Activation of Cytotoxic T Cells Proinflammatory Mediator Release

The Journal of Immunology

CD81 Controls Immunity to Listeria Infection through Rac-Dependent Inhibition of Proinﬂammatory Mediator Release and Activation of Cytotoxic T Cells

Gloria Martıńez del Hoyo,* Marta Ramı´rez-Huesca,* Shoshana Levy,† Claude Boucheix,‡ Eric Rubinstein,‡ Marıá Minguito de la Escalera,x Leticia Gonza´lez-Cintado,x Carlos Ardavıń,x Esteban Veiga,{ MarıáYań˜ez-Mo´,{ and Francisco Sańchez-Madrid*,‖

Despite recent evidence on the involvement of CD81 in pathogen binding and Ag presentation by dendritic cells (DCs), the molecular mechanism of how CD81 regulates immunity during infection remains to be elucidated. To investigate the role of CD81 in the regulation of defense mechanisms against microbial infections, we have used the Listeria monocytogenes infection model to explore the impact of CD81 deficiency in the innate and adaptive immune response against this pathogenic bacteria. We show that CD812/2 mice are less susceptible than wild-type mice to systemic Listeria infection, which correlates with increased numbers of inflammatory monocytes and DCs in CD812/2 spleens, the main subsets controlling early bacterial burden. Additionally, our data reveal that CD81 inhibits Rac/STAT-1 activation, leading to a negative regulation of the production of TNF-a and NO by inflammatory DCs and the activation of cytotoxic T cells by splenic CD8a+ DCs. In conclusion, this study demonstrates that CD81–Rac interaction exerts an important regulatory role on the innate and adaptive immunity against bacterial infection and suggests a role for CD81 in the development of novel therapeutic targets during infectious diseases. The Journal of Immunology, 2015, 194: 6090–6101.

etraspanins comprise a large superfamily of cell surface presentation through association with MHC class II (MHC-II) proteins that have an exceptional ability to associate with molecules, promoting an efficient naive T cell stimulation (4, 5). T each other and with other proteins, promoting the for- In this regard, several studies have emphasized the role of the tet- mation of supramolecular structures that regulate multiple bio- raspanin CD81 in promoting a potent costimulatory signal to T cells logical functions, including the modulation of immunity and/or (6) and in controlling sustained T cell signaling and molecular or- inflammatory processes (1, 2). In the immune system, tetraspanins ganization of the immune synapse (7). Additionally, whereas lym- expressed on APCs regulate Ag recognition through association phoid development in CD812/2 mice is normal (8–10), T cells from with C-type lectin receptors and Fc receptors (3) and regulate Ag CD812/2 mice present a hyperproliferative phenotype after anti-CD3 by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. stimulation and impaired development of Th2 responses (8, 11). Tetraspanins participate in the regulation of defense against in- *Departamento de Biologıá Vascular e Inflamacioń, Fundacioń Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain; †Division of Oncology, De- fection through their involvement in the binding and uptake of partment of Medicine, Stanford University School of Medicine, Stanford, CA 94035; pathogens and the subsequent initiation of the immune response. ‡ x INSERM, Universite´ Paris-Sud, Institut Andre´ Lwoff, 94807 Villejuif, France; De- CD81 is a ligand for hepatitis C virus in hepatocytes (12) and in T, partamento de Inmunologıá y Oncologıá, Centro Nacional de Biotecnologıá, Consejo Superior de Investigaciones Cientı´ficas, 28049 Madrid, Spain; {Unidad de Investiga- B, and dendritic cells (DCs) (3), and CD81 and CD9 can modulate cioń, Hospital Santa Cristina, Instituto de Investigacioń Sanitaria Princesa, 28009 ‖ HIV-1 membrane fusion and viral clustering at the viral synapse Madrid, Spain; and Servicio de Inmunologıá, Hospital de la Princesa, Instituto de (13). CD81 is also required for hepatocyte infection by Plasmo- Investigacioń Sanitaria Princesa, 28006 Madrid, Spain dium (14). The tetraspanins CD9, CD63, and CD81 were recently http://classic.jimmunol.org Received for publication November 25, 2014. Accepted for publication April 19, 2015. shown to be recruited to the bacterial entry site after infection of This work was supported by the Instituto de Salud Carlos III Grant PI11/00939 (to epithelial cells by Listeria (15). However, the molecular mecha- G.M.d.H.) and Spanish Ministry of Economy and Competitiveness Grant SAF2011- nisms underlying the regulatory role of CD81 during bacterial 25834 (to F.S.-M.). G.M.d.H. is supported by the Ramon y Cajal Program funded by infections remain to be elucidated. Spanish Ministry of Economy and Competitiveness Grant RYC-2010-05920. The Centro Nacional de Investigaciones Cardiovasculares is supported by the Spanish Listeria is a Gram-positive bacteria that causes severe infection Ministry of Economy and Competitiveness and the Pro–Centro Nacional de Inves- in immunocompromised individuals and is a widely used model to Downloaded from tigaciones Cardiovasculares Foundation. study innate and adaptive immunity to intracellular infection (16). Address correspondence and reprint requests to Dr. Gloria Martinez del Hoyo, De- During the first days of infection, professional phagocytic cells partamento de Biologıá Vascular e Inflamacioń, Fundacioń Centro Nacional de Inves- tigaciones Cardiovasculares, Calle de Melchor Fernandez Almagro 3, 28029 Madrid, trap bacteria from target organs, enabling control of bacterial Spain. E-mail address: [email protected] growth (17). After internalization by phagocytic cells, Listeria The online version of this article contains supplemental material. escapes from the phagosome into the cytoplasm, where it repli- Abbreviations used in this article: BHI, brain–heart infusion; BMDC, bone marrow– cates (18). Listeria can spread from cell to cell without leaving the derived DC; cDC, conventional DC; DC, dendritic cell; HKLM, heat-killed Listeria intracellular compartment, which is the main reason why a spe- monocytogenes; IFNAR, IFN-a/b receptor; iNOS, inducible NO synthase; IRF, IFN + regulatory factor; MHC-II, MHC class II; moi, multiplicity of infection; NOX, cific CD8 T cell response to Listeria is crucial for the control of NADPH oxidase; PAK, p21-activated kinase; ROS, reactive oxygen species; STING, the infection (19). The early control of Listeria burden largely stimulator of IFN gene; Tip-DC, TNF/inducible NO synthase–producing DC; WT, depends on the innate immune response occurring in the spleen, wild-type. which relies on a specialized subset of monocyte-derived DCs Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 called TNF/inducible NO synthase iNOS (iNOS)–producing DCs

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402957 The Journal of Immunology 6091

