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Human Dendritic CellsDifferentiation of CD40 Ligand-Activated Measles Virus Induces Abnormal

Daniel Hanau, Alain Fischer and Chantal Rabourdin-CombeBausinger, Serge Manié, Françoise Le Deist, Olga Azocar, Christine Servet-Delprat, Pierre-Olivier Vidalain, Huguette

http://www.jimmunol.org/content/164/4/1753doi: 10.4049/jimmunol.164.4.1753

2000; 164:1753-1760; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/164/4/1753.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Measles Virus Induces Abnormal Differentiation of CD40Ligand-Activated Human Dendritic Cells1

Christine Servet-Delprat,* Pierre-Olivier Vidalain,* Huguette Bausinger,† Serge Manie,‡

Francoise Le Deist,§ Olga Azocar,* Daniel Hanau,† Alain Fischer,§ andChantal Rabourdin-Combe2*

Measles virus (MV) infection induces a profound immunosuppression responsible for a high rate of mortality in malnourishedchildren. MV can encounter human dendritic cells (DCs) in the respiratory mucosa or in the secondary lymphoid organs. Thepurpose of this study was to investigate the consequences of DC infection by MV, particularly concerning their maturation andtheir ability to generate CD81 T cell proliferation. We first show that MV-infected Langerhans cells or monocyte-derived DCsundergo a maturation process similarly to the one induced by TNF-a or LPS, respectively. CD40 ligand (CD40L) expressed onactivated T cells is shown to induce terminal differentiation of DCs into mature effector DCs. In contrast, the CD40L-dependentmaturation of DCs is inhibited by MV infection, as demonstrated by CD25, CD69, CD71, CD40, CD80, CD86, and CD83 expres-sion down-regulation. Moreover, the CD40L-induced cytokine pattern in DCs is modified by MV infection with inhibition of IL-12and IL-1a/b and induction of IL-10 mRNAs synthesis. Using peripheral blood lymphocytes from CD40L-deficient patients, wedemonstrate that MV infection of DCs prevents the CD40L-dependent CD81 T cell proliferation. In such DC-PBL cocultures,inhibition of CD80 and CD86 expression on DCs was shown to require both MV replication and CD40 triggering. Finally, for thefirst time, MV was shown to inhibit tyrosine-phosphorylation level induced by CD40 activation in DCs. Our data demonstrate thatMV replication modifies CD40 signaling in DCs, thus leading to impaired maturation. This phenomenon could play a pivotal rolein MV-induced immunosuppression. The Journal of Immunology,2000, 164: 1753–1760.

M easles virus (MV)3 infection is responsible for anacute childhood disease which remains the fourthcause of infant mortality in the world. Paradoxically,

the development of the MV-specific response, which establishesefficient long-term immunity, is associated with a transient butprofound immunosuppression. The latter persists several weeksafter infection and contributes to the high frequency of opportu-nistic infections. MV infection has been involved in decrease of

tuberculin skin reactivity, inhibition of Ab response toSalmonellatyphi vaccine, reduced proliferation capacity of T and B lympho-cytes in response to mitogens, and dysregulation of cytokine re-sponses with a Th2 polarization (1). Moreover, in vitro studieshave suggested that both lymphocytes and APCs might be in-volved in MV-induced immunosuppression (2, 3). MV-infectedDCs become unable to induce both allogeneic and syngeneic T cellproliferation (4–6). MV infection of monocytes and dendritic cells(DCs) inhibits their ability to secrete IL-12 (3, 4). