labeled OT-I Ly5.1+ 3 5 the cell migration and Ag presentation machinery in DCs. In this T cells i.p. into recipient mice. Two days later, 5 10 LPS activated (1 mg/ml), SIINFEKL-pulsed (1 mg/ml, 1 h), and Cell Tracker Orange article, we show a central role for CD82 in DC function where (0.5 mM; Life Technologies)–labeled BMDCs were injected intradermally remarkably, it shows an effect opposite to CD37; CD82 is up- into the base of the tail. Seventy hours later, mice were culled, draining regulated upon bone marrow–derived dendritic cells (BMDC) lymph nodes harvested, and the number of T cell divisions and migrated activation, negatively regulates DC migration, but is required for BMDCs were measured by flow cytometry. The ratio was calculated by efficient activation of T cells. Thus, the data support a new concept enumerating the total number of T cell divisions and dividing this by the number of BMDCs that had migrated to the draining lymph nodes. that by regulating the expression of the functionally opposing tetraspanins CD37 and CD82 during activation, DCs coordinate In vitro T cell proliferation, Ag presentation, and costimulation the cellular machinery important for both Ag presentation and cell assays migration. T cell proliferation was performed as previously described (31). In brief, purified WT or CD822/2 T cells were stimulated with anti-CD3 in the presence or absence of anti-CD28. In vitro Ag presentation was performed Materials and Methods by coculturing of OVA-specific OT-I and OT-II T cells or B3Z hybridomas Mice with either splenic or WT, CD822/2, and CD372/2 BMDCs pulsed with either SIINFEKL or ISQAVHAAHAEINEAGR (OVA helper peptide). In All mice used were backcrossed 10 times to the C57BL/6J [wild-type (WT)] 2/2 costimulation assays, WT T cells were stimulated with anti-CD3, in the genetic background. CD37 mice were generated by homologous re- 2/2 2/2 2/2 presence or absence of WT, CD82 , or CD37 BMDCs. T cell pro- combination (29). CD82 mice were generated by cre-loxP recombi- 3 nation (30). Mice were bred from established colonies at either the Alfred liferation was measured by [ H]thymidine incorporation (Amersham). B3Z Medical Education and Research Precinct Animal Services (Prahran, hybridoma activation was measured by colorimetric determination of IL-2 Victoria, Australia), the Animal Research Laboratories (Clayton, Victoria, production (24). Australia), or the Radboud University Medical Center (the Netherlands). MHC class II maturation C57BL/6-OT I, C57BL/6-OT I Ly5.1+, and C57BL/6-OT II mice were obtained from the Walter and Eliza Hall Institute (Melbourne, Victoria, Maturation assays were performed on freshly isolated splenic DCs or Australia). All mice used were male, 6–12 wk of age, and age matched to BMDCs as previously described (32). In brief, DCs were serum staved relevant controls. All experiments were carried out under the ethics ap- before pulsing with [35S]methionine/cysteine before a chase of 4.5 h. proval of the Animal Ethics Committee at the Alfred Medical Education Levels of MHC maturation were measured via immunoprecipitation of and Research Precinct Animal Services. MHC class II (MHC II) (M5/114) and SDS-PAGE and exposure to film. PCR and testing of CD82 ablation DC–T cell conjugate formation Mice were tail or ear clipped upon weaning (14–21 d old), and genomic OT-I or OT-II cells were stained with CFSE (0.5 mM, 15 min, 37˚C; DNA was isolated via a DNA Isolation Kit (Promega). Oligonucleotides Invitrogen) or Cell Tracker Orange (0.5 mM, 15 min; Life Technologies). were custom orders (Geneworks). WT PCR was performed using the DCs were stained with either Cell Proliferation Dye eFluor 670 (referred to 980 TETRASPANINS CD82 AND CD37 REGULATE DC ACTIVATION in this article as CT670; 0.5mM, 15 min, 37˚C; eBioscience) or CFSE as before a Mouse Inflammation Cytometric Bead Array was performed as described earlier. For flow conjugation, BMDCs were preactivated for 18 h per the manufacturer’s guidelines (Catalog 552364; Becton Dickinson). with 1 mg/ml LPS (splenic DCs were not activated in vitro) and after staining immediately incubated with increasing concentrations of OVA Statistical analysis peptide [either SIINFEKL or ISQAVHAAHAEINEAGR (OVA helper All data are presented as mean 6 SEM. Comparisons between groups were peptide) for at least 2 h at 37˚C], after which cells were mixed together determined by Student t test, or, where appropriate, ANOVA. The p values are 3 g (1:1 T cell/DC ratio), centrifuged at 200 for 2 min, and incubated as follows: p . 0.05 not significant, *p , 0.05, **p , 0.01, ***p , 0.001. (37˚C; 15 min). Cells were placed on and immediately analyzed by flow cytometry. For live microscopy, BMDCs were allowed to adhere to glass slides in the presence of 1 mg/ml LPS for 18 h and were pulsed with SIINFEKL or CD4 helper for at least 2 h before being washed three times. Results Slides were then placed within a temperature- and CO2-controlled stage CD82 ablation dysregulates T cell responses and T cells were added for live microscopy. Slides were imaged every 30 s To determine whether the tetraspanin CD82 has a role in the cell- from 5 min after addition of T cells for between 20 and 60 min with a 2/2 3 mediated immune response, we studied a newly generated CD82 special-order Nikon A1r Plus Confocal (60 lens; driven by NIS-Elements 2 2 4.1 acquisition software; CFI Apochromat 40XWI). mouse. WT and CD82 / mice were challenged intradermally with gamma-irradiated B16-OVA cells. Two weeks later, the fre- Migration assays quency of T cell–specific IFN-g responses in the spleen was DC migration assays were performed as previously described (14). In brief, measured by peptide-specific ELISPOT. CD822/2 mice had sig- migration of dermal DCs out of ear explants was performed, where ears nificantly decreased Ag-specific CD8+ responses (Fig. 1A); how- were cultured in the presence or absence of CCL19. Transwell migration + was performed by the use of LPS-activated BMDCs in transwells mi- ever, CD4 -specific responses were not above background in a Downloaded from grating toward a CCL19 chemokine gradient. In vivo migration of intra- single immunization, as had been previously described in this dermally injected BMDCs to draining lymph nodes was performed as model (14). The reduced CD8 response could not be attributed to previously described (14) except 1 mM CT670 (eBioscience) was used in a defect intrinsic to CD822/2 T cells because IFN-g responses place of SNARF-1. in CD822/2 T cells stimulated with ConA were indistinguishable Cell-surface expression from WT (data not shown). Moreover, in vitro stimulation with mAbs against CD3 and CD28 revealed that T cells from CD822/2 BMDC basal expression and expression after activation with 1 mg/ml http://www.jimmunol.org/ LPS for 18 h was examined. Cells were stained with anti–CD11c- mice were hyperproliferative (Fig. 1B–E). This finding is con- allophycocyanin (BD Pharmingen, San Diego, CA) and either anti–MHC sistent with previous findings that T cells deficient in other II–FITC (M5/114), anti–CD80-PE, or anti–CD86-PE, anti–LFA-1 FITC, tetraspanins are also hyperproliferative (33), but does not explain anti–CD11b FITC, CD49dFITC (all generated in-house), anti–MHC I the poor in vivo cellular immunity observed in CD822/2 mice. biotin (BD Pharmingen), or anti-CD49e biotin (BD Pharmingen) plus streptavidin-FITC (BD Pharmingen), anti-CCR7 PE (eBioscience), anti– CD82 and CD37 expression in DCs have opposing effects on ICAM-1 PE (Molecular Probes), and examined under flow cytometry by the activation of naive T cells FACSCalibur (Becton Dickinson) or special-order LSR Fortessa (Becton 2/2 Dickinson). The impaired T cell responses observed in CD82 mice (Fig. 1) are reminiscent of mice deficient in the tetraspanin CD37, the

BMDC adhesion and cytoskeletal rearrangement by guest on September 26, 2021 closest phylogenetic relative to CD82 (25). In this article, the poor BMDC morphology and adhesion were assessed as previously described T cell responses induced by CD37 ablation were attributed to (14). In brief, BMDCs were stimulated with 1 mg/ml LPS, adhered to defects in DC biology, because CD37 both restrains Ag presen- fibronectin-coated slides in the presence of 50 ng/ml PMA before being tation and promotes DC migration (14, 24). We therefore hy- fixed and stained for phalloidin-FITC. Microscopy images were scored blind before the average size of the was calculated. For pothesized that CD82 also had an important role in DCs, and thus confocal microscopic analysis, cells were additionally stained with rabbit sought to compare CD37 and CD82 function in DCs. We first anti–b-tubulin Ab (Cell Signaling Technology) and then anti-rabbit Alexa compared the expression of CD82 and CD37 in WT mouse 647. Cells were imaged by confocal microscopy [Nikon A1r Plus Con- BMDCs, before and after activation, by quantitative PCR, because focal, driven by NIS-Elements 4.1 acquisition software; a 603 lens (CFI Apochromat LWD 40xWI) was used]. Images acquired consisted of a stack Abs are not available for murine CD82 and CD37. After 1 h of of 5 3 1 mm steps, and the fluorescence intensity of both phalloidin and activation, CD37 expression is moderately upregulated and CD82 b-tubulin staining was determined at the focal plane using ImageJ analysis. moderately downregulated. However, 18 h later, CD82 was markedly upregulated, whereas CD37 was downregulated and comparable with Small GTPase activation assay basal levels (Supplemental Fig. 1A). The data are broadly consistent Two million naive BMDCs were allowed to adhere to plastic tissue culture with published flow cytometry that confirmed a downregulation of wells for 18 h before being serum staved for 2 h. Rho/Rac-1/Cdc42 Ac- CD37 protein in human monocyte-derived DCs (MoDCs) upon LPS tivator I (CN04 toxin) was added for 2 h, before cells were lysed in situ for G protein linked immunosorbent assay. G-LISA RhoA Activation Assay activation (23) and with flow cytometry and confocal microscopy Biochem Kit (catalog no. BK124), G-LISA Rac-1 Activation Assay Bio- where we observed that CD82 protein is upregulated upon human chem Kit (catalog no. BK128), and G-LISA Cdc42 Activation Assay MoDC activation (Supplemental Fig. 1B, 1C). Biochem Kit (catalog no. BK127) were performed as per manufacturer’s Given that CD37 and CD82 molecularly interact with MHC in guidelines (all from Cytoskeleton, Denver, CO). APCs (16–18, 22, 28), we next investigated whether CD82 has a DC Ag uptake role in regulating the priming of naive T cells by DCs. WT, 2/2 2/2 + 2/2 CD82 , and CD37 BMDCs were pulsed with either CD4 - CD82 or WT splenic DCs were coincubated with FITC-labeled, + carboxylate-modified microspheres (Invitrogen-Molecular Probes, Carlsbad, or CD8 -specific peptides and cocultured with Ag-specific T cells. 2/2 CA) either at a DC/bead ratio of 500:1 for 40 nm FITC-coated beads or 10:1 In contrast with CD37 BMDCs, which showed a hyper- for 500 nm FITC-coated beads over 48 h at 37˚C. After 48 h, cells were stimulatory phenotype (Fig. 2A, 2B, 2D, 2E), in both temporal and washed three times, stained for CD11c expression (as described earlier), and dose responses, CD822/2 BMDCs showed a clear impairment in analyzed via flow cytometry. the ability to present Ag to either class I–restricted CD8+ OT-I Cytokine production upon LPS stimulation T cells (Fig. 2A, 2B) or class II–restricted CD4+ OT-II T cells (Fig. 2D, 2E). We observed a similar impairment of T cell acti- BMDCs were cultured at 1 million cells in 1 ml complete RPMI 1640 2/2 containing 1 mg/ml LPS for 24 h. Supernatant was then collected and vation in Ag-pulsed splenic CD82 DCs (Fig. 2C, 2F). We filtered through a 0.22-mm filter and spun twice to remove cellular debris, conclude that, unlike CD37, which restrains Ag presentation, The Journal of Immunology 981

small (7%) but reproducible and significant decrease in the for- mation of mature MHC/peptide complexes relative to WT cells (Fig. 2G, 2H). From these data, we conclude that CD82 does regulate MHC II maturation and that this regulation might con- tribute to the impairment of T cell activation induced by Ag-pulsed CD822/2 DCs. CD82 expression in DCs is critical for conjugate formation with T cells Because CD82 is involved in the regulation of cellular adhesion in nonimmune cells (26), we reasoned that CD82 might regulate cell–cell adhesion between DCs and T cells; therefore, we ex- amined whether CD822/2 DCs were able to form stable conju- gates with T cells. BMDCs from WT or CD822/2 mice were stained with CT670 and pulsed with increasing concentrations of OVA helper peptide. BMDCs were then cocultured for 15 min with CFSE-labeled OT-II T cells and analyzed by flow cytometry Downloaded from http://www.jimmunol.org/

FIGURE 1. CD82 ablation dysregulates in vivo and in vitro T cell 2/2 responses. (A) In vivo T cell responses from WT and CD82 mice by guest on September 26, 2021 were analyzed by ELISPOT 14 d after intradermal challenge with gamma- irradiated B16-OVA. Splenocytes were restimulated with SIINFEKL or OVA helper peptides to assess CD8+ and CD4+ responses, respectively, and the data were expressed as the frequency of IFN-g–producing T cells relative to ConA controls. Data were pooled from five independent ex- periments; WT: n = 28, CD822/2: n = 21. (B–E) Purified CD4+ or CD8+ T cells from WT and CD822/2 mice were stimulated with surface- adsorbed anti-CD3 mAbs in the presence or absence of soluble anti-CD28 mAbs. T cell proliferation was assessed by the incorporation of tritiated thymidine (mean 6 SEM). (B) CD4+ T cells (anti-CD3), (C) CD4+ T cells (anti-CD3 + anti-CD28), (D) CD8+ T cells (anti-CD3), (E) CD8+ T cells (anti-CD3 + anti-CD28). Representative data from three independent ex- periments. *p , 0.05, **p , 0.01, ***p , 0.001 relative to WT. FIGURE 2. CD37 and CD82 in DCs have opposing effects on the ac- 2/2 2/2 CD82 expression in DCs is essential for T cell activation, consistent tivation of naive T cells. WT, CD82 , and CD37 DCs were pulsed 2/2 either with SIINFEKL (A–C) and coincubated with OT-I T cells or with with the poor cellular immunity observed in CD82 mice caused D F 2/2 OVA helper peptide ( – ) and cocultured with OT-II T cells. T cell– by a failure of CD82 DCs to adequately stimulate naive T cells. proliferative responses were assessed by tritiated thymidine incorporation To understand the mechanism by which CD82 in DCs regulates (mean 6 SEM). (A) Temporal analysis of SIINFEKL-pulsed BMDCs, (B) T cell activation, we used classical assays of Ag presentation to dose response of SIINFEKL-pulsed BMDCs, (C) temporal analysis of T cell hybridomas, the upregulation of activation markers after SIINFEKL-pulsed splenic DCs, (D) temporal analysis of helper peptide- stimulation, costimulatory activity, and cytokine production. pulsed BMDCs, (E) dose response of helper peptide-pulsed BMDCs, and Even though we confirmed that CD372/2 BMDCs were hyper- (F) temporal analysis of helper peptide-pulsed splenic DCs. Data are stimulatory to T cell hybridomas (24), we could detect no dif- pooled from 2–4 independent experiments, groups of 4–10 mice. (G and H) 2/2 ferences between WT and CD822/2 BMDCs (Supplemental Fig. Pulse chase analysis of MHC II where WT and CD82 splenic DCs were 35 2A–E). CD82 is found in intracellular compartments, including incubated in [ S]-containing media for an hour, then returned to normal media for 4.5 h. MHC II was immunoprecipitated from lysates at the 0- phagosomes, and MHC II compartments together with MHC II and 4.5-h time points. (G) Representative SDS-PAGE analyses showing and HLA-DM and HLA-DO (16–18). Therefore, we next con- boiled (B) and nonboiled (NB) samples. The positions of the mature MHC/ sidered the possibility that CD82 plays a role in Ag uptake, pro- peptide complex