(Tip-DCs). Tip-DCs are characterized by their ability to produce counted after overnight incubation at 37˚C. To analyze splenic cell subsets, TNF-a and the enzyme iNOS, which leads to the production of NO spleens were digested with 0.5 mg/ml collagenase A and 40 mg/ml DNase-I (20). Another splenic DC subset, namely CD8a+ conventional DCs (Roche, Mannheim, Germany) for 10 min at 37˚C. RBCs were lysed and splenic cell suspensions were resuspended in PBS containing 5 mM (cDCs), is responsible for the final resolution of infection against EDTA, 0.5% BSA, and 5 mg/ml DNase-I. T and B cells were depleted by Listeria through the cross-priming of bacterial-derived Ags to magnetic cell sorting using MACS separation columns (Miltenyi Biotec, specific CD8+ T cells and the subsequent induction of the cytotoxic Bergisch Gladbach, Germany) and the cells obtained were analyzed by T cell response (21, 22). Interestingly, Ag cross-presentation in DCs flow cytometry. requires endosomal alkalinization through reactive oxygen species In vitro infection of BMDCs (ROS) production resulting from NADPH oxidase (NOX) 2 re- Listeria cruitment, a process dependent on the small GTPase Rac (23–25). BMDCs were incubated with and assessed for the gentamicin survival assay (30, 31). DCs were infected with Listeria at a multiplicity of infection Additionally, a direct interaction between CD81 and Rac was (moi) of 10 for 1 h at 37˚C. To determine the number of bacteria entering the revealed by proteomics analysis (26), suggesting that CD81 could cells, extracellular bacteria were killed by treatment with 100 mg/ml genta- modulate the cross-priming capacity of DCs. micin for an additional hour at 37˚C. Then, infected cells were washed with DC activation after encounter with Listeria essentially relies on PBS, counted, and lysed with 0.05% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in distilled water. Serial dilutions were seeded in BHI agar plates and the activation of two signaling pathways: a TLR-dependent path- CFUs were counted. way, resulting from cell surface and endosomal bacteria sensing, which leads to the activation of a MyD88-dependent response; and Phagocytosis of fluorescent beads by DCs a cytosolic pathway, triggered after Listeria escapes into the cytosol BMDCs were incubated with biotin-labeled yellow-green fluorescent by secreting listerolysin O, which activates the cytosolic sensor FluoSpheres 1 mm in diameter (Invitrogen/Life Technologies, Thermo stimulator of IFN genes (STING) by nucleotides. STING activation Fisher Scientific, Waltham, MA) and analyzed by flow cytometry. Extra- leads to IFN regulatory factor (IRF)3–dependent production of cellular beads were detected with fluorescently tagged streptavidin. IFN-b (27). IFN-b in turn activates an autocrine loop after binding ELISA to its cognate receptor, the heterodimer formed by the type I IFN receptor (IFNAR)-1 and IFNAR-2 receptors, promoting subse- Production of cytokines and NO was analyzed in the supernatants of BMDC cultures 16 h after stimulation with Listeria, Pam3Cys, heat-killed Listeria quently the triggering of the IFNAR signaling pathway, which monocytogenes (HKLM), flagellin, polyinosinic-polycytidylic acid (Invivo- controls the transcription of IFN target genes essential for antiviral Gen, San Diego, CA), or LPS from Escherichia coli (Sigma-Aldrich). TNF-a and antibacterial immunity (28). was analyzed with OptEIA ELISA kits (BD Biosciences, San Diego, CA) To investigate the role of CD81 in the modulation of defense andIL-6withthemouseELISAReady-SET-Go! kit from eBioscience (Affymetrix, San Diego, CA). NO was estimated from the nitrite concen- against bacterial infections, we have explored the impact of CD81 tration measured with a Griess reagent kit (Molecular Probes/Life Tech- deficiency in the innate and adaptive immune response after Listeria nologies, Thermo Fisher Scientific). infection in vivo. Our data reveal that CD812/2 mice are protected against Listeria infection and support the view that CD81 negatively Measurement of reactive oxygen production regulates the defense mechanisms to this bacterial infection by the Production of ROS was assayed by luminol-ECL, using the L012-luminol inhibition of Rac/STAT-1 activation that interferes with the pro- derivative (Wako Chemicals, Neuss, Germany). BMDCs were seeded in 96- 3 5 duction of proinflammatory mediators by DCs and the potential to well luminometer plates (1 10 cells/well) in medium containing 500 mM

by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. L012 with or without HKLM (moi of 50:1) or PMA (250 ng/ml). Chemilu- cross-present bacterial Ags of DCs to cytotoxic T cells. minescence was measured at 15-min intervals. Materials and Methods CD81 C-terminal peptides Mice TAMRA N-terminal–labeled peptides with the sequences RRRRRRR- 2 2 CCGIRNSSVY (CD81 peptide) or RRRRRRRYSVNICRGCSS (scram- CD81 / mice were described in Maecker and Levy (29). Experiments 2 2 bled peptide) were purchased from Life Tein (South Plainfield, NJ). were performed using CD81 / mice in a C57BL/6 genetic background (backcrossed .20 generations), and wild-type (WT) mice were littermates Pull-down assay sex and age matched. CD45.1 OT-I transgenic mice express a TCR specific 2 2 for OVA peptide 257–264. Mice were housed under specific pathogen-free Pull-down assays were carried out with WT and CD81 / BMDCs serum http://classic.jimmunol.org conditions at the Centro Nacional de Investigaciones Cardiovasculares, and starved for 20 h. For preferential loading of Rac with GDP or GTP, cells the experiments were approved by the Centro Nacional de Investigaciones were lysed in 5 mM EDTA containing lysis buffer and the lysates were Cardiovasculares Ethical Committee for Animal Welfare and by the incubated with 50 nM of GTP-gS or GDP at 30˚C for 5 min before Spanish Ministry of Agriculture, Food, and the Environment. Animal addition of 6 mM MgCl2. Alternatively, cells stimulated with Listeria for procedures conformed to European Union directive 2010/63EU and rec- different times were washed once with ice-cold PBS, lysed in the ommendation 2007/526/EC regarding the protection of animals used for presence of 2 mM MgCl2, incubated with Sepharose beads coated with p21- experimental and other scientific purposes, enforced in Spanish law under activated kinase (PAK)–GST, and analyzed by Western blotting. Rabbit Real Decreto 1201/2005. polyclonal anti-Rac Ab was from BD Biosciences, GST-PAK-Crib was Downloaded from provided by J.C. Collard (The Netherlands Cancer Institute, Amsterdam, Generation of bone marrow-derived DCs The Netherlands), and GTP-gS and GDP were from Sigma-Aldrich. Bone marrow-derived DCs (BMDCs) were obtained from bone marrow cell Western blot analysis suspensions after culture on nontreated culture 150-mm Petri dishes in complete RPMI 1640 medium supplemented with 10% FBS, 2 mM Total cell extracts from BMDCs stimulated with Listeria for different times L-glutamine, 100 mg/ml penicillin, 100 mg/ml streptomycin, 50 mM were prepared in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxy- 2-ME, and 20 ng/ml GM-CSF (PeproTech, London, U.K.). Cells were cholate, 0.1% SDS, 0.5% Triton X-100) and analyzed by Western blotting. collected at day 9 and BMDCs were characterized as CD11c+MHC-II+ Rabbit polyclonal anti-Stat1 and phospho-Stat1 (Tyr701) Abs were from Ly6G2 cells. Cell Signaling Technology (Leiden, The Netherlands). Listeria systemic infections Sorting of Listeria-infected splenic CD8a+ cDCs We used Listeria 10403S strain or the isogenic OVA-expressing strain, CD812/2 and WT mice were i.v. infected with Listeria, and after 15 h Listeria-OVA, provided by Dr. D. Brockstedt (Aduro Biotech, Berkeley, CA). splenic DC-enriched cell fractions were obtained by centrifugation in cold Mice were injected i.v. with Listeria (1 3 104 to 5 3 104 CFUs/mouse). To isosmotic OptiPrep solution (Nyegaard Diagnostics, Oslo, Norway). Cells determine bacterial load, spleens and livers were homogenized and serial were sorted using a BD InFlux cell sorter (BD Biosciences). The purity of dilutions were grown on brain–heart infusion (BHI) agar plates; CFUs were the subpopulations was .95%. 6092 CD81 MEDIATES SUSCEPTIBILITY TO LISTERIA INFECTION

Analysis of Listeria-specific CD8+ T cell response in vivo 3,and4dpostinfection(Fig.1A).TheseresultsshowthatCD812/2 Spleens and lymph nodes were obtained from OT-I CD45.1+ mice, and mice are able to control bacterial growth more efficiently than are CD8+ T cells were isolated by negative selection in an autoMACS Pro WT mice. 2 2 separator (Miltenyi Biotec) according to the manufacturer’s instructions. We next analyzed the survival of WT and CD81 / mice after + + Selected cells were .95% CD8a Va2 , and subsequently these cells were lethal Listeria infection. At a dose that caused 50% mortality in labeled with CellTrace Violet (Molecular Probes) and i.v. transferred into 2/2 2 2 WT mice after 10 d of infection, ∼75% of CD81 mice survived recipient WT and CD81 / mice. One day later, these recipient mice were infected with Listeria-OVA (1 3 104 CFUs per mouse). Three days later, (Fig. 1B), showing that the low bacterial burden observed in 2/2 splenocytes were obtained from infected mice to analyze the activation of CD81 mice correlates with reduced mortality. transferred OT-I cells by flow cytometry. Additionally, splenocytes from infected mice were also cultured in 96-well plates (0.2 3 105 cells/well) in Absence of CD81 allows effective differentiation and survival the presence of 1 mg/ml OVA peptide 257–264 (SIINFEKEL, purchased of inflammatory DCs after Listeria infection from GenScript, Piscataway,NJ), and 2 h later BD GolgiPlug (BD Bio- sciences) was added to block the secretion of cytokines overnight. Cells To understand the protective effect conferred by the absence of CD81 were washed and stained for detection of IFN-g expression, after fixation during Listeria infection, we analyzed the recruitment and differ- and permeabilization using a BD Cytofix/Cytoperm kit (BD Biosciences), entiation of cells that control the innate (monocytes, Tip-DCs, and using an anti-mouse IFN-g Ab (BD Biosciences). In parallel, endogenous + 2 + 2/2 neutrophils) and the adaptive (CD8a and CD8a cDCs) immune OVA-specific CD8 T cells were also analyzed in WT and CD81 mice 2/2 infected with Listeria-OVA for 7 d. At this time point, splenocytes were responses. Analysis of WT and CD81 mice before infection did obtained and Ag-specific CD8+ T cells were analyzed using SIINFEKL–H- not reveal differences in the numbers or percentages of these subsets 2b–PE tetramers, referred to as OVA tetramers (provided by Dr. D. Sancho, (Fig. 2A, day 0), indicating that the development of the different Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain). splenic subsets was not affected by CD81 deficiency. Between days Splenocytes were also cultured in the presence of OVA peptide 257–264 (1 mg/ml), and supernatants were collected to determine IFN-g production 2 and 4 postinfection, both genotypes increased the numbers of by ELISA (mouse ELISA Ready-SET-Go! kit, eBioscience) after over- inflammatory monocytes and Tip-DCs described upon Listeria in- 2 2 night culture; alternatively, splenocytes cultured with OVA peptide for 2 h fection (20). Importantly, in CD81 / mice this increase was sig- were incubated with BD GolgiPlug, and intracellular IFN-g was analyzed nificantly more pronounced compared with WT mice (Fig. 2A). by flow cytometry. cDC numbers increased from day 0 to day 2 in CD812/2 mice and Flow cytometry subsequently dropped to a similar level in both genotypes by day 4 postinfection (Fig. 2A); neutrophils displayed a comparable in- Splenic cell subsets in T/B cell–depleted fractions were analyzed by crease in number in WT and CD812/2 mice from day 0 to day 4 staining with FITC-conjugated anti-CD11b, allophycocyanin-conjugated anti-CD11c, biotin-conjugated anti–Ly-6C, biotin-conjugated anti–MHC- (Fig. 2A). Additionally, analysis of lymphocyte subpopulations II, and biotin-conjugated anti-CD81, followed by incubation with PerCP- revealed that T and B cell numbers were drastically reduced in the conjugated streptavidin, PE-conjugated anti-CD49b, and PE-conjugated spleen of both WT and CD812/2 mice during the first days of anti–Ly-6G (all Abs from BD Biosciences). For the detection of intra- Listeria infection (Supplemental Fig. 1B). Interestingly, the surface cellular iNOS, T/B cell–depleted splenocytes were fixed and permeabilized using a BD Cytofix/Cytoperm Kit (BD Biosciences) and stained with rabbit expression of CD81 was modulated by Listeria infection in the anti-iNOS (Cell Signaling Technology) followed by FITC-conjugated goat different splenic subsets analyzed of WT mice (Supplemental Fig. anti-rabbit Ab (Jackson ImmunoResearch Laboratories, West Grove, PA). 1C). Whereas the expression of CD81 was downregulated by in- Serum and splenic cytokines were measured using a BD cytometric bead flammatory monocytes, neutrophils, and cDCs, Tip-DCs increased by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. array mouse Th1/Th2/Th17 cytokine kit (BD Biosciences). BMDCs were CD81 expression levels during Listeria infection. To investigate analyzed after differentiation by incubation with PE-conjugated anti-CD11c, FITC-conjugated MHC-II, and allophycocyanin-conjugated Gr-1 (BD Bio- how CD81 controls early bacterial replication during Listeria in- 2/2 sciences). Splenic DC-enriched cell fractions were stained for sorting with fection, we analyzed Tip-DC activation in WT and CD81 mice, anti–CD8a-allophycocyanin, anti–CD11c-PE, anti–B220-FITC, and anti– because these cells have been claimed to be critical cells in limit- CD4-FITC (BD Biosciences). Live cells were identified by staining with ing Listeria burden owing to the production of the microbicidal DAPI (Molecular Probes). Flow cytometric analysis was performed on a mediators TNF-a and NO (20). Tip-DC activation, assessed by FACSCanto II (BD Biosciences), and data were analyzed using FlowJo 2/2 v8.8.5 (Tree Star) software. intracellular iNOS expression, was increased in CD81 mice compared with WT mice after 3 d of Listeria infection (Fig. 2B). Statistical analysis We next analyzed whether CD81 deficiency could also affect the http://classic.jimmunol.org Statistical analyses were performed using GraphPad Prism software. A production of inflammatory cytokines, as an early event of the in- 2 2 Student t test was used for individual comparisons of the mean between nate immune response. We found that CD81 / mice exhibited two groups. Differences were considered significant at p # 0.05 and p # increased serum levels of TNF-a, but comparable levels of serum 6 0.01. Data are shown in column bars representing the mean SD of at IL-6, than did WT mice after Listeria infection (Fig. 2C). Addi- least three independent experiments unless otherwise indicated. tionally, TNF-a production in the spleen of infected CD812/2 mice was also higher than WT mice production (Fig. 2C).

Downloaded from Results 2/2 Therefore, our results indicate that Listeria infection in the CD81 mice are resistant to a lethal dose of Listeria absence of CD81 leads to efficient differentiation of inflammatory To investigate the contribution of CD81 to immunity against Listeria monocytes and Tip-DCs, whose numbers and activation increase infection, we first analyzed the bacterial load in target organs at notably during the first days after infection, correlating with the different time points after sublethal infection of WT and CD812/2 observed protective phenotype. mice. We did not observe any significant differences between WT 2 2 a and CD81 / mice using a low bacterial infection dose (103 CFUs CD81 regulates Listeria-induced production of TNF- and NO per mouse; Supplemental Fig. 1A). When we used a higher infec- by infected DCs tion dose (2 3 104 CFUs per mouse), bacterial load in the spleen Early protective immunity against Listeria relies on the production was also similar in WT and CD812/2 mice at day 1 postinfection of proinflammatory cytokines, such as TNF-a and IL-6, mainly (Fig. 1A), and liver CFUs were below the detection limit of the driven by TLR2-dependent signaling, as well as microbicidal assay (data not shown). However, analysis of bacterial burden at mediators, such as NO, after STING-dependent signaling trig- longer times revealed that CD812/2 mice showed significantly gering the IFNAR autocrine pathway (27, 28). To investigate the lower CFUs than did WT mice in both the spleen and the liver at 2, role of CD81 in the production of microbicidal mediators in re- The Journal of Immunology 6093 by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd.

FIGURE 1. CD81 mediates susceptibility to lethal systemic Listeria infection. (A) Bacterial load in the spleen and the liver of WT and CD812/2 mice postinfection with 2 3 104 Listeria per mouse at the indicated time points (mean 6 SD; n = 5 mice/condition). Data are representative of three independent experiments with similar results. *p , 0.05 (unpaired t test). (B) Survival of WT and CD812/2 mice postinfection with 5 3 104 Listeria per mouse (n =10mice/ group). Data are representative of three independent experiments with similar results. *p , 0.05, log-rank test.

sponse to Listeria, we compared the capacity of WT and CD812/2 displayed a similar ability to internalize fluorescent beads, suggest- http://classic.jimmunol.org BMDCs to produce proinflammatory cytokines after Listeria in- ing that CD81 does not interfere with the phagocytic capacity of fection in vitro. According to the results obtained on cytokine DCs (Fig. 3F). Moreover, no differences were found in the capacity production in serum and spleen (Fig. 2C), infected CD812/2 of CD812/2 and WT BMDCs to kill Listeria, as revealed by the BMDCs produced higher levels of TNF-a than did WT BMDCs similardecreaseofCFUsobservedbetween4and6h(Fig.3D). (Fig. 3A), whereas the levels of IL-6 were similar in the two Importantly, both genotypes displayed a comparable viability 9 h genotypes (Fig. 3B). In parallel with the increased TNF-a pro- after in vitro infection (Fig. 3E). These data confirmed that the Downloaded from duction observed, CD812/2 BMDCs produced also higher levels higher TNF-a and NO production capacity of CD812/2 BMDCs did of NO (Fig. 3C). not result from a higher susceptibility to infection. The higher TNF-a and NO production potential displayed by We next addressed whether the differential production of TNF-a CD812/2 BMDCs compared with WT BMDCs could reflect a andNObyWTandCD812/2 BMDCs reflected that CD81 inter- differential bacteria internalization capacity and susceptibility to feres with Listeria-mediated DC activation. In this regard, in re- infection leading to a stronger DC activation or, alternatively, sponse to Listeria, the production of TNF-a and NO is controlled that CD81 directly modulates Listeria-induced DC activation both by TLR signaling and by cytosolic STING-mediated signal- and consequently the production of proinflammatory mediators. ing triggering the IFNAR autocrine pathway, whereas IL-6 pro- We first analyzed the capacity of BMDCs to internalize bacteria duction is not dependent on STING/IFNAR signaling. To determine in vitro by the gentamicin survival assay (30, 31). These experi- whether CD81 differentially affects Listeria-induced signaling ments revealed that WT and CD812/2 BMDCs had a comparable through the TLR2 versus STING/IFNAR-induced signaling, we susceptibility to infection, as assessed by the number of CFUs at analyzed the response of WT and CD812/2 BMDCs to HKLM, 2 h, indicating that both genotypes internalized Listeria with a sim- which activates the TLR2 signaling pathway but not the STING/ ilar efficiency (Fig. 3D). Accordingly, CD812/2 and WT BMDCs IFNAR-dependent signaling. Our data revealed that HKLM induced 6094 CD81 MEDIATES SUSCEPTIBILITY TO LISTERIA INFECTION by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. http://classic.jimmunol.org

