Influenza A Virus H3N2 Pneumonia T Cell Response during Acute + ...

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Influenza A Virus H3N2 Pneumonia T Cell Response during Acute+CD8

Control of Pulmonary Inflammation and Potential Role of Invariant NKT Cells in the

Mustapha Si-Tahar and François TrotteinBarba-Speath, Michel-René Huerre, Christelle Faveeuw, Julien Pothlichet, Catherine Vendeville, GiovannaBlanc, Muriel Pichavant, Joelle Renneson, Emilie Bialecki, Christophe Paget, Stoyan Ivanov, Josette Fontaine, Fany

ol.1002348http://www.jimmunol.org/content/early/2011/04/12/jimmun

published online 13 April 2011J Immunol

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The Journal of Immunology

Potential Role of Invariant NKT Cells in the Control ofPulmonary Inflammation and CD8+ T Cell Response duringAcute Influenza A Virus H3N2 Pneumonia

Christophe Paget,*,†,‡,x,{,1 Stoyan Ivanov,*,†,‡,x,{,1 Josette Fontaine,*,†,‡,x,{

Fany Blanc,‖,# Muriel Pichavant,*,†,‡,x,{ Joelle Renneson,*,†,‡,x,{ Emilie Bialecki,*,†,‡,x,{

Julien Pothlichet,‖,# Catherine Vendeville,*,†,‡,x,{ Giovanna Barba-Speath,‖,**

Michel-Rene Huerre,‖,†† Christelle Faveeuw,*,†,‡,x,{ Mustapha Si-Tahar,‖,#

and Francois Trottein*,†,‡,x,{

Influenza Avirus (IAV) infection results in a highly contagious respiratory illness leading to substantial morbidity and occasionally

death. In this report, we assessed the in vivo physiological contribution of invariant NKT (iNKT) lymphocytes, a subset of lipid-

reactive ab T lymphocytes, on the host response and viral pathogenesis using a virulent, mouse-adapted, IAV H3N2 strain. Upon

infection with a lethal dose of IAV, iNKT cells become activated in the lungs and bronchoalveolar space to become rapidly anergic

to further restimulation. Relative to wild-type animals, C57BL/6 mice deficient in iNKT cells (Ja182/2 mice) developed a more

severe bronchopneumonia and had an accelerated fatal outcome, a phenomenon reversed by the adoptive transfer of NKT cells

prior to infection. The enhanced pathology in Ja182/2 animals was not associated with either reduced or delayed viral clearance

in the lungs or with a defective local NK cell response. In marked contrast, Ja182/2 mice displayed a dramatically reduced IAV-

specific CD8+ T cell response in the lungs and in lung-draining mediastinal lymph nodes. We further show that this defective CD8+

T cell response correlates with an altered accumulation and maturation of pulmonary CD103+, but not CD11bhigh, dendritic cells

in the mediastinal lymph nodes. Taken together, these findings point to a role for iNKT cells in the control of pneumonia as well as

in the development of the CD8+ T cell response during the early stage of acute IAV H3N2 infection. The Journal of Immunology,

2011, 186: 000–000.

Influenza infection is one of the most important causes ofrespiratory tract diseases and is responsible for widespreadmorbidity and mortality. Annual epidemics typically affect 5–

15% of the population and are thought to result in 250,000–500,000 deaths annually (for review, see Ref. 1). Of the three

types of influenza viruses, the type A virus is the most virulent to

humans and is capable of infecting multiple mammalian and avian

species. Human influenza A virus (IAV) can be further divided

into different serotypes on the basis of the Ab response to the viral

surface glycoproteins hemagglutinin and neuraminidase (1). The

most important subtypes for humans are H1N1 and H3N2, the

latter being currently associated with more severe disease (1, 2).

The pulmonary inflammation that develops during IAV infection is

often due to deleterious host immune responses directed against

the pathogen. The initial reactions to IAV are directed by the in-

nate immune system and lead to the containment of viral repli-

cation without an excessive inflammatory response. The early

response to IAV, which occurs during the first 48 h after the onset

of primary infection, has been attributed to stromal cells (epithe-

lial cells, fibroblasts) and immune cells such as neutrophils,

macrophages, dendritic cells (DCs), and NK cells (3–6).Invariant NKT (iNKT) cells represent a population of “innate-

like” ab T lymphocytes expressing markers associated with the

NK lineage. These cells have the particularity of recognizing self

and exogenous lipid Ags presented by the MHC class I-like

molecule CD1d (for reviews, see Refs. 7–11). Upon lipid recog-

nition through their TCR, iNKT cells swiftly secrete cytokines

with opposing effects on immune responses. This functional

property establishes iNKT cells as innate immune effector cells

as well as regulators of adaptive immune responses. Numerous

studies have shown that, upon intentional or natural activa-

tion, iNKT cells either suppress or enhance immune-mediated

*Institut Pasteur de Lille, Centre d’Infection et d’Immunite de Lille, F-59019 Lille,France; †Universite Lille Nord de France, F-59000 Lille, France; ‡Centre National de laRecherche Scientifique, Unite Mixte de Recherche 8204, F-59021 Lille, France; xIN-SERM, U1019, F-59019 Lille, France; {Institut Federatif de Recherche 142, F-59019Lille, France; ‖Institut Pasteur, F-75015 Paris, France; #INSERM, U874, F-75015 Paris,France; **Centre National de la Recherche Scientifique, Unite de Recherche Associee3015, Unite de Virologie Structurale, F-75015 Paris, France; and ††Unite de Rechercheet d’Expertise Histotechnologie et Pathologie, F-75015 Paris, France

1C.P. and S.I. contributed equally to this work.

Received for publication July 13, 2010. Accepted for publication March 9, 2011.

This work was supported by INSERM, the Centre National de la Recherche Scienti-fique, the University of Lille Nord de France, the Pasteur Institute of Lille, and theFrench National Research Agency (under reference ANR-08-MIEN-021-01). C.P. andS.I. were recipients of a doctoral fellowship from the Conseil Regional Nord Pas deCalais/INSERM and from the Ministere de l’Education Nationale de la Recherche etTechnique, respectively. J.R. and F.B. were supported by a postdoctoral fellowshipfrom the French National Research Agency (ANR-08-MIEN-021-01 and ANR–07-MIME-018-01, respectively). C.F., M.P., and M.S.-T. were supported by INSERM,and F.T. was supported by the Centre National de la Recherche Scientifique.

Address correspondence and reprint requests to Dr. Francois Trottein, Center for In-fection and Immunity of Lille, INSERM U1019-Centre National de la RechercheScientifique, Unite Mixte de Recherche 8204, Institut Pasteur de Lille, 1, Rue duProfesseur Calmette, BP 245, 59019 Lille cedex, France. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: BAL, bronchoalveolar lavage; DC, dendritic cell;a-GalCer, a-galactosylceramide; IAV, influenza A virus; iNKT, invariant NKT; LN,lymph node; MDSC, myeloid-derived suppressor cell; MLN, mediastinal lymph node;MNC, mononuclear cell; p.i., postinfection; PMN, polymorphonuclear; WT, wild-type.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

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responses during inflammation, cancer, autoimmune diseases andinfection (for reviews, see Refs. 8–11). The natural role ofiNKT cells in antiviral immunity and in the control of viral rep-lication and pathology has been studied using Ja182/2 mice,which lack iNKT cells. It was reported that the role of iNKT cellsduring experimental viral infection can vary according to the virusand experimental conditions (for reviews, see Refs. 12, 13). Forinstance, during HSV type 1 and 2 and lymphocytic choriome-ningitis virus infections, iNKT cells play a positive role in theantiviral immune responses and virus-associated pathology (14–17), whereas they rather appear to be deleterious during Sendaivirus (18), HSV type 2 (only in aged mice) (19), and Dengue virusserotype 2 (J. Renneson, R. Guabiraba, I. Maillet, R.E. Marques,S. Ivanov, J. Fontaine, C. Paget, V. Quesniaux, C. Faveeuw,B. Ryffel, M.M. Teixeira, and F. Trottein, submitted for publica-tion) infections. Studies using CD1d-deficient mice, which not onlylack iNKT cells but also non-iNKT cells, have suggested thatNKT cells (either iNKT cells, non-iNKT cells, or both) positivelycontribute to the immune response to respiratory syncytial virus(20), encephalomyocarditis virus (21), murine CMV (22), andTheiler’s murine encephalomyelitis virus (23). In contrast, non-iNKT cells are rather deleterious during coxsackievirus infection(24). Although suspected (25–27), the role of iNKT cells in humanviral infections is not entirely clear.The consequences of endogenous and exogenous activation of

iNKT cells in the control of IAV infection represent an intense areaof investigation at the moment. For instance, in the mouse system,in vivo stimulation of iNKT cells by the synthetic glycolipida-galactosylceramide (a-GalCer) promotes an efficient immuneresponse in the lungs that culminates in the control of viralload and improved disease course (28–32). The natural role ofiNKT cells during experimental IAV H1N1 (PR8 strain) infectionhas been studied using CD1d2/2 mice (33–35) and Ja182/2 mice(34). An early study of Benton et al. (33) using CD1d2/2-deficientmice showed that NKT cells may contribute to, but are not re-quired for, cross-protection to influenza viruses of different sub-types. More recently, De Santo et al. (34) and Ishikawa et al. (35),using a sublethal dose of IAV H1N1, showed that CD1d2/2-deficient mice had an increased mortality rate relative to controlsand failed to limit viral replication in the lungs, a phenomenonascribed to a reduced IAV-specific CTL response. Using Ja182/2

mice, De Santo et al. (34) demonstrated that iNKT cells arephysiologically important in this setting. In this work, we studiedthe potential regulatory role of iNKT cells in the development ofviral pathogenesis and host responses during acute IAV H3N2infection. To address this issue, we deliberately chose a highlypathogenic, human-origin IAV strain (Scotland/20/74) that indu-ces severe pneumonia in mice (36). We showed that in this system,endogenous activation of iNKT cells leads to a beneficial effect onthe control of airway inflammation and to a delay in the fataloutcome. We also demonstrated that iNKT cells favor the initia-tion of the IAV-specific CD8+ T cell response, in part by pro-moting the accumulation and maturation of a particular subset(CD103+) of respiratory DCs in the lung-draining mediastinallymph nodes (MLNs). These findings suggest a new role foriNKT cells in DC functions and extend the concept that pulmo-nary iNKT cells might be exploited in the future to trigger im-mune responses and to avert immune pathology during IAV in-fection.

Materials and MethodsMice and viruses

Six- to eight-week-old male wild-type (WT) C57BL/6 (H-2Db) mice werepurchased from Janvier (Le Genest-St-Isle, France). Ja18 2/2 mice and

CD1d 2/2 mice, back-crossed at least 10 times in C57BL/6, were a giftfrom Dr. M. Taniguchi (RIKEN Institute, Yokohama, Japan) (37) and Dr.L. Van Kaer (Vanderbilt University, Nashville, TN) (38), respectively. OT-ITCR-transgenic mice were purchased from Charles River (St Germain surl’arbresle, France). For IAV infection, mice were maintained in a biosafetylevel 2 facility in the Animal Resource Center at the Pasteur Institute, Lille(Lille, France). All animal work conformed to the Pasteur Institute, Lille,Animal Care and Use Committee guidelines (agreement no. AF 16/20090from the Comite d’Ethique en Experimentation Animale Nord Pas-De-Calais). The IAV strain used in this study (Scotland/20/74, H3N2) wasgrown in 10-d embryonated hens eggs by standard procedures and titratedon Madin-Darby canine kidney cells as described in Ref. 39.

Reagents and Abs

a-GalCer was from Axxora Life Sciences (Coger S.A., Paris, France). TheIAV-derived (SSLENFRAYV) and OVA-derived (SIINFEKL) peptideswere obtained from the Institut de Biologie et Chimie des Proteines(Lyon, France). mAbs against mouse CD3 (allophycocyanin-conjugated),NK1.1 (PE- or PerCp-Cy5.5–conjugated), TCR-b (FITC-conjugated),CD69 (PE-conjugated), CD11b (PE- or PerCp-Cy5.5–conjugated), Gr1(allophycocyanin-conjugated), CD8 (allophycocyanin- or FITC-conjugated),CD19 (allophycocyanin-conjugated), CD103 (PE-conjugated), CD11c (allo-phycocyanin-conjugated), granzyme B (FITC-conjugated), CD107a (FITC-conjugated), IFN-g (PE-conjugated), CD86 (biotinylated), CD40 (bio-tinylated), and isotype controls were purchased from BD Pharmingen (LePont de Claix, France). Allophycocyanin-conjugated PBS-57 glycolipid-loaded CD1d tetramer was from the National Institute of Allergy and In-fectious Diseases Tetramer Facility (Emory University, Atlanta, GA), andPE-conjugated Pro5 MHC pentamer (H-2Db, PA224–233; SSLENFRAYV)was from ProImmune (Oxford, UK). Sterile OVA was kindly provided byDr. G. Randolph (Mount Sinai School of Medicine, New York, NY). OVAwas stained using a FluoReporter FITC Protein Labeling Kit from Mo-lecular Probes (Invitrogen, Cergy Pontoise, France). Alamar blue was fromAbD Serotec (Dusseldorf, Germany).

IAV infection and assessment of the pathology

For IAV infection, mice were anesthetized and intranasally administered50 ml PBS containing 600 PFU IAV (Scotland/20/74, H3N2). Mice werethen monitored daily for illness and mortality for a period of 16 d. Clinicalmanifestations appeared around day 8 and included arching of the back,ruffling of the fur, and slowing of activity. Within 9 and 12 d postinfection(p.i.), all mice demonstrated severe sickness and weight loss and death atday 16. Disease was assessed by measuring lung inflammation, viral loadin the lungs, and lethality. Mice found to be moribund were euthanized andconsidered to have died on that day. Mice were also sacrificed at variousintervals for sampling the lung lumen by bronchoalveolar lavage (BAL).The BAL fluid was collected in 3 3 1 ml washes. After light centrifuga-tion, total cell numbers per BAL was determined by manual countingunder the microscope. A morphology-based differential cell count wasconducted on cytospin preparations from the BAL fluid and stained withDiff-Quik solution (Sigma). For histopathologic examination, lungs werefixed by inflation and immersion in PBS 3.2% paraformaldehyde andembedded in paraffin. To evaluate airway inflammation, we subjected fixedlung slices (5-mm sections) to H&E staining. Evaluators who were blindedto genotype scored lung sections (0 [none] to 5 [extreme]) on the basisof edema, hemorrhages, recruitment of polymorphonuclear (PMN) cells,macrophages, lymphocytes, lesions of alveolitis and bronchitis, area offocal or diffuse consolidation, necrosis and metaplasia of pneumonocytes:grade 1, minimal or mild focal lesions combined with the perivascularrecruitment of a few inflammatory cells; grade 2, mild to moderate focallesions within the alveoli and bronchi, a few cells located in the peri-vascular and peribronchial areas; grade 3, mild to moderate focal anddiffuse lesions extended to a whole lobule with focal consolidation com-bined with perivascular and peribronchial recruitment of inflammatorycells; grade 4, severe lesions, focal and diffuse with large areas of lobularpneumopathy including severe lesions of alveolitis, bronchitis, and con-solidation (between 20 and 40% of normal lung); grade 5, destruction ofthe lungs with severe lesions of alveolitis, bronchitis, with necrosis andlarge areas of consolidation extended to several lobules (,20% of func-tional lung parenchyma).

Analysis of virus load and of genes associated with viralreplication by quantitative RT-PCR

Total RNA from lungs of naive or IAV-infected mice was extracted usingRNAse free beads for tissue lysis and RNeasy RNA isolation kit (Qiagen,

2 iNKT CELLS AS REGULATORS OF ACUTE INFLUENZA A INFECTION


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Courtaboeuf, France) subjected to DNase I treatment (Roche Diagnostics,Meylan, France), and a cDNA template was prepared from 1 mg by usingrandom hexamer primers and Moloney murine leukemia virus RNase Hminus RT (Promega, Charbonnieres, France) according to the manufac-turer’s instructions. PCR was carried out by using 1% of cDNA as startingmaterial in an ABI 7500 Thermocycler (Applied Biosystems, Foster City,CA). Quantitative PCR was carried out using 0.5 mM specific primersfor IAV M2 gene (59-AAGACCAATCCTGTCACCTCTGA-39 and 59-CAAAGCGTCTACGCTGCAGTCC-39) that complement 20 temporallyand spatially divergent influenza A matrix protein gene sequences, aspreviously described (36), and specific primers for mouse hprt (59-CAGGCCAGACTTTGTTGGAT-39 and 59-TTGCGCTCATCTTAGGCT-TT-39) with melting temperature (Tm) of 60˚C and 58˚C, respectively, andQuantiTect SYBR Green PCR Master Mix (Qiagen) according to themanufacturer’s instructions. IAV and hprt copies for each experimentalsample were quantified in duplicate using the standard curves obtained byPCR amplification on serial dilutions of purified PCR products. Viral loadis expressed as viral RNA normalized to hprt expression level. Primersspecific for gapdh (59-TGCCCAGAACATCATCCCTG-39 and 59-TCA-GATCCACGACGGACACA-39), ifn-b (59-CAGGTGGATCCTCCACGC-T-39 and 59-CATTCAGCTGCTCCAGGAGC-39), mx1 (59-TGCAGAGG-TCAGCAGGACATC-39 and 59-GGCAGTTTGGACCATCTCTGAA-39)were designed by the Primer Express Program (Applied Biosystems) andused for amplification in triplicate assays. PCR amplification of Gapdhwas performed to control for sample loading and to allow normalizationbetween samples. DCt values were obtained by deducting the raw cyclethreshold (Ct values) obtained for Gapdh mRNA, the internal standard,from the Ct values obtained for the investigated genes. For graphicalrepresentation, data are expressed as fold mRNA level increase comparedwith the expression level in lungs from naive mice.

Analysis of virus load by plaque assay

Briefly, lungs were homogenized, and homogenates were diluted beforebeing applied to 95% confluentMadin-Darby canine kidney cells. Virus wasadsorbed onto the cells for 1 h at room temperature before being washed offfour times with serum-free DMEM. Cells were then covered with serum-free DMEM containing trypsin and 0.8% agarose and incubated for 48 hat 37˚C with 5% carbon dioxide. Agarose was then removed, and themonolayer was stained with 1% gentian violet to allow for the visualiza-tion and counting of plaques.

Preparation of pulmonary cells and analysis of iNKT cell, NKcell, and CD8+ T lymphocyte activation in vivo

Pulmonary cells from naive or infected mice were prepared by classicalprocedures. Lungs were perfused with PBS, excised and finely minced,followed by enzymatic digestion for 20 min at 37˚C in RPMI 1640 con-taining 1 mg/ml collagenase type VIII (Sigma) and 1 mg/ml DNase type I(Sigma). After wash, lung homogenates were resuspended in a 35% Per-coll gradient and carefully layered onto 70% Percoll and centrifuged at2600 rpm, without brake, at 22˚C for 30 min. After centrifugation, twolayers of cells were formed. The layer in the interface between the twoPercoll concentrations was carefully aspirated and washed in PBS 2%FCS. RBCs were removed with lysis buffer (Sigma).

To analyze iNKTor NK cells, mononuclear cell (MNC) suspensions wereincubated with appropriate dilutions of allophycocyanin-conjugated PBS-57 glycolipid-loaded CD1d tetramer and FITC-labeled anti–TCR-b orallophycocyanin-conjugated CD5 and PerCP-Cy5.5–labeled NK1.1 (forNK cell analysis) and PE-conjugated anti-CD69 in some cases for 30 minin PBS containing 2% FCS and 0.01% NaN3. Cells were then fixed in PBS1% paraformaldehyde for 10 min, resuspended in PBS plus 2% FCS and0.1% saponin (permeabilization buffer), and incubated with PE-conjugatedmAb against IFN-g or control rat IgG1 mAb in permeabilization buffer.To study the CD8+ T cell response, lungs were extracted 4 and 7 d p.i.,and pulmonary cells were incubated with appropriate dilutions ofallophycocyanin-conjugated anti-CD19, FITC-labeled anti-CD8, and PE-conjugated Pro5 MHC pentamer H-2Db SSLENFRAYV. Alternatively,MLN cells were prepared 2, 3, and 4 d p.i. Cells were acquired and ana-lyzed on a FACSCalibur (Becton Dickinson, Rungis, France) cytometerusing the CellQuest software. To assess the functionality of virus-specificCD8+ T cells, 5 3 105 lung cells were seeded on 96-well plates and thenstimulated with a peptide specific for the viral polymerase 2 protein(PA224–233) and H-2Db (SSLENFRAYV; 10 mg/ml). To study iNKT cellanergy, lungs cells were stimulated with a-GalCer (100 ng/ml). Forty-eighthours later, the cell culture supernatants were collected and analyzed forthe IFN-g and IL-4 concentration by ELISA (R&D Systems). Bonemarrow-derived DCs were prepared as in Ref. 40.

Migration assay and analysis of DCs in the MLNs

Mice were infected with IAV, and 48 h later endotoxin-free OVA–FITC (25ml, 5 mg/ml) was administered intranasally as described (41). Twenty-fourhours later, MLNs were digested by enzymatic procedure for 20 min at37˚C in RPMI 1640 containing 1 mg/ml collagenase type VIII (Sigma) and1 mg/ml DNase type I (Sigma). MLNs were next mechanically homoge-nized with a 90-mm-pore filter. For DC subset analysis, DCs were dis-criminated into three subsets based on the expression of CD103 andCD11b markers as described (42). To analyze their maturation state, CD86and CD40 expression was assessed by FACS staining.

Isolation of CD11b+ Gr1+ cells and OT-I proliferation assay

Lungs were collected 4 d p.i. and prepared as described above. CD11b+

Gr1+ cells were sorted by flow cytometry (FACSAria; BD Biosciences).Purity of CD11b+ Gr1+ cells after sorting was consistently .98%. Spleencells from OT-I TCR transgenic mice were pulsed with 2 mg/ml SIINFEKLpeptide for 2 h at 37˚C and extensively washed. Purified CD11b+ Gr1+

cells from naive or infected mice (3 3 104 cells) were cocultured with 2 3105 OT-I splenocytes. Four days later, Alamar blue was added in each wellto assess cell proliferation, and DDO (570, 620 nm) was analyzed 24 hlater.

NKT purification and adoptive transfer

For adoptive transfer experiments, and to prevent activation of iNKT cells,NKT cells were purified from the livers of naive animals using CD5and NK1.1 Abs, but not on the basis of PBS-57 glycolipid-loaded CD1dtetramer and TCRb (or CD3) staining. Briefly, liver cells from WTdonor mice were stained with anti-NK1.1 (PE-conjugated) and anti-CD5(allophycocyanin-conjugated) mAbs (BD Pharmingen). Labeled cells wereisolated using a FACSAria and BD FACSDiva software (BD Biosciences).Ja182/2 recipient mice were inoculated intravenously either with 1 3 106

purified NKT cells or with the same volume of medium alone 24 h beforeIAV infection. CD5+ NK1.1+ cell purity after sorting was consistently.98%. About 90% of sorted hepatic CD5+ NK1.1+ cells also stainedpositive with PBS-57 glycolipid-loaded CD1d tetramer.

Statistical analysis

Results are expressed as the mean 6 SD or mean 6 SEM. The statisticalsignificance of differences between experimental groups was calculated bya one-way Anova with a Bonferroni posttest or an unpaired Student t test(GraphPad Prism 4 Software, San Diego, CA). The possibility to use theseparametric tests was assessed by checking if the population is Gaussianand the variance is equal (Bartlett’s test). Survival of mice was comparedusing Kaplan–Meier analysis and log-rank test. Results with a p value,0.05 were considered significant.

ResultsPulmonary iNKT cells become activated during IAV infection

The potential recruitment/expansion of iNKT cells in the lung aswell as their activation status have not yet been examined duringIAV infection. To investigate this, C57BL/6 mice were infectedwith IAV Scotland/20/74 (H3N2), and the absolute numberof pulmonary PBS-57 glycolipid-loaded CD1d tetramer+ TCRb+

(iNKT) cells was first determined during the early course of in-fection. As depicted in Fig. 1A (upper panel), the number of de-tectable iNKT cells in the lung tissue remained stable at all timepoints analyzed (from days 2 to 7 p.i.). This finding was confirmedby quantifying the genomic level of Va14Ja18 TCR gene rear-rangement (specific to iNKT cells) in the lung tissue usingquantitative PCR (data not shown). To investigate the activationstatus of pulmonary iNKT cells, we monitored the expression ofthe early activation marker CD69. As revealed in Fig. 1A (lowerpanel), and relative to mock-treated animals, the level of CD69expression on lung iNKT cells increased at days 2, 4, and 7 p.i.The potential accumulation and activation status of iNKT cells inthe alveolar space were also analyzed. Although iNKT cells werealmost undetectable in the BAL fluid of uninfected mice, weobserved an influx of iNKT cells into the airways at day 4 p.i.,with a peak at day 7 p.i. (Fig. 1B and data not shown). Finally,iNKT cells present in the alveolar space express a high level of

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CD69 (Fig. 1B, right panel). Thus, iNKT cells in lung parenchyma

and airways display clear signs of phenotypic activation during the

early phase of IAV infection.It is known that after primary activation, iNKT cells enter into

a state of nonresponsiveness and fail to produce cytokines upon

subsequent stimulation (43–45). We took advantage of this prop-

erty to confirm further that pulmonary iNKT cells become acti-

vated during IAV infection. To this end, lung cells from mock

or IAV-infected mice were stimulated with the iNKT cell super-

agonist a-GalCer, and cytokine release was quantified. As

expected, lung cells from uninfected mice produced copious

amounts of IFN-g (Fig. 1C) and IL-4 (data not shown) in response

to a-GalCer. In marked contrast, lung cells from mice infected 4

and 7 d earlier produced much lower amounts of cytokines upon

a-GalCer stimulation. To verify that the observed effect was not

dependent on decreased APC functions in the mixed lung cell

culture, lung cells from naive or infected mice were cocultured

with a-GalCer–pulsed bone marrow DCs. This procedure did not

restore cytokine release by lung cells isolated from infected ani-

mals (data not shown), indicating that the effect is intrinsic to

iNKT cells. Overall, IAV infection is accompanied by an activa-

tion of iNKT cells in the lungs followed by a state of non-

responsiveness.

Mice deficient in iNKT cells show significantly increasedmortality but not reduced or delayed viral clearance

Excessive inflammation in the lung is detrimental after a varietyof respiratory challenges. Infection of mice with IAV H3N2(Scotland/20/74) causes acute bronchiolitis and pneumonia thateventually leads to death. It is known, in the mouse system, thatiNKT cells are versatile controllers of inflammatory responses. Toaddress the potential role of iNKT cells in the development and/or control of pulmonary immunopathology, WT and Ja182/2

(iNKT cell-deficient) mice were first used in survival studies witha lethal dose (600 PFU) of virus. In WT animals, the first clinicalmanifestations appeared on day 7 p.i. and within 8 and 12 d p.i.,all animals demonstrated severe sickness ending in death (85%mortality at day 16) (Fig. 2A, left panel). Notably, Ja182/2 micedeveloped accelerated clinical manifestations and succumbedmore rapidly to infection relative to WT animals. No survival wasobserved in infected Ja182/2 mice past day 11 after viral chal-lenge (p , 0.05). The role of iNKT cell in disease regulation wasconfirmed using CD1d2/2 animals, which lack iNKT cells as wellas non-iNKT cells (Supplemental Fig.1). These data are in linewith those from De Santo et al. and Ishikawa et al. who reportedenhanced mortality in IAV (H1N1)-infected Ja182/2 (34) andCD1d2/2 (34, 35) mice relative to controls and strongly suggest

FIGURE 1. Total number and activation status

of iNKT cells in the lung tissue and in the BAL

fluids in IAV-infected mice. A, The lungs were

harvested from uninfected (mock) mice or at 2, 4,

and 7 d p.i., and iNKT (PBS-57 glycolipid-loaded

CD1d tetramer+ TCRb+) cells were analyzed by

FACS staining. A representative dot plot is depic-

ted (mock). In the upper panel, the average num-

ber 6 SEM of iNKT cells in the lung tissue is

indicated for each time point. In the lower panel,

the mean fluorescence intensity (MFI) of CD69

expression on gated iNKT cells is represented. A

representative histogram is shown (7 d p.i.). B, The

BAL fluids were collected, and iNKT cells were

analyzed by FACS staining (data shown are at 7 d

p.i.). C, Lung MNCs isolated from either mock-

inoculated or infected mice were stimulated for

48 h with a-GalCer (100 ng/ml), and afterward

IFN-g production was quantified by ELISA. A–C,

Data represent the mean 6 SEM (n = at least 9).

**p , 0.01, ***p , 0.001 (two-tailed Student t

test).



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that iNKT cells are beneficial during acute IAV infection. Todemonstrate further the involvement of iNKT cells in diseaseregulation during acute IAV H3N2 infection, Ja182/2 mice werereconstituted with NKT cells (.90% iNKT cells) purified fromnaive animals and then challenged with a lethal dose of IAV. Asshown in Fig. 2A (right panel), symptoms of illness (data notshown) and mortality developed more slowly in the reconstitutedanimal group than in nonreconstituted Ja182/2 mice. In thiscondition, .30% of reconstituted mice survived the infection(p , 0.05).To determine whether the accelerated clinical signs and mortal-

ity in infected Ja182/2 mice are due to the impairment of viralclearance after influenza administration, the viral load was mon-itored at day 4 (the peak of viral replication) and day 7 p.i. byquantitative RT-PCR and plaque assay. At day 4 p.i., the viral loadin the lungs in WT and Ja182/2 mice was identical (Fig. 2B). Atday 7 p.i., both WT and Ja182/2 mice had a lower and similarviral load in their lungs. In agreement with this, the transcriptlevels of genes associated with viral replication (ifnb, mx1) werenot significantly different between infected WT and Ja182/2 mice(Fig. 2C). Thus, in our experimental system, iNKT cell deficiencydoes not lead to an impaired containment and clearance of IAV in

the lungs during infection. This suggests that, in this infectioussystem, inflammatory injury, rather than uncontrolled viral repli-cation, is an important determinant of the more rapid mortalityobserved in Ja182/2 mice.