and a- and b-chains are indicated. (H) Densitometric cessing, and MHC maturation. Even though CD82 ablation did not analyses from data pooled from three independent experiments (n =5) affect the phagocytosis of fluorescent beads (Supplemental Fig. where mature MHC is normalized to WT controls. *p , 0.05, **p , 0.01, 2F, 2G), pulse chase analyses of MHC II maturation did show a ***p , 0.001 relative to WT. 982 TETRASPANINS CD82 AND CD37 REGULATE DC ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. CD82 is essential for conjugate formation between T cells and DCs. (A–F) WT CD822/2 and CD372/2 DCs were labeled with CT670, pulsed with titrated doses of either SIINFEKL or OVA helper peptide, and cocultured with CFSE-labeled OT-I or OT-II T cells. Stable conjugates were identified as double-positive events by flow cytometry. (A) Representative flow cytometry depicting the impaired conjugate formation between OVA helper peptide–pulsed CD822/2 BMDCs and OT-II T cells. Quantification of conjugates between (B) OT-I T cells and WT, CD822/2 or CD372/2 BMDCs pulsed with SIINFEKL; (C) OT-II T cells and WT, CD822/2 or CD372/2 BMDCs pulsed with OVA helper peptide (n = 6–12, 2–3 independent experiments). (D and E) T cell/DC conjugate assays performed with splenic DCs (D) pulsed with SIINFEKL, (E) pulsed with OVA helper peptide (n = 6–7, 2 independent experiments). (F–J) T cell/DC conjugate formation was also analyzed by live microscopy comparing WT and CD822/2 BMDCs. (F) Representative frames depicting interactions at various time points among Ag-pulsed WT BMDCs (fluorescently labeled blue), CD822/2 BMDCs (green), and OT-I T cells (orange). The durations (min) of the recording (top left), a representative T cell/WT DC interaction (bottom left), and a representative T cell/CD822/2 DC interaction (bottom right) interaction are depicted. Quantification of the duration of T cell/DC interactions: (G) histogram showing the interaction times between SIINFEKL-pulsed BMDCs and OT-I T cells and (H) between OVA helper peptide-pulsed BMDCs and OT-II T cells. (I) Histogram showing the interaction times classified into 5-min bins, as a percentage of total interactions, for WT (black) and CD822/2 (white) BMDCs (pooled data for both SIINFEKL and helper peptides). (J) Percentage static interactions (as a readout of stability) for WT (black) and CD822/2 (white) with OT-I or OT-II T cells. Individual data points show mean for each biological sample, with group mean and SEM also shown. n . 130 interactions for each strain and peptide, n = 6 mice, 3 independent experiments. Data include all color swap experiments. *p , 0.05, **p , 0.01, ***p , 0.001 relative to WT. to enumerate DC–T cell conjugates defined as viable double- BMDCs to form stable conjugates with Ag-specific T cells color–positive events (Fig. 3A, top right quadrant). Representative (Fig. 3A). Quantitative analyses demonstrate that increasing flow cytometry data show a clear defect in the ability of CD822/2 peptide concentration significantly increased conjugate formation The Journal of Immunology 983 for all strains, but CD822/2 BMDCs formed significantly fewer hypermigratory phenotype affected the efficiency of CD822/2 conjugates with T cells than WT BMDCs (or CD372/2 BMDCs). DCs (which are poor presenters of Ag to T cells; Figs. 2, 3) to Indeed, conjugate formation was attenuated by up to 40% at high initiate T cell activation in vivo, WT and CD822/2 BMDCs were peptide concentrations (Fig. 3B, 3C). The same phenotype was fluorescently labeled, pulsed with SIINFEKL, and injected intra- observed in primary CD822/2 splenic DCs (Fig. 3D, 3E). To dermally into WT recipients, where both the migration of BMDCs examine the kinetics of conjugate formation, we used a live cell to draining lymph nodes and the proliferation of adoptively imaging approach where BMDCs were labeled with either CT670 transferred, CFSE-labeled OT-I T cells were measured and or CFSE, and cocultured for 18 h with LPS and 2 h with peptide expressed as a ratio. Although the total amount of T cell divisions before being imaged for up to 1 h with either OT-I or OT-II T cells induced by CD822/2 BMDCs did not differ from their WT stained with Cell Tracker Orange. An example (shown in Fig. 3F) counterparts (data not shown), the efficiency of in vivo Ag pre- demonstrates that although WT BMDCs interacted with T cells sentation per migrated CD822/2 BMDCs was significantly im- for several minutes (see blue DCs interacting with orange T cell paired (Fig. 4F). Finally, the expression of cell-surface molecules from 5 to 15 min in Fig. 3F), the interactions between CD822/2 that are involved in DC migration and adhesion, such as the BMDCs and T cells were transient (see green DCs interacting with CCL19 receptor CCR7 and integrins, were assessed by flow T cell from 2 to 5 min, but by 7.5 min the interaction is not cytometry; we could detect no significant differences between WT maintained in Fig. 3F; see also Supplemental Video 1 for another and CD822/2 BMDCs (Fig. 4G). We conclude that in contrast example). Quantification of the interactions revealed that the av- with CD37, which is required for optimal DC migration, CD82 erage duration of the interactions between CD822/2 BMDCs and negatively regulates DC migration.

T cells was shorter than WT counterparts for both MHC class Downloaded from I– and class II–restricted Ags (Fig. 3G, 3H), with the CD822/2 CD82 and CD37 control DC cytoskeletal dynamics via a BMDCs having a much larger proportion of short-lived interac- differential regulation of Rho GTPases tions relative to WT (Fig. 3I). Moreover, when assessing the na- Tetraspanin CD82 negatively regulates DC migration yet pro- ture of the interaction, we noted that the proportion of interactions motes Ag presentation. Conversely, its closest phylogenetic relative between DC and T cells that were static (as opposed to interac- among the tetraspanins, CD37, opposes CD82 as it promotes DC

tions where the T cell crawled over the DC surface) was .2-fold migration but negatively regulates Ag presentation. Given that http://www.jimmunol.org/ higher in WT BMDCs compared with CD822/2 BMDCs (Fig. 3J). adhesion-dependent and -independent DC migration are both re- We therefore conclude that CD822/2 DCs are poor stimulators of liant on cytoskeletal rearrangement, and that the cytoskeleton plays T cells mainly because of their inability to form long, stable, a key role in organizing the immunological synapse and promoting static, cellular interactions with T cells. Ag presentation in DCs (34, 35), we reasoned that ablation of the tetraspanins CD37 and CD82 in DCs might induce a dysregula- CD37 and CD82 have opposing effects on DC migration tion in cytoskeletal function. To assess cytoskeletal function in We have recently published that CD37 is important for optimal BMDCs, we first analyzed their ability to spread upon adhesion to DC migration from the periphery to the draining lymph node the integrin fibronectin. The morphology of BMDCs in all (14). Given the extensive data from cancer cell biology document- three strains was heterogeneous, and three types of cell mor- by guest on September 26, 2021 ing a role for CD82 in suppressing migration and metastases (27), phologies were observed: 1) a small unspread morphology that we hypothesized that CD82 might also regulate DC migration. lacked cellular projections, 2) a large spread cell that lacked Therefore, we assessed migration of WT, CD822/2,andCD372/2 cellular projections, and 3) a classical dendritic morphology DCs using a series of in vivo and in vitro assays. First, in a model (Fig. 5A). Quantitative assessment of spreading confirms our where skin DCs migrate out of mouse ear explants in response to previous findings (14) that CD372/2 BMDCs tend to be of cell the chemokine CCL19, we confirmed our previous observations type 1, in that they spread poorly on fibronectin and have a lack that CD372/2 DCsshowedimpairedmigration(14).Bycontrast, of cellular projections. CD822/2 BMDCs also lacked projections, CD822/2 DCs showed a striking hypermigratory phenotype but by contrast were predominantly of cell type 2, large spread (Fig. 4A). This finding could not be attributed to a DC devel- cells without projections (Fig. 5B). This was confirmed by mea- opmental defect, because the total number of CD11c+ DCs in surement of surface area of BMDC spreading on fibronectin, CD822/2 ear tissue, enumerated by enzymatic digestion and re- which was exaggerated in the absence of CD82 (Fig. 5C). lease, was comparable with WT mice (Fig. 4B). To determine Fibronectin-adherent BMDCs were permeabilized, costained with whether the dysregulated migration induced by CD82 ablation was phalloidin (FITC) and Abs against b-tubulin (red), and analyzed intrinsic to DCs, or might be explained by defects in CD822/2 by confocal microscopy to further analyze the cytoskeleton. microanatomy, BMDCs were loaded into the upper chamber of Representative images depicting the cytoskeletal network in WT transwells with or without CCL19 in the lower chamber. CD822/2 BMDCs, large spread CD822/2 BMDCs, and small unspread BMDCs migrated toward CCL19 in much greater numbers than CD372/2 BMDCs are depicted (Fig. 5D–F). To quantify the tu- WT BMDCs, and in contrast with the hypomigratory CD372/2 bulin and polymerized networks, we measured the mean fluores- BMDCs (Fig. 4C). To determine whether the dysregulated mi- cence intensity of phalloidin and b-tubulin. The data show a gration induced by CD82 ablation affected the ability of DCs to defect in the amount of polymerized actin measured in both traffic to draining lymph nodes, we differentially labeled BMDCs CD822/2 and CD372/2 BMDCs (Fig. 5H), whereas a poor ex- from WT, CD822/2, or CD372/2 mice with CFSE or CT670 and pression of b-tubulin was evident in the CD372/2 BMDCs (the coinjected intradermally. Forty-eight hours later, draining lymph increased expression of b-tubulin in CD822/2 BMDCs did not nodes were analyzed by flow cytometry. In these experiments, reach statistical significance, p = 0.1; Fig. 5G). CD822/2 BMDCs displayed a markedly increased ability to mi- To understand how tetraspanin deficiency could lead to a dys- grate to draining lymph nodes (Fig. 4D). This was confirmed by regulation of cytoskeletal rearrangement, we assessed whether determining the migration index calculated relative to an internal CD82 or CD37 influenced the activation of the key regulators of the CT670-stained WT control. CD822/2 BMDCs showed, on av- cytoskeleton, the Rho GTPases. This was done by measuring Rho erage, 6-fold greater numbers arriving in the draining lymph GTPase activation in adherent BMDCs in response to the small nodes than WT BMDCs (Fig. 4E). To determine how this GTPase-activating toxin CN04. The data show a clear dysregu- 984 TETRASPANINS CD82 AND CD37 REGULATE DC ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. CD37 and CD82 have opposing effects on DC migration. Ears were removed from WT, CD822/2, or CD372/2 mice and (A) placed in media with or without CCL19 for 24 h or (B) digested with collagenase. Skin DCs were then enumerated by flow cytometry (CD11c and MHC II; pooled data from n = 9–14 mice, 6 independent experiments). (C) BMDCs were placed in the upper chamber of transwells and allowed to migrate for 2 h with or without CCL19. DCs in the lower chamber were counted by flow cytometry and compared with input numbers (pooled data from n = 6–7 mice, 4 in- dependent experiments). (D and E) WT, CD822/2, or CD372/2 BMDCs were stained with CFSE and coinjected with a WT CT670-stained internal control intradermally. After 48 h, draining lymph nodes were removed and the number of CD11c+ migrating DCs was assessed by flow cytometry for CFSE or CT670. (D) Representative two-dimensional dot plots comparing migrated WT and CD822/2 DCs and (E) migration index relative to WT-CT670 (pooled experiments, n = 4–18 mice, 7 independent experiments). (F) WT and CD822/2 BMDCs were fluorescently labeled (Cell Tracker Orange), activated by LPS, pulsed with SIINFEKL, and injected into separate WT recipients. The migration of BMDCs to draining lymph nodes and the proliferation of adoptively transferred CFSE-labeled OT-I T cells were measured by flow cytometry and expressed as a ratio (pooled data from 2 independent experiments from n = 9–14 mice). (G)WT and CD822/2 BMDCs were activated by LPS and surface stained for adhesion molecules, a5, CD11a, CD11b, a4, and ICAM-1, as well as the CCL19 receptor CCR7, and assessed by flow cytometry. Experiments were representative of n . 3. *p , 0.05, **p , 0.01, ***p , 0.001 relative to WT. lation in the activity of these signaling molecules in tetraspanin- portant article by Faure-Andre´ et al. (36) identified the invariant deficient BMDCs (Fig. 5I–K). After activation with CN04, chain as a key molecular link between the Ag presentation ma- CD82- and CD37-deficient DCs both showed significant ele- chinery and the cytoskeleton. The invariant chain molecularly vations in the amount of active Cdc42 relative to WT. In contrast, interacts with MHC II and plays an important role in the traf- RhoA activation was significantly elevated only in CD822/2 ficking and maturation of MHC II complexes. However, in cell BMDC lysates, whereas the activation of Rac-1 was impaired only migration, it acts as a negative regulator via its ability to modulate in the absence of CD37 (Fig. 5I–K). We conclude that CD82 and the phosphorylation of myosin L chain and inhibit actinomyosin CD37 control DC biology through their opposing regulation of contraction. In this study, we identify the tetraspanins CD82 and cytoskeletal dynamics. CD82 is a negative regulator of RhoA, CD37 as novel proteins that also molecularly interact with MHC whereas CD37 promotes Rac-1 activation. and play a role in DC biology in linking and coordinating Ag processing and presentation with cell migration. Discussion CD82 expression in mouse BMDCs and human MoDCs is The mechanisms that DCs use to coordinate the simultaneous upregulated upon activation (Supplemental Fig. 1); these data modulation of Ag processing and presentation, and cellular mi- agree with published microarray analyses that show that CD82 gration, during their activation are not fully understood. An im- expression is upregulated upon activation of both mouse splenic The Journal of Immunology 985

FIGURE 5. CD82 and CD37 reg- ulate cytoskeletal rearrangement and Rho GTPase signaling. (A–C)WT, CD822/2, or CD372/2 BMDCs were allowed to adhere to fibronectin and then fixed and stained with phalloidin- FITC. (A and B) Slides were scored blind (n = 9, 3 independent experi- ments) for morphology. (A) Image illustrating the three categories of cells observed: 1) unspread, 2) spread, and 3) dendritic. (B)PercentageofWT, CD822/2,andCD372/2 BMDCs in each category of the three categories is shownin(A), and in (C), the cell area of each biological replicate was cal- culated. (D–H) Confocal microscopic analysis of fibronectin-adhered WT, CD822/2, or CD372/2 BMDCs, fixed and costained with anti–b-tubulin Downloaded from (Alexa 647, red) and phalloidin-FITC. (D–F) Representative confocal images depicting the cytoskeleton in (D) WT, (E)CD822/2, and (F)CD372/2 BMDCs. Scale bars, 10 mm. (G and H) Quantitation of (G) tubulin and http://www.jimmunol.org/ (H) phalloidin staining. Data were pooled from three experiments, six to eight cells analyzed per experi- ment. (I–K) Rho GTPase activation in WT, CD822/2, and CD372/2 BMDCs; BMDCs were allowed to adhere before being serum starved for 2 h. CN04 was added for 2 h, before cells were lysed in situ and the amount of active (I) RhoA, (J) by guest on September 26, 2021 Rac-1, and (K) Cdc42 in 4 mglysate was assessed via G protein linked immunosorbent assay. Pooled data from n = 9–14 mice, 3 independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 relative to WT.

DCs (37) and human MoDCs (38) (data are accessible at National The inability of Ag-pulsed CD822/2 splenic DCs and BMDCs Center for Biotechnology Information Expression Omnibus to stimulate T cells suggests that CD82 expression in DC pro- database (39), accession numbers GDS352 and GDS2221). In motes Ag presentation and productive interactions with Ag-spe- this study, the increased CD82 expression modifies DC biology in cific T cells. In this study, we observed that CD82 plays an im- two ways: first, CD82 inhibits DC migration; and second, CD82 portant role in both the processing of MHC II (Fig. 2G, 2H) and is required for DCs to activate naive T cells. In a tissue explant particularly the promotion of physical interaction of DCs and model of skin DC migration and in in vitro chemotaxis and an T cells (Fig. 3). CD82 molecularly interacts with MHC II both at in vivo migration of BMDCs from the periphery to the drain- the cell surface and in MHC II compartments (15–17, 28), and ing lymph node, CD822/2 BMDCs were notably hypermigratory although we can detect no differences in cell-surface expression of (Fig. 4). This finding is in accordance with the well-established MHC in CD822/2 DCs, pulse chase analyses show a significant, role of CD82 as a metastasis suppressor gene in nonimmune albeit small, defect in the maturation of MHC/peptide complexes cancers (26, 27). A dysregulation of the Rho GTPases is likely to (Fig. 2G, 2H). The latter change might reflect the association be a key mechanism that drives this enhancement in DC migra- between CD82 with HLA-DM and HLA-DO (16, 28). The im- tion. We observed increased RhoA and Cdc42 phosphorylation in paired ability of CD822/2 DCs to physically interact with T cells, assays of activated CD822/2 BMDCs, and cell-spreading assays like the dysregulation in cell migration, is also likely driven by the demonstrated an increase in cytoskeletal rearrangement. CD822/2 dysregulation of cytoskeletal rearrangement and aberrant RhoA BMDCs spread to a greater extent than WT BMDCs and have and Cdc42 activation. Upon specific interactions between the a relative inability to produce membrane protrusions (Fig. 5). TCR and peptide-MHC, activated DCs rapidly polarize their cyto- This observation agrees with studies of that also skeleton, a process that is essential for immunological synapse showed a negative regulation of RhoA by CD82 (40), and with formation and the optimal activation of T cells (34, 35, 42). The signaling studies of T cells that show that CD82 cross-linking failure of CD822/2 BMDCs to form an adequate number of stable leads to RhoA activation (41). conjugates with T cells (Fig. 3), their increased spreading on fi- 986 TETRASPANINS CD82 AND CD37 REGULATE DC ACTIVATION bronectin, and their dysregulation of Rho GTPase signaling tems (11), in contrast with CD37 ablation that dramatically re- (Fig. 5) all demonstrate a critical role for CD82 in mobilizing and duces the ability of DCs to migrate from tissues to draining lymph regulating cytoskeletal rearrangements in DCs during interactions nodes (14). with T cells. That this impairment in cytoskeletal dynamics affects Thus, when considering the difference in biology in DCs before immune responses is supported by the poor cell-mediated im- and after they are activated by danger signals, the expression of the munity induced in CD822/2 mice (Fig. 1A). Indeed, this poor MHC-interacting tetraspanins CD37 and CD82 is of critical im- cell-mediated immunity is consistent with our observations that portance. Our analyses of BMDCs show that an early activated DC CD82 ablation affects not only the duration (Fig. 3G–I) of the expresses low levels of CD82, a negative regulator of RhoA ac- T cell/DC interaction but also whether the interaction is static or tivation, but high levels of CD37, a positive regulator of Rac-1, kinetic (Fig. 3J). Static conjugate formation is required to form an resulting in a highly motile cell with a limited ability to activate initial immunological synapse, which within the OVA system naive T cells. By contrast, a late activated DC expresses high levels must be at least 30 s (43). A stable, static conjugate influences of CD82 and low levels of CD37 and, therefore, has modified its asymmetry within the T cell, and in turn fate, including differ- migratory, cytoskeletal, and Ag presentation machinery to become entiation into Th1, Th2, or Th17 for naive CD4+ cells, or memory a cell superbly adapted to activating naive T cells. The next or effectors for naive CD8+ cells. Interactions between DCs and challenge is to determine how strongly the controlled expression T cells, which are transient (44), or where the T cells crawl over of CD37 and CD82 contributes to the coordinated regulation of the DC surface may lead to tolerance and anergy (45). migration and Ag presentation in classical migratory DCs. A major question in the tetraspanin field concerns functional overlap between family members. Given that tetraspanins often Acknowledgments Downloaded from molecularly interact with one another within TEMs, it is not We thank the staff at Clayton and Alfred Medical Research and Education surprising that often tetraspanins share overlapping functions, a Precinct animal houses for animal care, and Stephen Cody and the staff of classical example being the ability of at least five tetraspanins to Monash Micro Imaging for assistance with setup of live cell microscopy. negatively regulate T cell hyperproliferation (33). In this article, We thank Dr. Greg Moseley and Aaron Brice for advice on tubulin staining, we show that, at least in splenic DCs and BMDCs, the closely and Dr. Katrina Binger for critical reading of the manuscript.

related tetraspanins CD37 and CD82, rather than displaying http://www.jimmunol.org/ functional overlap, counteract one another in function. Unlike Disclosures CD82, which is upregulated upon DC activation, CD37 is The authors have no financial conflicts of interest. expressed predominantly in immature DCs (Supplemental Fig. 1) (23) [see also published microarray analyses that show that CD37 expression is downregulated upon activation in both mouse References 1. Steinman, R. M. 1991. The dendritic cell system and its role in immunogenicity. splenic DCs (37) and human MoDCs (38); data accessible at Annu. Rev. Immunol. 9: 271–296. National Center for Biotechnology Information Gene Ex- 2. Villadangos, J. A., P. Schnorrer, and N. S. Wilson. 2005. Control of MHC class II pression Omnibus database (39), accession numbers GDS352 antigen presentation in dendritic cells: a balance between creative and destruc- tive forces. Immunol. Rev. 207: 191–205. 2/2 by guest on September 26, 2021 and GDS2221]. Analyses of CD37 BMDCs suggest that CD37 3. Benvenuti, F., S. Hugues, M. Walmsley, S. Ruf, L. Fetler, M. Popoff, expression in immature DCs impairs Ag presentation (Fig. 2) (24) V. L. Tybulewicz, and S. Amigorena. 2004. Requirement of Rac1 and Rac2 but promotes cell migration (Fig. 4) (14). Although both tetra- expression by mature dendritic cells for T cell priming. Science 305: 1150–1153. 4. West, M. A., R. P. Wallin, S. P. Matthews, H. G. Svensson, R. Zaru, spanins molecularly interact with MHC (16, 22), the mechanisms H. G. Ljunggren, A. R. Prescott, and C. Watts. 2004. Enhanced dendritic cell by which CD37 regulates Ag presentation differs from that of antigen capture via toll-like receptor-induced actin remodeling. Science 305: 1153–1157. CD82. Although CD37 clearly regulates the cytoskeleton, as 5. Acton, S. E., J. L. Astarita, D. Malhotra, V. Lukacs-Kornek, B. Franz, P. R. Hess, evidenced by an impairment in cell spreading and the formation of Z. Jakus, M. Kuligowski, A. L. Fletcher, K. G. Elpek, et al. 2012. Podoplanin- membrane protrusions observed in CD372/2 BMDCs (Fig. 5), this rich stromal networks induce dendritic cell motility via activation of the C-type 2/2 lectin receptor CLEC-2. Immunity 37: 276–289. does not affect stable adhesion with T cells because CD37 DCs 6. Ma, J., J. H. Wang, Y. J. Guo, M. S. Sy, and M. Bigby. 1994. In vivo treatment 2 2 are able to form conjugates normally (Fig. 3). In contrast, CD37 / with anti-ICAM-1 and anti-LFA-1 inhibits contact sensitization- DCs have a dysregulation in the presentation of peptide/MHC as induced migration of epidermal Langerhans cells to regional lymph nodes. Cell. Immunol. 158: 389–399. evidenced by their hyperstimulation of T cell hybridomas, possi- 7. Maddaluno, L., S. E. Verbrugge, C. Martinoli, G. Matteoli, A. Chiavelli, Y. Zeng, bly through the display of MHC clusters (24) (Supplemental Fig. E. D. Williams, M. Rescigno, and U. Cavallaro. 2009. The adhesion molecule regulates transendothelial migration and trafficking of dendritic cells. J. Exp. 2A). With regard to cell migration and cytoskeletal signaling, in Med. 206: 623–635. adherent BMDCs, CD37 is essential for the adequate phosphor- 8. La¨mmermann, T., B. L. Bader, S. J. Monkley, T. Worbs, R. Wedlich-So¨ldner, ylation and activation of Rac-1 (Fig. 5J). The impaired cell mi- K. Hirsch, M. Keller, R. Fo¨rster, D. R. Critchley, R. Fa¨ssler, and M. Sixt. 2008. 2/2 Rapid leukocyte migration by integrin-independent flowing and squeezing. gration and dendrite formation in the CD37 BMDCs is similar Nature 453: 51–55. to observations of Rac-deficient DCs (3). However, the inability of 9. Hemler, M. E. 2005. Tetraspanin functions and associated microdomains. Nat. Rac-12/2 DCs to activate naive T cells is in stark contrast with the Rev. Mol. 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