FIGURE 2. Recruitment and differentiation of inflammatory monocytes, splenic DC subsets, and neutrophils after Listeria infection. Analysis of splenic cell subpopulations from WT and CD812/2 mice at days 0, 2, and 4 postinfection with 2 3 104 Listeria per mouse. (A) Dot plots show identification of monocytes, Tip-DCs, and cDCs based on CD11b and CD11c expression, after immunomagnetic depletion of T and B cells and gating out neutrophils and NK cells, in WT and CD812/2 noninfected mice. Bar charts represent absolute cell numbers of monocytes, Tip-DCs, cDCs, and neutrophils at indicated times after Listeria infection (mean 6 SD of four mice per condition). Data are representative of three independent experiments with similar results. Downloaded from (B) Representative FACS plots show iNOS expression by Tip-DCs [gated as in (A)] at day 3 after Listeria infection. Quantification of total numbers and frequencies of iNOS-expressing cells in Tip-DCs from noninfected and Listeria-infected WT and CD812/2 mice (mean 6 SD of five mice per condition). Data are representative of two independent experiments with similar results. (C) Mice were infected with Listeria and serum levels of TNF-a and IL-6, and splenic levels of TNF-a were analyzed at the indicated time points postinfection. Data are representative of two independent experiments with similar results (mean 6 SD of five mice per condition). *p , 0.05, **p , 0.01, unpaired t test.

a signiﬁcantly lower production of TNF-a andIL-6thandidlive CD81 deﬁciency (Fig. 3H). Because BMDCs stimulated with Listeria, and NO was not produced after HKLM stimulation HKLM or TLR2 ligands induced lower TNF-a production com- (Fig. 3G). Therefore, the production of TNF-a, IL-6, and NO pared with BMDCs infected with live Listeria, we performed a in response to HKLM was not affected in CD812/2 BMDCs dose-response curve to determine TNF-a production by Listeria- (Fig. 3G). Interestingly, similar results were obtained in response to infected DCs at different doses to compare similar ranges of TNF-a the synthetic ligand Pam3Cys, which activates TLR2 signaling but production. We found that TNF-a production of Listeria-infected not STING/IFNAR signaling (Fig. 3H), although Pam3Cys induced DCs were higher than HKLM- or Pam3Cys-stimulated DCs, even at similar IL-6 levels than did live Listeria that were not affected by the lower infection rate used (moi of 1; Supplemental Fig. 2A). The The Journal of Immunology 6095

FIGURE 3. Production of proin- ﬂammatory cytokines and NO by WT and CD812/2 DCs after Listeria infection. WT and CD812/2 BMDCs were infected with Listeria and 16 h later TNF-a (A), IL-6 (B), and NO (C) were analyzed in noninfected or Listeria-infected DCs. Data are presented as the mean 6 SD and are representative of four independent experiments with similar results. Fold differences in TNF-a and NO production normalized to WT BMDC production after Listeria stimulation are also shown, and data are pooled from four independent experiments. *p , 0.05, unpaired t test. (D) Analysis of Listeria infection by gentamicin survival assay after incubation of WT and CD812/2 BMDCs with Listeria (moi of 10) for the indicated times. The chart shows the numbers of CFUs present in 5 3 104 DCs (mean 6 SD). Data are pooled from four independent experiments. (E)WTandCD812/2 BMDCs were incubated with Listeria (moi of 10) for 9 h and DC viability was determined by staining with propidium

by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. iodide and 7-aminoactinomycin D. The chart shows the percentage of viable DCs (mean 6 SD; representative data from three independent experiments). (F) Phagocytosis of fluorescent beads by DCs. WT and CD812/2 BMDCs were incubated with fluorescent microspheres, and phagocytized particles were analyzed by flow cytometry. Data are presented http://classic.jimmunol.org as the percentage of DCs with internalized beads (mean 6 SD; representative data from four independent experiments). WT and CD812/2 BMDCs were stimulated with HKLM (G) or Pam3Cys (H) for 16 h, and TNF-a, IL-6, and NO were deter- Downloaded from mined. Data are presented as the mean 6 SD and are representative of three independent experiments with similar results. 6096 CD81 MEDIATES SUSCEPTIBILITY TO LISTERIA INFECTION

TLR ligands ﬂagellin (TLR5 ligand), polyinosinic-polycytidylic acid (TLR3 ligand), and LPS (TLR4 ligand) also induced similar levels of TNF-a,IL-6,andNOinWTandCD812/2 BMDCs (Supplemental Fig. 2B, 2C, and 2D, respectively). These results demonstrate that the observed differences in TNF-a and NO production were dependent on live Listeria and suggest that CD81 regulates TNF-a and NO production in Listeria-infected DCs by interfering with STING/IFNAR signaling. Therefore, it can be hy- pothesized that the higher survival and lower splenic bacterial load observed in CD812/2 mice is, at least in part, due to a higher production of TNF-a and NO by splenic inﬂammatory Tip-DCs. To further investigate the involvement of CD81 in the STING/ IFNAR signaling in response to Listeria, we analyzed the expression of IFN-b by Listeria-infected BMDCs. Listeria triggers a STING-driven cytosolic signaling pathway after bacterial nu- cleotide sensing in DCs that leads to the production of IFN-b (27). Our results showed that IFN-b mRNA was expressed by BMDCs at3hofListeria infection and this expression was increased at 5 h postinfection (Supplemental Fig. 3). However, comparable levels of IFN-b were observed in infected WT and CD812/2 BMDCs (Supplemental Fig. 3). Our results show that CD81 does not affect the STING-mediated production of IFN-b in response to Listeria infection, but these results are consistent with the possibility that CD81 interferes with the activation of the autocrine IFNAR signaling pathway, which controls the transcription of IFN-regulated genes essential for antiviral and antibacterial immunity (28). CD81 downregulates IFNAR signaling by controlling Rac activation and STAT-1 phosphorylation in Listeria-infected DCs IFN triggers an autocrine loop after engagement of its cognate receptor IFNAR promoting the activation of STAT-1, a central mediator of IFN responses that is crucial for IFN-induced target genes, such as the production of NO (32). Based on the reported role of Rac1 in the regulation of STAT-1 activation during IFN

by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. signaling (33) and on the described ability of CD81 to regulate Rac activity (26), we investigated whether CD81 could interfere with IFNAR signaling by regulating the activation of STAT-1. FIGURE 4. CD81 downregulates Rac activation and STAT-1 phosphorylation in Listeria-infected DCs. (A) WT and CD812/2 BMDCs were For this purpose, we first analyzed the activation of the small 2/2 serum-starved for 24 h and incubated with Listeria for the indicated times. GTPase Rac after Listeria infection of WT and CD81 BMDCs. Rac activation was analyzed by pull-down assay with GST-PAK–Cdc42/ We assessed Rac activation in Listeria-infected BMDCs by pull- Rac interactive binding domain. Western blots are from a representative down of the active (GTP-bound) form of Rac with GST-PAK– experiment out of four performed. The bar chart shows the fold induction 2/2 Cdc42/Rac interactive binding domain. CD81 BMDCs con- of PAK-GST band density normalized to WT BMDCs (mean 6 SD of four tained higher levels of active Rac than did WT cells 30 min after independent experiments). (B) Western blot analysis of total and phos- 2 2 http://classic.jimmunol.org Listeria infection, and these levels were maintained almost con- phorylated STAT-1 in cell extracts of WT and CD81 / BMDCs treated stant in CD812/2 DCs at 45 min postinfection (Fig. 4A). These with Listeria as indicated. Tubulin was used as loading control of the Western blot to confirm that total STAT-1 levels are similar in WT and results suggest that CD81 negatively regulates Rac activation in 2/2 Listeria-infected DCs. CD81 BMDCs. A representative experiment out of three performed is shown. The bar chart shows the fold induction of phosphorylated STAT-1 We next analyzed whether the higher levels of active Rac dis- 6 2/2 band density normalized to WT BMDCs (mean SD of three independent played by CD81 BMDCs after Listeria infection correlated experiments). *p , 0.05, unpaired t test. Downloaded from with a stronger STAT-1 activation. In this regard, NO production by DCs is driven by IFN-b–mediated triggering of the IFNAR autocrine pathway (34). Thus, we investigated whether CD81 Listeria-infected DCs involves a downregulation of Rac activity could interfere with IFNAR signaling by regulating the activation resulting from direct CD81–Rac interaction, we performed in vitro of STAT-1 in BMDCs after Listeria infection. Phosphorylation of infection assays in the presence of a cell-permeable peptide corre- STAT-1 was evident in DCs 3 h after in vitro infection with Lis- sponding to the C-terminal sequence of CD81. This CD81 peptide teria, and it was enhanced in CD812/2 DCs compared with WT prevents Rac–CD81 association, leading to an increase in Rac ac- DCs (Fig. 4B). These data support that CD81 exerts a negative tivation (26). For this purpose, we incubated WT DCs with fluo- regulation on the production of NO and other proinflammatory rescently labeled CD81 peptide or with a control peptide resulting mediators such as TNF-a in Listeria-infected DCs by interfering from a scrambled combination of the same eight amino acids. These with IFNAR signaling through the inhibition of the Rac/STAT-1 peptideswereinternalizedbyDCsand1hafterincubation,100% activation pathway. of the DCs were fluorescently labeled. The mean fluorescence in- To demonstrate that the mechanism responsible for the inhibi- tensityofthecellsincreasedupto4handdeclinedthereafter,al- tion of NO and proinflammatory cytokine production by CD81 in though 100% of the cells retained detectable fluorescence after 20 h The Journal of Immunology 6097

(Fig.5A).Onceweconfirmedthat DCs internalized CD81 or mediated recruitment of NOX2 to the endosomal compartment. scrambled peptides, we assessed the capacity of Listeria-infected The recruitment of NOX2 to the phagosomes leads to the gener- DCs to produce TNF-a and NO after the treatment with the pep- ation of ROS in CD8a+ cDCs, which are also dependent on Rac2 tides. As shown in Fig. 5B, WT DCs treated with the CD81 peptide (25). Our data revealing that CD81 inhibits Rac activation through but not with the control peptide increased the production of TNF-a direct association prompted us to investigate whether CD81 defi- to levels comparable to those displayed by CD812/2 DCs. Like- ciency interferes with the production of ROS in DCs during Lis- wise, CD81 peptide-treated WT DCs induced a significantly higher teria infection. For this purpose, ROS production was assessed production of NO than did those treated with control peptide by WT and CD812/2 BMDCs stimulated with HKLM, because live (Fig. 5C). Interestingly, the production of TNF-a and NO was not Listeria presents inhibitory mechanisms of ROS production, or affected in Listeria-infected CD812/2 DCs treated with the CD81 PMA, during a 180-min period using a chemiluminiscence assay. peptide. These data show that inhibition of Rac–CD81 interaction CD812/2 BMDCs induced higher ROS levels than did WT BMDCs results in a significantly higher production of TNF-a and NO by after stimulation with either HKLM or PMA (Fig. 6A), suggesting Listeria-infected WT DCs, further supporting that CD81 exerts that CD81 could exert a negative effect on the microbicidal po- a negative regulatory role during the DC-mediated anti-Listeria tential of BMDCs through the regulation of Rac-mediated ROS immune response through the inhibition of Rac activity. production.

+ Presentation of Listeria-derived Ags during Listeria infection has In vivo priming of effector CD8 T cells is more efficient in + 2/2 been claimed to rely essentially on the CD8a cDC subset. Splenic CD81 than in WT mice in response to systemic Listeria CD8a+ cDCs are the first infected subset in the spleen during the infection early phases of Listeria infection (35). Listeria-infected CD8a+ DCs are highly efficient in Ag cross-presentation mainly due to cDCs in the spleen are essential not only for CD8+ T cell generation phagosomal alkalinization, which is mainly regulated by Rac2- but also for Listeria expansion and dissemination within the host,

FIGURE 5. Inhibition of CD81–Rac interaction

by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. results in increased production of TNF-a and NO by Listeria-infected DCs. (A) BMDCs were incubated with 1 mM ﬂuorescently labeled cell-permeable peptide with the sequence of the C-terminal cytoplasmic domain of CD81 or a scrambled version for the indicated times. The internalization of the peptide by DCs was analyzed by ﬂow cytometry. (B and C)WT or CD812/2 BMDCs were incubated for 2 h with 1 mM permeable peptides containing the sequence of the C-terminal cytoplasmic domain of CD81 or the http://classic.jimmunol.org scrambled version. After this treatment, BMDCs were infected with Listeria and 16 h later the production of TNF-a (B)andNO(C) was analyzed. Data are presented as the mean 6 SD and are representative of three independent experiments with similar results. Fold differences in TNF-a and NO production normalized to BMDC production after Listeria stimula- Downloaded from tion in the absence of peptides are also shown, and data are pooled from three independent experiments. *p , 0.05, **p , 0.01 (unpaired t test). 6098 CD81 MEDIATES SUSCEPTIBILITY TO LISTERIA INFECTION

FIGURE 6. Susceptibility of splenic CD8a+ cDCs to in vivo infection and induction of T cell responses in WT and CD812/2 mice. (A) WT and CD812/2 BMDCs were stimulated with PMA or heat-killed Listeria and ROS were assayed by luminol-ECL. Data are presented as mean 6 SD and are representative 2 2 2 by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. of two independent experiments with similar results. (B) WT and CD81 / mice were infected with Listeria and 15 h later CD8a+ and CD8a cDCs were sorted and the purified populations were Triton X-100 lysed. The dot plot shows the identification of the two subsets of CD8a+ and CD8a2 cDC, defined on the basis of CD8a expression after gating on CD11c+ cells. The charts show the numbers of live intracellular Listeria present in 5 3 104 cells after lysis and dilution plating on BHI agar (mean 6 SD of four mice per condition). Data are representative of two independent experiments with similar results. (C) CellTrace Violet–labeled CD45.1+ OT-I cells were adoptively transferred into WT or CD812/2 mice. After 24 h, mice were infected with Listeria-OVA and activation of transferred OT-I cells was analyzed in infected spleens. Graphs show OT-I absolute cell numbers among total splenocytes and cell proliferation determined by loss of CellTrace Violet, assessing the number of OT-I cells that had divided at 72 h after Listeria-OVA infection. (D) CD25 expression levels in OT-I cells are shown. (E) Absolute numbers of IFN-g+ cells in OT-I cells after OVA peptide restimulation of splenocytes from Listeria-OVA–infected WT or CD812/2 mice. (F) WT or CD812/2 mice were infected with Listeria-OVA and 7 d later mice were analyzed for the presence of endogenous Listeria- OVA–specific CD8+ T cell in spleens. Dot plots show the proportion of tetramer+CD44+ cells, and graph shows absolute cell numbers of Listeria-OVA– http://classic.jimmunol.org specific CD8+ T cells in infected WT or CD812/2 mice. (G) Splenocytes from infected WT or CD812/2 mice were restimulated in the presence of OVA peptide, and IFN-g production was analyzed by flow cytometry, assessing the number of IFN-g+ cells in CD8+ T cells or by ELISA determining IFN-g production in cell supernatants. Data are presented as mean 6 SD (five mice per condition, three independent experiments with similar results). *p , 0.05, **p , 0.01, unpaired t test.

enabling the establishment of protective T cell responses (36). We Induction of Listeria-specific effector CD8+ T cells is crucial for Downloaded from therefore analyzed whether infection of splenic CD8a+ cDCs in vivo bacterial clearance and the induction of protective immunity. Thus, wasaffectedbyCD81deficiencyinthe early postinfection period. we analyzed whether Rac–CD81 interaction could affect the ca- We sorted CD11chighCD8a+ and CD11clow to CD11chigh CD8a2 pacity of DCs to activate cytotoxic T cells. For this purpose, we splenic DC subsets 15 h after systemic Listeria infection of CD812/2 used OVA-expressing Listeria (Listeria-OVA) to assess the activa- and WT mice and determined the number of viable Listeria re- tion of Listeria-specific effector T cells during infection. WT coverable from these cells. Consistent with previous reports, our and CD812/2 mice were adoptively transferred with OVA-specific results revealed that infection was readily detectable in CD8a+ cDCs transgenic OT-I CD8+ T cells. Twenty-four hours later, mice were at 15 h postinfection before bacteria spread to CD11chighCD8a2 or systemically infected with Listeria-OVA. In this system, splenic CD11clowCD8a2 Tip-DCs (Fig. 6B). Interestingly, CD8a+ cDCs CD8a+ cDCs are the main DC subset involved in the presentation isolated from spleens of infected CD812/2 mice presented signifi- of Listeria-derived Ag to CD8+ T cells. Proliferation and activation cantly more CFUs than did WT CD8a+ cDCs at 15 h postinfection of OT-I cells transferred to WT or CD812/2 mice were analyzed at in vivo, indicating a higher infection rate (Fig. 6B) and providing an 72 h postinfection (Fig. 6C–E). The expansion of OT-I cells in the additional explanation for the more efficient immune response spleen was higher in CD812/2 than in WT mice, as assessed by against Listeria observed in CD812/2 mice. the analysis of their absolute cell number at 72 h postinfection The Journal of Immunology 6099

(Fig. 6C). Correspondingly, the number of cells in proliferation was pathways during infection (27). TLR engagement by Listeria higher in the spleen of CD812/2 mice when analyzed by the induces a MyD88-dependent pathway leading to the synthesis of number of cells that had undergone three, four, and five divisions at proinflammatory cytokines. Additionally, Listeria is detected by 72 h postinfection (Fig. 6C). Additionally, activation, assessed by cytosolic sensors that trigger the production of type I IFN through CD25 surface expression (Fig. 6D) and intracellular expression of the STING/IRF3-dependent pathway. Our results show that CD81 IFN-g (Fig. 6E) in OT-I cells, was significantly higher in CD812/2 does not affect the STING-dependent production of type I IFN in than in WT mice. In parallel, we also analyzed the activation of Listeria-infected DCs, but is able to negatively regulate the IFN- endogenous OVA-specific CD8+ T cells postinfection of WT and induced Rac/STAT-1 signaling through the triggering of the CD812/2 mice with Listeria-OVA. According to our data on the IFNAR autocrine loop. It has been claimed that Rac is involved in activation of adoptively transferred transgenic OT-I cells, the ex- the activation of STATs in response to a variety of inflammatory pansion of endogenous OVA-specific effector CD8+ T cells was mediators. Rac deficiency leads to attenuation of IFN-g–induced higher in CD812/2 than in WT mice as assessed by staining with inflammatory responses, with cells expressing a Rac dominant- OVA tetramers (Fig. 6F). Likewise, these CD8+ T cells produce negative mutant presenting reduced expression of several IFN- higher levels of IFN-g (Fig. 6G), demonstrating that OVA-specific g–responsive genes such as MCP-1, ICAM-1, and phosphorylated CD8+ T cells were functionally more active in CD812/2 than in STAT-1 and STAT-3 (33). Conditional STAT-1 ablation reveals the WT mice. Taken together, our results reveal that CD81 exerts importance of IFN signaling for immunity to Listeria infection a negative regulation on the immune response against Listeria (42), although the effect is cell type–dependent (42, 43). Type I through the inhibition of Rac/STAT-1 activation that leads to a re- IFN is important for host defense against viral and bacterial duction of the production of proinflammatory mediators and the infections, although the outcome of the IFN response highly activation of effector CD8+ Tcells. depends on the timing of production, the cellular context, and type of pathogen (44). In this sense, it has been shown that low levels of Discussion type I IFN are required at an early stage during Listeria infection In this study, we report that CD81 regulates the anti-Listeria immune for the resolution of the infection (45). Type I IFN promotes DC response by limiting the differentiation and recruitment of inflam- activation, increasing the expression of costimulatory molecules, matory monocytes and Tip-DCs to infected spleens and ultimately IL-12 secretion, and their potential to activate T cells (46). Con- inhibiting the induction of a protective anti-Listeria Tcellresponse. versely, type I IFN can also exert detrimental effects during Listeria The synthesis by DCs of TNF-a and NO, critical microbicidal infection. The main mechanism described to this harmful role relies mediators in the response to Listeria infection, was more efficient in on the induction of apoptosis, particularly of lymphocytes as well as the absence of CD81. This functional effect involves the direct macrophages (47, 48). It has been shown, using IFN-b reporter association of CD81 C-term cytoplasmic domain with Rac that mice, that Tip-DCs are an important source of IFN-b during Lis- regulates Rac/STAT-1. CD81 thus mediates susceptibility to Listeria teria infection (49) as are CD11b+ DCs (50). This finding might infection, reducing the survival rate postinfection (Supplemental suggest that type I IFN production can act as a mechanism of self- Fig. 4). regulation by immune cells, which could be subverted by Listeria Different mechanisms underlie tetraspanin involvement during for its own profit. However, whether Tip-DCs as well as CD11b+ microbial infection. Interestingly, our data show that CD81 controls DCs are targets of type I IFN–induced cell death remains unclear by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. different aspects of the immune response that take place in the course (44). The IFN-b–dependent autocrine loop, initiated by interaction of in vivo Listeria infection. The low bacterial load in the spleens of IFN-b with IFNAR receptors, activates the transcription factor of CD812/2 mice would allow efficient bacterial elimination by STAT-1 through IRF1, regulating the induction of inducible NO phagocytic cells as well as an enhanced differentiation and re- synthase and NO production (34). TNF-a activates an IFN-b–de- cruitment of the inflammatory monocytes and Tip-DCs that are pendent autocrine loop, leading to sustained expression of STAT-1– crucial for the initial control of bacterial replication. Recent studies dependent type I IFN (32). The high levels of TNF-a and NO have demonstrated that failed recruitment of Tip-DCs reduces host produced by CD812/2 BMDCs after Listeria infection seem to be survival after Listeria infection (20) in a process that depends on specific for live Listeria, and notably signaling through other TLRs cDCs and NK cells (37). Furthermore, recruitment of Tip-DCs to was unaffected in CD812/2 DCs. Other studies have described the http://classic.jimmunol.org gut-associated tissues has also been described during infection with role played by tetraspanins in preventing signaling downstream of Salmonella typhimurium (38) and is critical for the control of pathogen receptors. For example, CD37 regulates antifungal im- Toxoplasma gondii infection (39). munity by stabilizing the C-type lectin receptor dectin-1 on the cell Our results show that CD81 regulates Rac activity in DCs after surface of APCs and controlling dectin-1–mediated IL-6 production Listeria infection, but this activity does not impair internalization in response to fungal infection (51). Moreover, CD9 acts as a neg- of bacteria by CD812/2 DCs. In DCs, phagocytosis requires a dy- ative regulator of CD14/TLR4 complex formation and signaling in Downloaded from namic regulation of the actin cytoskeleton, and Rac is involved in macrophages, as shown by the increased macrophage infiltration this function (40, 41). Moreover, tetraspanins exhibit a remarkable and TNF-a production in LPS-treated CD92/2 mice (52). Addi- capacity to form functional complexes with microbial receptors in tionally, CD9 associates with ADAM17 and negatively regulates the the host cell membrane during infection. Recent data have dem- sheddase activity of ADAM17 against its substrate TNF-a (53). onstrated the recruitment of tetraspanins CD9, CD63, and CD81 to Rac plays a well-established role in the development and function the Listeria entry site during in vitro invasion of nonphagocytic of CD8a+ cDCs (23). Despite the critical role of CD8a+ cDCs in target cells, highlighting the role of CD81 in this step. However, initiating T cell–mediated immunity, they are also a required entry CD81 does not affect the recruitment of Met, the Listeria receptor point for productive Listeria infection (21, 35). Thus, Batf32/2 in epithelial cells, to the bacterial entry site, suggesting that CD81 mice, which lack CD8a+ cDCs, exhibit an enhanced resistance to acts as a membrane organizer of signaling events at Listeria entry Listeria (21). Our results show that the efficient early infection of sites (15). CD8a+ cDCs in CD812/2 mice increases survival. Unlike other Tetraspanin microdomains facilitate the clustering of membrane phagocytes, such as neutrophils, macrophages, and CD8a2 cDCs, receptors and signaling molecules for the regulation of signal CD8a+ cDCs are highly efficient at Ag cross-presentation, due in transduction events. Listeria is detected by multiple signaling part to their delayed phagosomal acidification and their limited 6100 CD81 MEDIATES SUSCEPTIBILITY TO LISTERIA INFECTION

proteolytic activity in endosomes. This efficient cross-presentation 13. Gordoń-Alonso, M., M. Yan˜ez-Mo´, O. Barreiro, S. Alvarez, M. A. Munõz- a+ Fernańdez, A. Valenzuela-Fernańdez, and F. Sańchez-Madrid. 2006. Tetraspa- relies on Rac, because in the absence of Rac, CD8 cDCs behave nins CD9 and CD81 modulate HIV-1-induced membrane fusion. J. Immunol. like other phagocytes in terms of phagosomal acidification and cross- 177: 5129–5137. presentation (23, 25). Contrasting observations that Rac activity is 14. Silvie, O., E. Rubinstein, J. F. Franetich, M. Prenant, E. Belnoue, L. Reńia, 2/2 2/2 L. Hannoun, W. Eling, S. Levy, C. Boucheix, and D. Mazier. 2003. Hepatocyte impaired in CD81 DCs (54), our data show that CD81 DCs CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite exhibit persistent Rac activation, which could account for the high infectivity. Nat. Med. 9: 93–96. capacity of these cells to present bacterial Ags to specific CD8+ 15. Tham, T. N., E. Gouin, E. Rubinstein, C. Boucheix, P. Cossart, and J. Pizarro- Cerda. 2010. Tetraspanin CD81 is required for Listeria monocytogenes invasion. T cells during Listeria infection in vivo. Cross-presentation of ex- Infect. Immun. 78: 204–209. ogenous Ags has been recognized as a major mechanism of induc- 16. Pamer, E. G. 2004. Immune responses to Listeria monocytogenes. Nat. Rev. tion of CD8+ T cell responses against virus and tumor Ags; however, Immunol. 4: 812–823. 17. Tam, M. A., and M. J. Wick. 2004. Dendritic cells and immunity to Listeria: the contribution of this mechanism on T cell immunity to bacteria TipDCs are a new recruit. Trends Immunol. 25: 335–339. in vivo is not well established. In this sense, it has been recently 18. Pizarro-Cerda´, J., and P. Cossart. 2009. Listeria monocytogenes membrane trafficking and lifestyle: the exception or the rule? Annu. Rev. Cell Dev. Biol. 25: shown that DC cross-priming is essential for immune responses to 649–670. Listeria, because mice presenting a defective cross-priming capacity 19. Condotta, S. A., M. J. Richer, V. P. Badovinac, and J. T. Harty. 2012. Probing are severely compromised in their ability to generate Listeria-specific CD8 T cell responses with Listeria monocytogenes infection. Adv. Immunol. 113: + 51–80. CD8 T cell responses in vivo (55). 20. Serbina, N. V., T. P. Salazar-Mather, C. A. Biron, W. A. Kuziel, and E. G. Pamer. In summary, our data reveal that CD81 plays a regulatory role 2003. TNF/iNOS-producing dendritic cells mediate innate immune defense during systemic Listeria infection by controlling different aspects against bacterial infection. Immunity 19: 59–70. 21. Edelson, B. T., T. R. Bradstreet, K. Hildner, J. A. Carrero, K. E. Frederick, of immune cell invasion through the modulation of downstream W. Kc, R. Belizaire, T. Aoshi, R. D. Schreiber, M. J. Miller, et al. 2011. CD8a+ signaling pathways and the regulation of the presentation activity dendritic cells are an obligate cellular entry point for productive infection by of DCs, which lead to the T cell–protective immunity. Our results Listeria monocytogenes. Immunity 35: 236–248. 22. Jung, S., D. Unutmaz, P. Wong, G. Sano, K. De los Santos, T. Sparwasser, S. Wu, suggest that tetraspanins could play a prominent role as novel S. Vuthoori, K. Ko, F. Zavala, et al. 2002. In vivo depletion of CD11c+ dendritic therapeutic targets in the treatment of infectious diseases. cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 17: 211–220. 23. Joffre, O. P., E. Segura, A. Savina, and S. Amigorena. 2012. Cross-presentation Acknowledgments by dendritic cells. Nat. Rev. Immunol. 12: 557–569. We thank Simon Bartlett for assistance with English editing. We are grateful 24. Kerksiek, K. M., F. Niedergang, P. Chavrier, D. H. Busch, and T. Brocker. 2005. Selective Rac1 inhibition in dendritic cells diminishes apoptotic cell uptake and to the Centro Nacional de Investigaciones Cardiovasculares’ Cellomics and cross-presentation in vivo. Blood 105: 742–749. Comparative Medicine Units and the Centro Nacional de Investiga- 25. Savina, A., A. Peres, I. Cebrian, N. Carmo, C. Moita, N. Hacohen, L. F. Moita, ciones Oncolo´gicas’ Flow Cytometry Unit. We thank Dr. P. Domıńguez, and S. Amigorena. 2009. The small GTPase Rac2 controls phagosomal alka- + M. Torres-Torresano, and S. Lo´pez-Martıń for assistance, Dr. V. Rocha- linization and antigen crosspresentation selectively in CD8 dendritic cells. Immunity 30: 544–555. Perugini for discussions and critical review of the manuscript, and 26. Tejera, E., V. Rocha-Perugini, S. Lo´pez-Martıń, D. Pe´rez-Hernańdez, Drs. D. Sancho and S. Iborra for discussions and sharing reagents. A. I. Bachir, A. R. Horwitz, J. Va´zquez, F. Sańchez-Madrid, and M. Yań˜ez-Mo. 2013. CD81 regulates cell migration through its association with Rac GTPase. Mol. Biol. Cell 24: 261–273. Disclosures 27. Witte, C. E., K. A. Archer, C. S. Rae, J. D. Sauer, J. J. Woodward, and D. A. Portnoy. 2012. Innate immune pathways triggered by Listeria mono- The authors have no financial conflicts of interest. cytogenes and their role in the induction of cell-mediated immunity. Adv. by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. Immunol. 113: 135–156. 28. Honda, K., A. Takaoka, and T. Taniguchi. 2006. Type I interferon gene induction References by the interferon regulatory factor family of transcription factors. Immunity 25: 1. Hemler, M. E. 2005. Tetraspanin functions and associated microdomains. Nat. 349–360. Rev. Mol. Cell Biol. 6: 801–811. 29. Maecker, H. T., and S. Levy. 1997. Normal lymphocyte development but delayed 2. Yań˜ez-Mo´, M., O. Barreiro, M. Gordon-Alonso, M. Sala-Valde´s, and F. Sańchez- humoral immune response in CD81-null mice. J. Exp. Med. 185: 1505–1510. Madrid. 2009. Tetraspanin-enriched microdomains: a functional unit in cell 30. Cruz-Adalia, A., G. Ramirez-Santiago, C. Calabia-Linares, M. Torres-Torresano, plasma membranes. Trends Cell Biol. 19: 434–446. L. Feo, M. Galań-Dıéz, E. Fernańdez-Ruiz, E. Pereiro, P. Guttmann, M. Chiappi, 3. van Spriel, A. B., and C. G. Figdor. 2010. The role of tetraspanins in the et al. 2014. T cells kill bacteria captured by transinfection from dendritic cells pathogenesis of infectious diseases. Microbes Infect. 12: 106–112. and confer protection in mice. Cell Host Microbe 15: 611–622. 4. Kropshofer, H., S. Spindeldreher, T. A. Ro¨hn, N. Platania, C. Grygar, N. Daniel, 31. Vaudaux, P., and F. A. Waldvogel. 1979. Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes. Antimicrob. Agents Chemo- http://classic.jimmunol.org A. Wo¨lpl, H. Langen, V. Horejsi, and A. B. Vogt. 2002. Tetraspan microdomains distinct from lipid rafts enrich select peptide-MHC class II complexes. Nat. ther. 16: 743–749. Immunol. 3: 61–68. 32. Yarilina, A., K. H. Park-Min, T. Antoniv, X. Hu, and L. B. Ivashkiv. 2008. TNF 5. Unternaehrer, J. J., A. Chow, M. Pypaert, K. Inaba, and I. Mellman. 2007. The activates an IRF1-dependent autocrine loop leading to sustained expression of tetraspanin CD9 mediates lateral association of MHC class II molecules on the chemokines and STAT1-dependent type I interferon-response genes. Nat. dendritic cell surface. Proc. Natl. Acad. Sci. USA 104: 234–239. Immunol. 9: 378–387. 6. Levy, S. 2014. Function of the tetraspanin molecule CD81 in B and T cells. 33. Park, E. J., K. A. Ji, S. B. Jeon, W. H. Choi, I. O. Han, H. J. You, J. H. Kim, Immunol. Res. 58: 179–185. I. Jou, and E. H. Joe. 2004. Rac1 contributes to maximal activation of STAT1 and 7. Rocha-Perugini, V., M. Zamai, J. M. Gonza´lez-Granado, O. Barreiro, E. Tejera, STAT3 in IFN-g-stimulated rat astrocytes. J. Immunol. 173: 5697–5703. Downloaded from M. Yan˜ez-Mo´, V. R. Caiolfa, and F. Sanchez-Madrid. 2013. CD81 controls 34. Domıńguez, P. M., M. Lo´pez-Bravo, U. Kalinke, and C. Ardavıń. 2011. Statins sustained T cell activation signaling and defines the maturation stages of cognate inhibit iNOS-mediated microbicidal potential of activated monocyte-derived den- immunological synapses. Mol. Cell. Biol. 33: 3644–3658. dritic cells by an IFN-b-dependent mechanism. Eur. J. Immunol. 41: 3330–3339. 8. Maecker, H. T., M. S. Do, and S. Levy. 1998. CD81 on B cells promotes in- 35. Neuenhahn, M., K. M. Kerksiek, M. Nauerth, M. H. Suhre, M. Schiemann, terleukin 4 secretion and antibody production during T helper type 2 immune F. E. Gebhardt, C. Stemberger, K. Panthel, S. Schro¨der, T. Chakraborty, et al. responses. Proc. Natl. Acad. Sci. USA 95: 2458–2462. 2006. CD8a+ dendritic cells are required for efficient entry of Listeria mono- 9. Miyazaki, T., U. Muller,€ and K. S. Campbell. 1997. Normal development but cytogenes into the spleen. Immunity 25: 619–630. differentially altered proliferative responses of lymphocytes in mice lacking 36. Yang, X., X. Zhang, Y. Sun, T. Tu, M. L. Fu, M. Miller, and Y. X. Fu. 2014. A CD81. EMBO J. 16: 4217–4225. BTLA-mediated bait and switch strategy permits Listeria expansion in CD8a+ 10. Tsitsikov, E. N., J. C. Gutierrez-Ramos, and R. S. Geha. 1997. Impaired CD19 DCs to promote long-term T cell responses. Cell Host Microbe 16: 68–80. expression and signaling, enhanced antibody response to type II T independent 37. Kang, S. J., H. E. Liang, B. Reizis, and R. M. Locksley. 2008. Regulation of antigen and reduction of B-1 cells in CD81-deficient mice. Proc. Natl. Acad. Sci. hierarchical clustering and activation of innate immune cells by dendritic cells. USA 94: 10844–10849. Immunity 29: 819–833. 11. Deng, J., R. H. Dekruyff, G. J. Freeman, D. T. Umetsu, and S. Levy. 2002. 38. Rydstro¨m, A., and M. J. Wick. 2007. Monocyte recruitment, activation, and Critical role of CD81 in cognate T-B cell interactions leading to Th2 responses. function in the gut-associated lymphoid tissue during oral Salmonella infection. Int. Immunol. 14: 513–523. J. Immunol. 178: 5789–5801. 12. Pileri, P., Y. Uematsu, S. Campagnoli, G. Galli, F. Falugi, R. Petracca, 39. Dunay, I. R., R. A. Damatta, B. Fux, R. Presti, S. Greco, M. Colonna, and A. J. Weiner, M. Houghton, D. Rosa, G. Grandi, and S. Abrignani. 1998. Binding L. D. Sibley. 2008. Gr1+ inflammatory monocytes are required for mucosal re- of hepatitis C virus to CD81. Science 282: 938–941. sistance to the pathogen Toxoplasma gondii. Immunity 29: 306–317. The Journal of Immunology 6101

40. Benvenuti, F., S. Hugues, M. Walmsley, S. Ruf, L. Fetler, M. Popoff, IFN sensitizes macrophages to cell death induced by Listeria monocytogenes. J. V. L. Tybulewicz, and S. Amigorena. 2004. Requirement of Rac1 and Rac2 Immunol. 169: 6522–6529. expression by mature dendritic cells for T cell priming. Science 305: 1150–1153. 49. Dresing, P., S. Borkens, M. Kocur, S. Kropp, and S. Scheu. 2010. A fluorescence 41. West, M. A., A. R. Prescott, E. L. Eskelinen, A. J. Ridley, and C. Watts. 2000. reporter model defines “Tip-DCs” as the cellular source of interferon b in murine Rac is required for constitutive macropinocytosis by dendritic cells but does not listeriosis. PLoS ONE 5: e15567. control its downregulation. Curr. Biol. 10: 839–848. 50. Stockinger, S., R. Kastner, E. Kernbauer, A. Pilz, S. Westermayer, B. Reutterer, 42. Kernbauer, E., V. Maier, D. Stoiber, B. Strobl, C. Schneckenleithner, V. Sexl, D. Soulat, G. Stengl, C. Vogl, T. Frenz, et al. 2009. Characterization of the U. Reichart, B. Reizis, U. Kalinke, A. Jamieson, et al. 2012. Conditional Stat1 interferon-producing cell in mice infected with Listeria monocytogenes. PLoS ablation reveals the importance of interferon signaling for immunity to Listeria Pathog. 5: e1000355. monocytogenes infection. PLoS Pathog. 8: e1002763. 51. van Spriel, A. B., M. Sofi, K. H. Gartlan, A. van der Schaaf, I. Verschueren, 43. Archer, K. A., J. Durack, and D. A. Portnoy. 2014. STING-dependent type I IFN R. Torensma, R. A. Raymakers, B. E. Loveland, M. G. Netea, G. J. Adema, et al. 2009. The tetraspanin protein CD37 regulates IgA responses and anti-fungal production inhibits cell-mediated immunity to Listeria monocytogenes. PLoS immunity. PLoS Pathog. 5: e1000338. Pathog. 10: e1003861. 52. Suzuki, M., I. Tachibana, Y. Takeda, P. He, S. Minami, T. Iwasaki, H. Kida, 44. McNab, F., K. Mayer-Barber, A. Sher, A. Wack, and A. O’Garra. 2015. Type I S. Goya, T. Kijima, M. Yoshida, et al. 2009. Tetraspanin CD9 negatively reg- interferons in infectious disease. Nat. Rev. Immunol. 15: 87–103. ulates lipopolysaccharide-induced macrophage activation and lung inflamma- 45. Pontiroli, F., O. Dussurget, I. Zanoni, M. Urbano, O. Beretta, F. Granucci, tion. J. Immunol. 182: 6485–6493. P. Ricciardi-Castagnoli, P. Cossart, and M. Foti. 2012. The timing of IFNb 53. Gutie´rrez-Lo´pez, M. D., A. Gilsanz, M. Yań˜ez-Mo´, S. Ovalle, E. M. Lafuente, production affects early innate responses to Listeria monocytogenes and deter- C. Domıńguez, P. N. Monk, I. Gonza´lez-Alvaro, F. Sańchez-Madrid, and mines the overall outcome of lethal infection. PLoS ONE 7: e43455. C. Cabanãs. 2011. The sheddase activity of ADAM17/TACE is regulated by the 46. Montoya, M., G. Schiavoni, F. Mattei, I. Gresser, F. Belardelli, P. Borrow, and tetraspanin CD9. Cell. Mol. Life Sci. 68: 3275–3292. D. F. Tough. 2002. Type I interferons produced by dendritic cells promote their 54. Quast, T., F. Eppler, V. Semmling, C. Schild, Y. Homsi, S. Levy, T. Lang, phenotypic and functional activation. Blood 99: 3263–3271. C. Kurts, and W. Kolanus. 2011. CD81 is essential for the formation of mem- 47. Carrero, J. A., B. Calderon, and E. R. Unanue. 2004. Type I interferon brane protrusions and regulates Rac1-activation in adhesion-dependent immune sensitizes lymphocytes to apoptosis and reduces resistance to Listeria infection. cell migration. Blood 118: 1818–1827. J. Exp. Med. 200: 535–540. 55. Reinicke, A. T., K. D. Omilusik, G. Basha, and W. A. Jefferies. 2009. Dendritic 48. Stockinger, S., T. Materna, D. Stoiber, L. Bayr, R. Steinborn, T. Kolbe, H. Unger, cell cross-priming is essential for immune responses to Listeria monocytogenes. T. Chakraborty, D. E. Levy, M. Muller,€ and T. Decker. 2002. Production of type I PLoS ONE 4: e7210. by guest on September 30, 2021. Copyright 2015 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from