Mice deficient in iNKT cells develop increased pneumopathy

To compare the extent of pulmonary inflammation, lungs fromWTand Ja182/2 mice were harvested for histology during the acute-phase response. Relative to infected WTanimals, infected Ja182/2

mice developed a marked increase in lung inflammation (Fig. 3).This was characterized by a more marked infiltration of neu-trophils and macrophages, but not lymphocytes, in the lung tissue(Fig. 3, bottom panel) and in the BALs (data not shown). Histo-logical scoring of H&E-stained lung sections confirmed the sig-nificant enhancement of airway inflammation in Ja182/2 mice(Fig. 3, bottom panel). In WT mice, mild to moderate lesions ofacute pneumopathy were observed including small and confluentareas of alveolitis, bronchiolitis, and one small focus of limitedconsolidation in ∼40% of mice. No necrosis of alveoli nor ofbronchus epithelia was observed in WT infected mice. By con-trast, lesions in Ja182/2 mice were more severe showing a largerspectrum of inflammatory lesions. In particular, severe lesions of

FIGURE 2. Survival rates and viral load in WT and iNKT cell-deficient mice infected with a lethal dose of IAV. A, Age-matched WT or Ja182/2 mice

were infected with 600 PFU IAV Scotland/20/74/H3N2 strain and then followed for mortality. In the left panel, the survival of Ja182/2 mice was monitored

and compared with that of WT mice (n = at least 15 mice/group). Log-rank test for comparisons of Kaplan–Meier survival curves indicated a significant

increase in the mortality of Ja182/2 mice compared with that of WT animals. *p , 0.05. In the right panel, Ja182/2 mice were i.v. injected with 1 3 106

cell-sorted NKT cells or with PBS 24 h before IAV challenge. The survival of reconstituted Ja182/2 mice was monitored and compared with that of

Ja182/2 mice injected with PBS alone (n = 10 to 15/group). *p, 0.05. B, Analysis of the viral load in the lung of IAV-infected WTor Ja182/2 mice. In the

left panel, on the stated days p.i., IAV M2 mRNA levels were measured by quantitative RT-PCR. Data are normalized to expression of hprt. Shown are IAV

M2/hprt mRNA expression ratios. Data represent the mean 6 SEM of three independent experiments performed in triplicate (n = 12/group/day). In the

right panel, the viral load, expressed as PFU/mg of lung tissue, was determined by plaque assay 4 d p.i. Shown is the mean 6 SEM of one representative

experiment of two (n = 5). C, mx1 and ifnb mRNA copy numbers were determined by quantitative real-time PCR. Data are normalized to expression of

gapdh and are expressed as fold increase over average gene expression in naive mice. Data represent the mean 6 SEM of three independent experiments

performed in triplicate (n = 12/group/day).



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bronchiolitis with necrosis of epithelia were observed. Finally, thelungs of ∼80% of Ja182/2 mice showed large areas of lobularconsolidation. iNKT cell deficiency thus leads to a significant in-crease in IAV-associated airway inflammation.

Mice deficient in iNKT cells show normal NK cell activationbut an altered IAV-specific CD8+ T cell response in the lungs

In vivo activation of iNKT cells triggers downstream stimulation ofvarious immune cells including NK cells and CD8+ T cells, knownto be important during IAV infection. For instance, activatediNKT cells can stimulate NK cells to produce IFN-g. To study therole of iNKT cells on the transactivation of NK cells in the contextof IAV infection, we first compared the frequency of IFN-g+ NKcells in the lungs of IAV-infected WT and Ja182/2 animals. Asshown in Fig. 4A (left panel), pulmonary NK cells from WTinfected mice labeled positively for IFN-g (but not IL-4, data notshown) at days 4 and 7 p.i., as judged by intracellular FACSstaining. Notably, iNKT cell deficiency did not reduce the fre-quency of IFN-g+ NK cells and even significantly increased it atday 4 p.i. Similarly, the expression of CD107a (Fig. 4A, rightpanel) and granzyme B (not shown), two molecules known toparticipate in cellular cytotoxicity, was also enhanced on NK cells

from Ja182/2 mice relative to that of WT mice at day 4 p.i. Thus,iNKT cells are not necessary to activate NK cells in our experi-mental IAV infection model.IAV infection leads to a rapid recruitment, expansion, and ac-

tivation of CD8+ T lymphocytes in pneumonic lungs. The virus-specific CD8+ T cell response was thus compared in terms of cellnumber and IFN-g production. For this, cells specific for animmunodominant Db-restricted CD8+ T epitope derived from theviral polymerase 2 protein (PA224–233) (46) were analyzed in thelungs of infected WT and Ja182/2 mice. As measured by MHCclass I pentamer staining, CD8+ DbPA224–233

+ cells accumulated inthe lungs of infected WT animals as early as 4 d p.i. and theirabsolute number was higher at day 7 p.i. (Fig. 4B). Notably, rel-ative to WT controls, Ja182/2 mice had a dramatically decreasednumber of IAV-specific CD8+ T cells in the lungs at days 4 and 7p.i. This phenomenon was also observed in the BAL (Supple-mental Fig. 2). As depicted in Fig. 4C, the absolute number ofIAV-specific CD8+ T cells was partially (∼40%), but significantly,restored after adoptive transfer of NKT cells into Ja182/2 mice(shown is day 4 p.i.). Finally, upon restimulation with PA224–233,the release of IFN-g by lung cells isolated from infected Ja182/2

mice was dramatically reduced compared with that from WTanimals (Fig. 4D). These results demonstrate that iNKT cell de-ficiency strongly affects the early IAV-specific CD8+ T cell re-sponse that develops in the lungs, a finding in agreement with Ref.34.

iNKT cells do not control the recruitment and the suppressiveactivity of CD11b+ Gr1+ cells in the lung during the early stepof IAV infection

During H1N1 IAV infection, it appears that the defective IAV-specific CD8+ T cell response observed in Ja182/2 mice is dueto an enhanced recruitment and an increased suppressive activityof a population of myeloid cells expressing Gr1 (34). We thusinvestigated whether this is also the case in our experimentalsystem using a pathogenic H3N2 IAV strain. To do this, we firstquantified the frequency and the number of CD11b+ Gr1+ cells,defined as myeloid-derived suppressor cells (MDSCs) in mice(47). As shown in Fig. 5A, and relative to noninfected animals,CD11b+ Gr1+ cells accumulated in the lungs of infected mice atdays 4 and 7 p.i. (by ∼3.6- and 7.9-fold in terms of absolutenumbers, respectively). However, no difference was observed, interms of frequency and cell number, between IAV-infected WTand Ja182/2 mice (Fig. 5A). To analyze potential differences intheir suppressive activity, CD11b+ Gr1+ cells were sorted andtested for their ability to reduce the proliferation of OT-I cellsupon OVA peptide stimulation. As seen in Fig. 5B, CD11b+ Gr1+

cells from naive mice significantly inhibited OVA-specific T cellproliferation (by ∼35%), but this effect was not enhanced withcells sorted from IAV-infected mice. Furthermore, the suppressiveactivity of CD11b+ Gr1+ purified from IAV-infected WT andJa182/2 animals was not different. These data suggest that thedefective IAV-specific CD8+ T cell response in the lungs ofiNKT cell-deficient mice is not due to a heightened suppressiveactivity of CD11b+ Gr1+ cells in this organ.

Ja182/2 mice show impaired IAV-specific CD8+ T cell primingin MLNs

These findings prompted us to investigate the possibility that thediminished CD8+ T cell response in the lung could be caused byan altered primary stimulation in the MLNs. As shown in Fig. 6A,CD8+ DbPA224–233

+ cells accumulate in the MLNs of infected WTanimals as early as 3 d p.i. In marked contrast, the absolutenumber of pentamer-positive CD8+ T cells was dramatically lower

FIGURE 3. Lung injury in infected iNKT cell-deficient mice. Age-

matched WT or Ja182/2 mice were infected with 600 PFU IAV Scotland/

20/74. Lungs were harvested 4 d p.i., and sections through the main

bronchiole of the left lobe were stained with H&E (original magnification

340 and 3100). In the top panel, representative lung sections are shown:

A, perivascular and peribronchic infiltrates combined with mild lesions of

alveolitis without consolidation; B, perivascular and peribronchic infil-

trates combined with severe lesions of alveolitis with consolidation; C,

PMN cells and MNCs surrounding the bronchi and vessels; D, PMN cells

and MNCs surrounding the bronchi and vessels combined with alveolitis

and consolidation. In the bottom panel, sections were scored blindly for

levels of immunopathology. The percentages of bronchial epithelium ne-

crosis are indicated. Results of a representative experiment of three re-

peated experiments are shown. Data represent the mean 6 SEM (n = 10

mice/group). Of note, immunohistological analysis using anti-H3N2 Abs

revealed no differences in the infection rate of bronchial epithelial cells

and alveolar macrophages between WT or Ja182/2 mice. *p , 0.05,

**p , 0.01 (two-tailed Student t test).



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in infected Ja182/2 animals. Finally, the IAV-specific CD8+ Tresponse was fully restored in Ja182/2 mice reconstituted withNKT cells (Fig. 6B). Together, these data strongly suggest that the

altered pulmonary CD8+ T cell response observed in iNKT cell-deficient animals is due to a defected priming in the lung-draininglymph nodes (LNs).

FIGURE 4. Characterization of the NK cell and CD8+ T cell responses in IAV-infected WT and Ja182/2 mice. Four and seven days p.i., lung cells from

infected mice were recovered and analyzed for NK cell and CD8+ T cell responses. A, In the upper panel, gated CD32 NK1.1+ cells from mock and

infected (4 d p.i.) WT or Ja182/2 mice were analyzed for intracellular IFN-g (and granzyme, not shown) production and for cell surface CD107a ex-

pression. The average percentages 6 SEM of pulmonary CD32 NK1.1+ cells positive for IFN-g (left panel) and CD107a (right panel) are represented (n =

9 mice/group). Of note, the total number of pulmonary NK cells is identical in influenza-infected WTand Ja182/2 mice (data not shown). B, Representative

dot plots of gated PA224–233-specific CD8+ T cells from mock and IAV-infected (7 d p.i.) mice are shown. C, In the left panel, the number of PA224–233-

specific CD8+ T cells represents the average (6SEM) of results obtained in three independent experiments performed (n = 10 mice/group). In the right

panel, Ja182/2 mice were reconstituted or not with NKT cells and 4 d p.i., the number of PA224–233-specific CD8+ T cells in the lung tissue was determined

by flow cytometry (n = 6/group). D, Lung cells were restimulated with PA224–233 peptide (10 mg/ml) for 3 d. IFN-g production was measured by ELISA

(n = 12). Of note, lung cells from naive WT and Ja182/2 mice produce equal amounts of cytokines after anti-CD3 stimulation (not shown). Although

increased compared with noninfected animals, the number of cells, including total CD8+ T cells, in the lungs was not different between IAV-infected WT

and Ja182/2 mice (data not shown). A and B, Differences in mean were analyzed using the two-tailed Student t test. C, A one-way ANOVA was used to

analyze the variance followed by a Bonferroni multiple comparison test to compare all groups. *p , 0.05, **p , 0.01, ***p , 0.001.



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The accumulation of CD103+, but not CD11bhigh, pulmonaryDCs to the MLNs is reduced in IAV-infected Ja182/2 mice

The migration of virus Ag-loaded pulmonary DCs to the MLNsplays a key role in the induction of the CD8+ T cell response earlyafter IAV infection (42, 48–50). We thus hypothesized that thedefective CD8+ T cell response observed in Ja182/2 mice couldbe due to an altered accumulation of respiratory DCs in theMLNs. To investigate this, endotoxin-free FITC-conjugated OVAwas inoculated 2 d after IAV infection, and 24 h later the numberof DCs positive for FITC was quantified in MLNs from WTand Ja182/2 mice. Because two predominant populations of re-spiratory DCs, which are mostly CD8a2 (51), have been de-scribed (52, 53), FITC+ DCs were discriminated on the basis ofCD11b and CD103 expression. Respiratory DCs undergo spon-taneous migration to secondary lymphoid tissues via afferentlymphatics (50, 54). In agreement with this, inoculation of FITC-conjugated OVA in noninfected animals resulted in the accumu-lation of both FITC+ CD11bhighCD103neg (here referred to asCD11bhigh) and CD11bintCD103+ (here referred to as CD103+)DCs in the MLNs (Fig. 7). In this setting, resident LN DCs(mostly CD1032 CD11b2) were not FITC+. Relative to un-infected animals, the number of FITC+CD11bhigh and FITC+

CD103+ DCs strongly augmented in the MLNs of IAV-infectedWT animals (∼5.5- and 6.5-fold increase over controls, re-spectively). Notably, the increased number of emigrating FITC+

CD103+ DCs in infected Ja182/2 mice was less substantial(∼3.4-fold increase compared with mock-treated Ja182/2 mice),whereas the fold increase of FITC+CD11bhigh DC number was ofequal amplitude relative to that observed in infected WT animals(∼4.8-fold increase). Thus, the lack of iNKT cells results in a di-

minished accumulation of pulmonary CD103+ DCs in the MLNsduring IAV infection.

iNKT cell deficiency leads to a reduced maturation ofrespiratory DCs in the MLNs

The maturation of DCs is a crucial step in the induction of T cellactivation and polarization. In this setting, iNKT cells may serve asimportant components (55, 56). We thus compared the maturationstatus of DCs in the MLNs of infected WT and Ja182/2 mice(4 d p.i.). Before this, the number of lung-derived DCs, as well asMLN resident DCs, was quantified. Of note, relative to infectedWT animals, the IAV-associated enhancement of CD103+ DCnumber in the MLNs of Ja182/2 mice was of lower amplitude,whereas that of CD11bhigh and CD1032 CD11b2 (LN resident)DCs was not significantly affected (Fig. 8A). Of interest, the re-duced accumulation of CD103+ DCs observed in Ja182/2 micewas partially (∼60%) overcome by NKT cell transfer (Fig. 8B).Infection with IAV induced an increased synthesis of CD86 andCD40 on CD103+ DCs from WT mice. In contrast, CD11bhigh andLN resident DCs had an immature phenotype. Of interest was theobservation that CD103+ DCs from Ja182/2 mice express lessCD86 and CD40, relative to DCs from WT infected animals.Thus, iNKT cell deficiency not only affects the accumulation ofCD103+ DCs but also their maturation in the MLNs. It is likelythat these phenomena are responsible, at least in part, for the re-duced priming of CD8+ T cells in iNKT cell-deficient animals.

DiscussionIn some, but not all, experimental models of viral infections,iNKT cells have been shown to exert positive or negative responses,

FIGURE 5. Analysis of CD11b+ Gr1+ frequency/number and suppressive activity in infected WT and Ja182/2 mice. A, Percentages and total cell

numbers of pulmonary CD11b+ Gr1+ within lung MNCs of IAV-infected mice. The lungs were harvested 4 and 7 d p.i., and the relative proportions (left

panel) and cell numbers (right panel) of CD11b+ Gr1+ cells were calculated by FACS staining. Control refers to mock-inoculated mice. Data represent the

mean percentage 6 SEM of four independent experiments (n = 12). B, Analysis of in vitro suppressive activity of CD11b+ Gr1+ cells. OT-I splenocytes

were cultured in the presence (or not) of the OVA peptide SIINFEKL (2 mg/ml) with or without pulmonary CD11b+ Gr1+ cells purified from mock or IAV-

infected mice (day 4). Data represent the average6 SD of n = 5 mice/group. A, Differences in mean were analyzed using the two-tailed Student t test. B, A

one-way ANOVAwas used to analyze the variance followed by a Bonferroni multiple comparison test to compare all groups. *p, 0.05, **p, 0.01, ***p,0.001.



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either controlling viral replication or contributing to immunopa-thogenesis (for reviews, see Refs. 12, 13). In this study, usinga mouse model of acute pneumonia triggered by IAV (H3N2)infection, we suggest that iNKT cells play a part in the control ofimmunopathology and susceptibility to mortality. We also suggestthat iNKT cells are important in the development of the local IAV-specific CD8+ T cell response, a phenomenon due in part to aninadequate accumulation and maturation of lung CD103+ DCs inMLNs.Viral infection of the respiratory tract can lead to the activation of

pulmonary NKT cells, and this may strongly impact the controlof the local immune response (18, 20). The activation status ofpulmonary iNKT cells as well as their potential recruitment/expansion in the lung during IAV infection have not yet beenthoroughly evaluated, although a recent study reported that theirfrequency in the lung is increased 1 d p.i. (28). Using a lethal doseof IAV, our data show that no massive expansion or, on the con-trary, numeric disappearance of iNKT cells occurs in the lungtissue during the early phase of infection. In contrast, iNKT cellsaccumulated in the alveolar space as early as 4 d p.i. Notably, atdays 4 and 7 p.i., the expression of the early activation marker

CD69 was strongly increased on pulmonary (tissue and BAL)iNKT cells. Finally, upon in vitro stimulation with the super-agonist a-GalCer, lung iNKT cells from IAV-infected mice failedto produce cytokines suggesting that they acquired a hypores-ponsive phenotype, a phenomenon that occurs after primo-activation (43–45). The mechanisms by which lung iNKT cellsbecome activated during H3N2 infection are still unknown. In thissetting, activation of iNKT cells might be triggered by cytokinesproduced from APC, by TCR ligation with self-lipids, or both (forreview, see Ref. 57). Several viruses, including human CMV andHSV type 1 (low dose), have been shown to upregulate CD1dsurface expression on APC (58, 59), whereas others, includingKaposi sarcoma-associated herpesvirus, vesicular stomatitis virus,vaccinia virus, HSV type 1 (high dose), and HIV 1, reduce itssynthesis to escape iNKT activation (59–65). In the case of IAV,this evasion mechanism does not appear to occur, at least in vitro(Ref. 34, and our unpublished data). The mechanisms (role ofthe CD1d molecule and/or inflammatory cytokines) by whichiNKT cells become activated during H3N2 IAV infection arecurrently being studied.

FIGURE 6. Analysis of the CD8+ T cell response in the MLNs from

IAV-infected WT and Ja182/2 mice. A, Two, three, and four days p.i.,

MLN cells from infected WT and Ja182/2 mice were recovered and the

number of PA224–233-specific CD8+ T cells were determined. The dot plot

shown corresponds with day 4 p.i. The average (6SEM) numbers are

indicated (n = 9 mice/group). Of note, the number of total CD8+ T cells in

the MLNs was not different between IAV-infected WT and Ja182/2 mice.

B, Ja182/2 mice were i.v. injected with 1 3 106 cell-sorted NKT cells or

with PBS 24 h before IAV challenge. Shown is the number 6 SEM of

PA224–233-specific CD8+ T cells 4 d p.i. (n = 6/group). A and B, Differences

in mean were analyzed using the two-tailed Student t test. *p , 0.05,

**p , 0.01.

FIGURE 7. Comparison of the migratory capacity of lung DCs into

MLNs between IAV-infected WT and Ja182/2 mice. Two days after IAV

infection, endotoxin-free FITC–OVA was inoculated intranasally, and the

number of FITC+ DCs was determined in WT or Ja182/2 mice (24 h post

FITC–OVA inoculation). FITC* DCs were discriminated on the basis of

CD11b and CD103 expression. At this time point, few FITC+ resident DCs

(CD11b2 were detected. In the top panel are shown the percentages of

CD11bhiCD103neg (referred to as CD11bhigh), CD11bintCD103+ (referred

to as CD103+), and CD1032 CD11b2 (resident LN) total and FITC+ DCs

in the MLNs of mock-treated or IAV-infected WT mice (one representative

animal/group). Data in the bottom panel represent the mean percentage 6SD (n = 5). One representative experiment of three is shown. Of note, the

total number of cells in the MLNs was not significantly different between

infected WT and Ja182/2 mice (4.6 6 0.93 3 106 and 3.9 6 0.79 3 106,

respectively, at day 4 p.i.). *p , 0.05, ***p , 0.001.



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The clinical response to IAV infection ranges from mild diseaseto severe pneumonia, according to IAV strains and inoculationdoses. In our experimental system, mice died at times after the peakof virus replication in the lung suggesting that inflammatory in-jury, rather than uncontrolled viral replication, is an importantdeterminant of the fatal outcome. Physiologically, iNKT cells canaugment or inhibit inflammatory responses through a variety ofmechanisms depending on the context (i.e., sterile or nonsterileinflammation) and the targeted organ. Several experimental modelshave highlighted the detrimental role of iNKT cells in lung in-flammation including allergic reaction, airway hyperreactivity, and(virus-induced) chronic obstructive pulmonary disease (18, 66,67). In this study, we evaluated their potential regulatory functionin IAV-induced pathology. In multiple repeated experiments, weconsistently observed that mice lacking iNKT cells, both Ja182/2

and CD1d2/2 mice, died more rapidly compared with infectedWT animals, a phenomenon fully reversed by the adoptive transferof FACS-purified NKT cells into Ja182/2 mice before IAV in-fection. Histological examination revealed that, compared withWT mice, infected Ja182/2 developed a more acute pneumopathywith severe alveolitis, bronchiolitis, and focal consolidation. Thiswas accompanied by a more pronounced infiltration of neutrophilsand macrophages, but not lymphocytes, in the lung tissue. Thus,iNKT cell deficiency accelerates lung injury and the fatal outcomein our system. This finding is in agreement with other studiesreporting beneficial effects of NKT cells, including iNKT cells,during acute respiratory viral infection (20, 34, 35) and suggeststhat iNKT cells play a part in the control of airway inflammationand susceptibility to IAV (H3N2)-associated mortality. The exactmechanisms by which iNKT cells attenuate IAV pathogenesis areyet to be defined. It is possible that they act through the synthesis

of factors able to control lung injury directly or indirectly.Quantitative RT-PCR analysis of whole lungs, however, revealedno major change in the synthesis of inflammatory (i.e., IL-6,TNF-a, inducible NO synthase, IFN-g) or of anti-inflammatory(i.e., IL-10, TGF-b) factors between infected WT and Ja182/2

mice (data not shown). It is also possible that the reduced numberof IAV-specific CD8+ T cells in Ja182/2 mice, or to the contrary,the enhanced infiltration of neutrophils and/or macrophages playa part in this phenomenon. Finally, iNKT cells might also in-fluence lung repair processes for instance to counteract epithelialdamage (i.e., necrosis). Attempts are now under way to investigatethese issues and to identify more subtle functions of iNKT cells inIAV-associated pneumonia.In contrast with Ref. 34 (H1N1 strain, sublethal dose),

iNKT cell deficiency did not lead to an impaired containment andclearance of IAV in the lungs in our experimental system (H3N2strain, lethal dose). This finding is not without precedent asiNKT cells have been shown to be dispensable to the control of theviral load in some experimental systems (22, 68, 69). In line withthis observation, the lack of iNKT cells did not reduce the NK cellresponse. First, NK cell accumulation in the lung was almostidentical in WT and Ja182/2 mice (data not shown), indicatingthat NK cell trafficking/expansion is unaffected by the absence ofiNKT cells. Second, the frequency of NK cells positive for IFN-gwas not reduced (even increased at day 4 p.i.) in infected Ja182/2

mice. Finally, the IAV-triggered expression of granzyme B andCD107a on/in NK cells was not affected (even increased at day 4p.i.) by the absence of iNKT cells. Thus, at least during acuteIAV infection, iNKT cells do not seem to favor NK cell activity.It is likely that triggering of NK activating receptors, includingNKG2D, NKp46, and NKp44, by ligands expressed on infected

FIGURE 8. Comparison of the maturation state of DCs in the MLNs between IAV-infected WT and Ja182/2 mice. A, The total number of CD103+,

CD11bhigh, and resident LN DCs (CD1032 CD11b2) are shown at day 4 p.i. Of note, relative to noninfected WT animals, the number of CD103+ DCs was

reduced, albeit not in a significant manner, in noninfected Ja182/2 mice. Data represent the mean percentage 6 SEM (n = 10) (three experiments per-

formed). B, Ja182/2 mice were reconstituted or not with NKT cells, and the number of CD103+ LN DCs (6SEM) was quantified 4 d p.i. (n = 8) (two

experiments performed). C, Four days p.i, the surface expression of CD86 and CD40 on DCs was analyzed by flow cytometry. Data represent the mean

fluorescence intensity (MFI) values 6 SEM (n = 10) (three experiments performed). *p , 0.05, **p , 0.01, ***p , 0.001.



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cells and/or the local IL-12 production are sufficient to activateNK cells (70, 71). Of note, it is possible that the enhanced NK cellresponse in Ja182/2 mice at day 4 p.i. may exacerbate the diseaseby damaging the affected tissues through direct cytotoxicity or bythe release of inflammatory cytokines and chemokines (72). Wenext examined the magnitude of the early IAV-specific CD8+

T cell response in the lungs of infected Ja182/2 mice. In agree-ment with Ref. 34, which focused on a distinct immunodominantepitope (NP366–374), we observed that Ja182/2 mice exhibiteda much less robust pulmonary CD8+ T cell response than that ofWT animals as assessed by PA224–233 epitope-specific CD8

+ T cellnumber and IFN-g production (Fig. 4B). Moreover, the frequencyof granzyme B and CD107a-expressing CD8+ T cells was alsoreduced by ∼50% in Ja182/2 mice (data not shown). Together,these data indicate that iNKT cell deficiency leads to a dramati-cally reduced CD8+ T cell response in the lung, an effect that isnot associated with a lack of control of viral replication as in Ref.34 (H1N1 strain, sublethal dose). This difference can be explainedby the fact that IAV-specific CD8+ T lymphocytes confer pro-tection against low (sublethal) dose, but not high dose (our study),viral challenge (73). In our system, the increased NK cell responsein concert with the remaining CD8+ T cell response may be suf-ficient to control the lung viral load in infected Ja182/2 mice. It isalso possible that other cells of the immune system cooperativelycontrol IAV replication.We next attempted to better understand how iNKT cells cripple

the magnitude of the lung CD8+ T cell response during IAV in-fection. A recent report has suggested that iNKT cells not onlycontrol the recruitment of MDSCs in the lungs but also theirsuppressive function on the CD8+ T cell response (34). The role ofiNKT cells on the functions of MDSCs has recently been con-firmed in the context of antitumor immunity (74). In our experi-mental system, the relatively low suppressive activity of CD11b+

Gr1+ cells isolated from noninfected mice was not enhanced p.i.,whatever the mouse genotype (iNKT cell competent or deficientanimals). Thus, unlike in Ref. 34, our data do not support a rolefor iNKT cells in the trafficking (and/or expansion) of CD11b+

Gr1+ cells into the lungs or in their immunosuppressive functions.Although the precise reasons for this discrepancy remain unclear,it may be due to experimental differences. For instance, the pre-vious study used a sublethal dose of H1N1 IAV (34), whereas weused a lethal dose of H3N2 IAV. The time points when the cellswere sorted (day 6 p.i. versus day 4 p.i.) as well as the basis of thesorting (Gr1+ versus CD11b+ Gr1+ cells) may also explain thisdiscrepancy. Our data also suggest that iNKT cell deficiency doesnot modulate the frequency/number of CD4+ CD25+ Foxp3+ cellsin the lungs during infection (data not shown). These findingsprompted us to investigate the possibility that the diminishedCD8+ T cell response in the lung could be caused by alteredprimary stimulation in the lung-draining MLNs rather than in-hibition of T cell expansion in the lung tissue. Indeed, it is knownthat secondary restimulation of IAV-specific CD8+ T responsetakes place in the lungs (as early as 4 d p.i.) and that it is de-pendent on the recruitment of TNF and inducible NO synthase-producing DCs (so called TipDCs) (75–77). However, we didnot observe differences in the frequency and number of TipDCsbetween infected WT and Ja182/2 mice (data not shown). Ourdata show that the IAV-specific CD8+ T response that normallydevelops in the MLNs of WT animals is strongly reduced inJa182/2 mice. This supports the hypothesis that iNKT cells act onthe primary stimulation of CD8+ T cells in the MLNs during IAVinfection. It is known that DCs and iNKT cells can potentiallyinfluence their mutual stimulation/maturation, including duringinfection (55, 56). DCs act during the priming phase of iNKT cell

activation, and iNKT cells promote DC maturation and cytokineproduction, in particular through IFN-g production. Migration ofrespiratory DCs from the lungs to the regional MLNs is a key stepin the initiation of virus-specific CD8+ T cell responses after in-fection (48, 49). Our data show that the defective CD8+ T cellresponse observed in infected Ja182/2 mice is associated witha reduced accumulation of respiratory CD103+ DCs, but notCD11bhigh DCs, in the MLNs. This phenomenon may be due toa lack of (iNKT cell-derived) factors able to promote the selectiveemigration of this DC subset. The reduced number of CD103+

DCs in the lung tissue of naive Ja182/2 mice (∼30% reductioncompared with that in WT animals, data not shown) may also, inpart, explain our observation. Recent findings have revealed thatthe CD103+ DC subset is of crucial importance in the promotionof the CD8+ T cell response during respiratory viral infection,including vaccinia virus and IAV (42, 78), although other DCsubsets also play a role in the latter system (49). In parallel, ourdata show that CD103+ DCs present in the MLNs express lessCD86 and CD40 in infected Ja182/2 mice compared with that inWT animals. Thus, the lack of iNKT cells not only negativelyaffects the selective accumulation of respiratory CD103+ DCs tothe MLNs but also their maturation process. This latter finding isin line with a recent study reporting the preferential modulatingeffect of iNKT cells on DCs (in this case the CD8a+ DC subset)during infection (56). Thus, along with other recently describedmechanisms (55, 56, 79, 80), our data suggest a new mechanism(promotion of DC emigration) by which iNKT cells may controlthe development of CD8+ T cells in vivo. This observation may berelevant in many pathological situations.Overall, our data suggest a key and early role for iNKT cells in

an acute model of pneumonia triggered by an H3N2 IAV strain.During IAV infection, both endogenous and exogenous (a-GalCer)iNKT cell activation appear beneficial to promote immune re-sponses and to control pulmonary inflammation. It is possible thata better understanding of the mode of in vivo iNKT cell activationand the precise functions they exert during IAV infection will bebeneficial to better harness these cells for prophylactic (vaccine)and/or therapeutic purposes in the future.

AcknowledgmentsWe acknowledge the generous support from the National Institute of

Allergy and Infectious Diseases Tetramer Facility (Emory University,

Atlanta, GA) for supplying CD1d tetramers. We thank Drs. T. Nakayama

and M. Taniguchi (RIKEN Institute, Yokohama, Japan) for the gift of

Ja182/2 C57BL/6 mice and Dr. L. Van Kaer (Vanderbilt University,

Nashville, TN) for the gift of CD1d2/2 C57BL/6 mice. We also thank

Drs. C. Jakubzick and G. Randolph (Mount Sinai School of Medicine,

New York, NY) for the gift of endotoxin-free OVA. M. Huot Khun (Unite

de Recherche et d’Expertise Histotechnologie et Pathologie, Pasteur In-

stitute, Paris, France) is acknowledged for the immunohistological anal-

ysis. We express our gratitude to Dr. L. Brossay (Brown University,

Providence, RI), M. Salio, F. Platt, A. Speak, and V. Cerundolo (Oxford

University, Oxford, U.K.) for helpful discussions. Dr. R. Pierce (Centre

d’Infection et d’Immunite de Lille, Lille, France) is acknowledged for

critical reading of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

References1. Palese, P. 2004. Influenza: old and new threats. Nat. Med. 10(12, Suppl): S82–

S87.2. Kaji, M., A. Watanabe, and H. Aizawa. 2003. Differences in clinical features

between influenza A H1N1, A H3N2, and B in adult patients. Respirology 8:231–233.



http://ww

w.jim

munol.org/

Dow

nloaded from


3. Cheung, C. Y., L. L. Poon, A. S. Lau, W. Luk, Y. L. Lau, K. F. Shortridge,S. Gordon, Y. Guan, and J. S. Peiris. 2002. Induction of proinflammatorycytokines in human macrophages by influenza A (H5N1) viruses: a mechanismfor the unusual severity of human disease? Lancet 360: 1831–1837.

4. Fujisawa, H. 2008. Neutrophils play an essential role in cooperation with anti-body in both protection against and recovery from pulmonary infection withinfluenza virus in mice. J. Virol. 82: 2772–2783.

5. Kobasa, D., S. M. Jones, K. Shinya, J. C. Kash, J. Copps, H. Ebihara, Y. Hatta,J. H. Kim, P. Halfmann, M. Hatta, et al. 2007. Aberrant innate immune responsein lethal infection of macaques with the 1918 influenza virus. Nature 445: 319–323.

6. McGill, J., J. W. Heusel, and K. L. Legge. 2009. Innate immune control andregulation of influenza virus infections. J. Leukoc. Biol. 86: 803–812.

7. Barral, D. C., and M. B. Brenner. 2007. CD1 antigen presentation: how it works.Nat. Rev. Immunol. 7: 929–941.

8. Bendelac, A., P. B. Savage, and L. Teyton. 2007. The biology of NKT cells.Annu. Rev. Immunol. 25: 297–336.

9. Cerundolo, V., J. D. Silk, S. H. Masri, and M. Salio. 2009. Harnessing invariantNKT cells in vaccination strategies. Nat. Rev. Immunol. 9: 28–38.

10. Godfrey, D. I., and M. Kronenberg. 2004. Going both ways: immune regulationvia CD1d-dependent NKT cells. J. Clin. Invest. 114: 1379–1388.

11. Van Kaer, L., and S. Joyce. 2005. Innate immunity: NKT cells in the spotlight.Curr. Biol. 15: R429–R431.

12. Diana, J., and A. Lehuen. 2009. NKT cells: friend or foe during viral infections?Eur. J. Immunol. 39: 3283–3291.

13. Tessmer, M. S., A. Fatima, C. Paget, F. Trottein, and L. Brossay. 2009. NKT cellimmune responses to viral infection. Expert Opin. Ther. Targets 13: 153–162.

14. Ashkar, A. A., and K. L. Rosenthal. 2003. Interleukin-15 and natural killer andNKT cells play a critical role in innate protection against genital herpes simplexvirus type 2 infection. J. Virol. 77: 10168–10171.

15. Diana, J., T. Griseri, S. Lagaye, L. Beaudoin, E. Autrusseau, A. S. Gautron,C. Tomkiewicz, A. Herbelin, R. Barouki, M. von Herrath, et al. 2009. NKT cell-plasmacytoid dendritic cell cooperation via OX40 controls viral infection ina tissue-specific manner. Immunity 30: 289–299.

16. Grubor-Bauk, B., J. L. Arthur, and G. Mayrhofer. 2008. Importance of NKT cellsin resistance to herpes simplex virus, fate of virus-infected neurons, and level oflatency in mice. J. Virol. 82: 11073–11083.

17. Grubor-Bauk, B., A. Simmons, G. Mayrhofer, and P. G. Speck. 2003. Impairedclearance of herpes simplex virus type 1 from mice lacking CD1d or NKT cellsexpressing the semivariant V alpha 14-J alpha 281 TCR. J. Immunol. 170: 1430–1434.

18. Kim, E. Y., J. T. Battaile, A. C. Patel, Y. You, E. Agapov, M. H. Grayson,L. A. Benoit, D. E. Byers, Y. Alevy, J. Tucker, et al. 2008. Persistent activation ofan innate immune response translates respiratory viral infection into chronic lungdisease. Nat. Med. 14: 633–640.

19. Stout-Delgado, H. W., W. Du, A. C. Shirali, C. J. Booth, and D. R. Goldstein.2009. Aging promotes neutrophil-induced mortality by augmenting IL-17 pro-duction during viral infection. Cell Host Microbe 6: 446–456.

20. Johnson, T. R., S. Hong, L. Van Kaer, Y. Koezuka, and B. S. Graham. 2002. NKT cells contribute to expansion of CD8(+) T cells and amplification of antiviralimmune responses to respiratory syncytial virus. J. Virol. 76: 4294–4303.

21. Exley, M. A., N. J. Bigley, O. Cheng, A. Shaulov, S. M. Tahir, Q. L. Carter,J. Garcia, C. Wang, K. Patten, H. F. Stills, et al. 2003. Innate immune response toencephalomyocarditis virus infection mediated by CD1d. Immunology 110: 519–526.

22. Wesley, J. D., M. S. Tessmer, D. Chaukos, and L. Brossay. 2008. NK cell-likebehavior of Valpha14i NK T cells during MCMV infection. PLoS Pathog. 4:e1000106.

23. Tsunoda, I., T. Tanaka, and R. S. Fujinami. 2008. Regulatory role of CD1d inneurotropic virus infection. J. Virol. 82: 10279–10289.

24. Huber, S., D. Sartini, and M. Exley. 2003. Role of CD1d in coxsackievirus B3-induced myocarditis. J. Immunol. 170: 3147–3153.

25. Levy, O., J. S. Orange, P. Hibberd, S. Steinberg, P. LaRussa, A. Weinberg,S. B. Wilson, A. Shaulov, G. Fleisher, R. S. Geha, et al. 2003. Disseminatedvaricella infection due to the vaccine strain of varicella-zoster virus, in a patientwith a novel deficiency in natural killer T cells. J. Infect. Dis. 188: 948–953.

26. Nichols, K. E., J. Hom, S. Y. Gong, A. Ganguly, C. S. Ma, J. L. Cannons,S. G. Tangye, P. L. Schwartzberg, G. A. Koretzky, and P. L. Stein. 2005. Reg-ulation of NKT cell development by SAP, the protein defective in XLP. Nat.Med. 11: 340–345.

27. Rigaud, S., M. C. Fondaneche, N. Lambert, B. Pasquier, V. Mateo, P. Soulas,L. Galicier, F. Le Deist, F. Rieux-Laucat, P. Revy, et al. 2006. XIAP deficiency inhumans causes an X-linked lymphoproliferative syndrome. Nature 444: 110–114.

28. Ho, L. P., L. Denney, K. Luhn, D. Teoh, C. Clelland, and A. J. McMichael. 2008.Activation of invariant NKT cells enhances the innate immune response andimproves the disease course in influenza A virus infection. Eur. J. Immunol. 38:1913–1922.

29. Kamijuku, H., Y. Nagata, X. Jiang, T. Ichinohe, T. Tashiro, K. Mori,M. Taniguchi, K. Hase, H. Ohno, T. Shimaoka, et al. 2008. Mechanism ofNKT cell activation by intranasal coadministration of alpha-galactosylceramide,which can induce cross-protection against influenza viruses. Mucosal Immunol.1: 208–218.

30. Ko, S. Y., H. J. Ko, W. S. Chang, S. H. Park, M. N. Kweon, and C. Y. Kang.2005. alpha-Galactosylceramide can act as a nasal vaccine adjuvant inducingprotective immune responses against viral infection and tumor. J. Immunol. 175:3309–3317.

31. Kopecky-Bromberg, S. A., K. A. Fraser, N. Pica, E. Carnero, T. M. Moran,R. W. Franck, M. Tsuji, and P. Palese. 2009. Alpha-C-galactosylceramide as anadjuvant for a live attenuated influenza virus vaccine. Vaccine 27: 3766–3774.

32. Youn, H. J., S. Y. Ko, K. A. Lee, H. J. Ko, Y. S. Lee, K. Fujihashi, P. N. Boyaka,S. H. Kim, T. Horimoto, M. N. Kweon, and C. Y. Kang. 2007. A single intranasalimmunization with inactivated influenza virus and alpha-galactosylceramideinduces long-term protective immunity without redirecting antigen to the cen-tral nervous system. Vaccine 25: 5189–5198.

33. Benton, K. A., J. A. Misplon, C. Y. Lo, R. R. Brutkiewicz, S. A. Prasad, andS. L. Epstein. 2001. Heterosubtypic immunity to influenza A virus in micelacking IgA, all Ig, NKT cells, or gamma delta T cells. J. Immunol. 166: 7437–7445.

34. De Santo, C., M. Salio, S. H. Masri, L. Y. Lee, T. Dong, A. O. Speak,S. Porubsky, S. Booth, N. Veerapen, G. S. Besra, et al. 2008. Invariant NKT cellsreduce the immunosuppressive activity of influenza A virus-induced myeloid-derived suppressor cells in mice and humans. J. Clin. Invest. 118: 4036–4048.

35. Ishikawa, H., K. Tanaka, E. Kutsukake, T. Fukui, H. Sasaki, A. Hata, S. Noda,and T. Matsumoto. 2010. IFN-g production downstream of NKT cell activationin mice infected with influenza virus enhances the cytolytic activities of both NKcells and viral antigen-specific CD8+ T cells. Virology 407: 325–332.

36. Le Goffic, R., V. Balloy, M. Lagranderie, L. Alexopoulou, N. Escriou, R. Flavell,M. Chignard, and M. Si-Tahar. 2006. Detrimental contribution of the Toll-likereceptor (TLR)3 to influenza A virus-induced acute pneumonia. PLoS Pathog. 2:e53.

37. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki,M. Kanno, and M. Taniguchi. 1997. Requirement for Valpha14 NKT cells in IL-12-mediated rejection of tumors. Science 278: 1623–1626.

38. Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, and L. VanKaer. 1997. CD1d1 mutant mice are deficient in natural T cells that promptlyproduce IL-4. Immunity 6: 469–477.

39. Guillot, L., R. Le Goffic, S. Bloch, N. Escriou, S. Akira, M. Chignard, and M. Si-Tahar. 2005. Involvement of toll-like receptor 3 in the immune response of lungepithelial cells to double-stranded RNA and influenza A virus. J. Biol. Chem.280: 5571–5580.

40. Paget, C., T. Mallevaey, A. O. Speak, D. Torres, J. Fontaine, K. C. Sheehan,M. Capron, B. Ryffel, C. Faveeuw, M. Leite de Moraes, et al. 2007. Activation ofinvariant NKT cells by toll-like receptor 9-stimulated dendritic cells requirestype I interferon and charged glycosphingolipids. Immunity 27: 597–609.

41. Jakubzick, C., J. Helft, T. J. Kaplan, and G. J. Randolph. 2008. Optimization ofmethods to study pulmonary dendritic cell migration reveals distinct capacitiesof DC subsets to acquire soluble versus particulate antigen. J. Immunol. Methods337: 121–131.

42. Kim, T. S., and T. J. Braciale. 2009. Respiratory dendritic cell subsets differ intheir capacity to support the induction of virus-specific cytotoxic CD8+ T cellresponses. PLoS ONE 4: e4204.

43. Chiba, A., C. C. Dascher, G. S. Besra, and M. B. Brenner. 2008. Rapid NKT cellresponses are self-terminating during the course of microbial infection. J.Immunol. 181: 2292–2302.

44. Kim, S., S. Lalani, V. V. Parekh, T. L. Vincent, L. Wu, and L. Van Kaer. 2008.Impact of bacteria on the phenotype, functions, and therapeutic activities ofinvariant NKT cells in mice. J. Clin. Invest. 118: 2301–2315.

45. Parekh, V. V., M. T. Wilson, D. Olivares-Villagomez, A. K. Singh, L. Wu,C. R. Wang, S. Joyce, and L. Van Kaer. 2005. Glycolipid antigen induces long-term natural killer T cell anergy in mice. J. Clin. Invest. 115: 2572–2583.

46. Belz, G. T., W. Xie, J. D. Altman, and P. C. Doherty. 2000. A previously un-recognized H-2D(b)-restricted peptide prominent in the primary influenza Avirus-specific CD8(+) T-cell response is much less apparent following secondarychallenge. J. Virol. 74: 3486–3493.

47. Gabrilovich, D. I., and S. Nagaraj. 2009. Myeloid-derived suppressor cells asregulators of the immune system. Nat. Rev. Immunol. 9: 162–174.

48. Belz, G. T., C. M. Smith, L. Kleinert, P. Reading, A. Brooks, K. Shortman,F. R. Carbone, and W. R. Heath. 2004. Distinct migrating and nonmigratingdendritic cell populations are involved in MHC class I-restricted antigen pre-sentation after lung infection with virus. Proc. Natl. Acad. Sci. USA 101: 8670–8675.

49. GeurtsvanKessel, C. H., M. A. Willart, L. S. van Rijt, F. Muskens, M. Kool,C. Baas, K. Thielemans, C. Bennett, B. E. Clausen, H. C. Hoogsteden, et al.2008. Clearance of influenza virus from the lung depends on migratory langerin+CD11b- but not plasmacytoid dendritic cells. J. Exp. Med. 205: 1621–1634.

50. Legge, K. L., and T. J. Braciale. 2003. Accelerated migration of respiratorydendritic cells to the regional lymph nodes is limited to the early phase ofpulmonary infection. Immunity 18: 265–277.

51. Dunne, P. J., B. Moran, R. C. Cummins, and K. H. Mills. 2009. CD11c+CD8alpha+ dendritic cells promote protective immunity to respiratory infectionwith Bordetella pertussis. J. Immunol. 183: 400–410.

52. del Rio, M. L., J. I. Rodriguez-Barbosa, E. Kremmer, and R. Forster. 2007.CD103- and CD103+ bronchial lymph node dendritic cells are specialized inpresenting and cross-presenting innocuous antigen to CD4+ and CD8+ T cells. J.Immunol. 178: 6861–6866.

53. Sung, S. S., S. M. Fu, C. E. Rose, Jr., F. Gaskin, S. T. Ju, and S. R. Beaty. 2006. Amajor lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cellpopulation expressing Langerin and tight junction proteins. J. Immunol. 176:2161–2172.

54. Lambrecht, B. N., M. De Veerman, A. J. Coyle, J. C. Gutierrez-Ramos,K. Thielemans, and R. A. Pauwels. 2000. Myeloid dendritic cells induce Th2responses to inhaled antigen, leading to eosinophilic airway inflammation. J.Clin. Invest. 106: 551–559.



http://ww

w.jim

munol.org/

Dow

nloaded from


55. Fujii, S., K. Shimizu, H. Hemmi, and R. M. Steinman. 2007. Innate Valpha14(+)natural killer T cells mature dendritic cells, leading to strong adaptive immunity.Immunol. Rev. 220: 183–198.

56. Joyee, A. G., H. Qiu, Y. Fan, S. Wang, and X. Yang. 2008. Natural killer T cellsare critical for dendritic cells to induce immunity in Chlamydial pneumonia. Am.J. Respir. Crit. Care Med. 178: 745–756.

57. Tupin, E., Y. Kinjo, and M. Kronenberg. 2007. The unique role of natural killerT cells in the response to microorganisms. Nat. Rev. Microbiol. 5: 405–417.

58. Raftery, M. J., F. Winau, T. Giese, S. H. Kaufmann, U. E. Schaible, andG. Schonrich. 2008. Viral danger signals control CD1d de novo synthesis andNKT cell activation. Eur. J. Immunol. 38: 668–679.

59. Raftery, M. J., F. Winau, S. H. Kaufmann, U. E. Schaible, and G. Schonrich.2006. CD1 antigen presentation by human dendritic cells as a target for herpessimplex virus immune evasion. J. Immunol. 177: 6207–6214.

60. Chen, N., C. McCarthy, H. Drakesmith, D. Li, V. Cerundolo, A. J. McMichael,G. R. Screaton, and X. N. Xu. 2006. HIV-1 down-regulates the expression ofCD1d via Nef. Eur. J. Immunol. 36: 278–286.

61. Cho, S., K. S. Knox, L. M. Kohli, J. J. He, M. A. Exley, S. B. Wilson, andR. R. Brutkiewicz. 2005. Impaired cell surface expression of human CD1d by theformation of an HIV-1 Nef/CD1d complex. Virology 337: 242–252.

62. Renukaradhya, G. J., T. J. Webb, M. A. Khan, Y. L. Lin, W. Du, J. Gervay-Hague, and R. R. Brutkiewicz. 2005. Virus-induced inhibition of CD1d1-mediated antigen presentation: reciprocal regulation by p38 and ERK. J.Immunol. 175: 4301–4308.

63. Sanchez, D. J., J. E. Gumperz, and D. Ganem. 2005. Regulation of CD1d ex-pression and function by a herpesvirus infection. J. Clin. Invest. 115: 1369–1378.

64. Webb, T. J., R. A. Litavecz, M. A. Khan, W. Du, J. Gervay-Hague,G. J. Renukaradhya, and R. R. Brutkiewicz. 2006. Inhibition of CD1d1-mediatedantigen presentation by the vaccinia virus B1R and H5R molecules. Eur. J.Immunol. 36: 2595–2600.

65. Yuan, W., A. Dasgupta, and P. Cresswell. 2006. Herpes simplex virus evadesnatural killer T cell recognition by suppressing CD1d recycling. Nat. Immunol.7: 835–842.

66. Lisbonne, M., S. Diem, A. de Castro Keller, J. Lefort, L. M. Araujo, P. Hachem,J. M. Fourneau, S. Sidobre, M. Kronenberg, M. Taniguchi, et al. 2003. Cuttingedge: invariant V alpha 14 NKT cells are required for allergen-induced airwayinflammation and hyperreactivity in an experimental asthma model. J. Immunol.171: 1637–1641.

67. Pichavant, M., S. Goya, E. H. Meyer, R. A. Johnston, H. Y. Kim,P. Matangkasombut, M. Zhu, Y. Iwakura, P. B. Savage, R. H. DeKruyff, et al. 2008.Ozone exposure in a mouse model induces airway hyperreactivity that requires thepresence of natural killer T cells and IL-17. J. Exp. Med. 205: 385–393.

68. Spence, P. M., V. Sriram, L. Van Kaer, J. A. Hobbs, and R. R. Brutkiewicz. 2001.Generation of cellular immunity to lymphocytic choriomeningitis virus is in-dependent of CD1d1 expression. Immunology 104: 168–174.

69. van Dommelen, S. L., H. A. Tabarias, M. J. Smyth, and M. A. Degli-Esposti.2003. Activation of natural killer (NK) T cells during murine cytomegalovirusinfection enhances the antiviral response mediated by NK cells. J. Virol. 77:1877–1884.

70. Draghi, M., A. Pashine, B. Sanjanwala, K. Gendzekhadze, C. Cantoni,D. Cosman, A. Moretta, N. M. Valiante, and P. Parham. 2007. NKp46 andNKG2D recognition of infected dendritic cells is necessary for NK cell acti-vation in the human response to influenza infection. J. Immunol. 178: 2688–2698.

71. Mandelboim, O., N. Lieberman, M. Lev, L. Paul, T. I. Arnon, Y. Bushkin,D. M. Davis, J. L. Strominger, J. W. Yewdell, and A. Porgador. 2001. Recog-nition of haemagglutinins on virus-infected cells by NKp46 activates lysis byhuman NK cells. Nature 409: 1055–1060.

72. Maghazachi, A. A. 2010. Role of chemokines in the biology of natural killercells. Curr. Top. Microbiol. Immunol. 341: 37–58.

73. Moskophidis, D., and D. Kioussis. 1998. Contribution of virus-specific CD8+cytotoxic T cells to virus clearance or pathologic manifestations of influenzavirus infection in a T cell receptor transgenic mouse model. J. Exp. Med. 188:223–232.

74. Ko, H. J., J. M. Lee, Y. J. Kim, Y. S. Kim, K. A. Lee, and C. Y. Kang. 2009.Immunosuppressive myeloid-derived suppressor cells can be converted intoimmunogenic APCs with the help of activated NKT cells: an alternative cell-based antitumor vaccine. J. Immunol. 182: 1818–1828.

75. Aldridge, J. R., Jr., C. E. Moseley, D. A. Boltz, N. J. Negovetich, C. Reynolds,J. Franks, S. A. Brown, P. C. Doherty, R. G. Webster, and P. G. Thomas. 2009.TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenzavirus infection. Proc. Natl. Acad. Sci. USA 106: 5306–5311.

76. McGill, J., and K. L. Legge. 2009. Cutting edge: contribution of lung-residentT cell proliferation to the overall magnitude of the antigen-specific CD8 T cellresponse in the lungs following murine influenza virus infection. J. Immunol.183: 4177–4181.

77. McGill, J., N. Van Rooijen, and K. L. Legge. 2008. Protective influenza-specificCD8 T cell responses require interactions with dendritic cells in the lungs. J.Exp. Med. 205: 1635–1646.

78. Beauchamp, N. M., R. Y. Busick, and M. A. Alexander-Miller. 2010. Functionaldivergence among CD103+ dendritic cell subpopulations following pulmonarypoxvirus infection. J. Virol. 84: 10191–10199.

79. Semmling, V., V. Lukacs-Kornek, C. A. Thaiss, T. Quast, K. Hochheiser,U. Panzer, J. Rossjohn, P. Perlmutter, J. Cao, D. I. Godfrey, et al. 2010. Alter-native cross-priming through CCL17-CCR4-mediated attraction of CTLs towardNKT cell-licensed DCs. Nat. Immunol. 11: 313–320.

80. Taraban, V. Y., S. Martin, K. E. Attfield, M. J. Glennie, T. Elliott, D. Elewaut,S. Van Calenbergh, B. Linclau, and A. Al-Shamkhani. 2008. Invariant NKT cellspromote CD8+ cytotoxic T cell responses by inducing CD70 expression ondendritic cells. J. Immunol. 180: 4615–4620.



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Corrections

Paget, C., S. Ivanov, J. Fontaine, F. Blanc, M. Pichavant, J. Renneson, E. Bialecki, J. Pothlichet, C. Vendeville, G. Barba-Speath,M.-R. Huerre, C. Faveeuw, M. Si-Tahar, and F. Trottein. 2011. Potential role of invariant NKT cells in the control of pulmonary in-flammation and CD81 T cell response during acute influenza A virus H3N2 pneumonia. J. Immunol. 186: 5590–5602.

The tenth author’s name was published incorrectly. The correct name is Giovanna Barba-Spaeth.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1190034

Copyright � 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

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Influenza A Virus H3N2 Pneumonia T Cell Response during Acute + ...

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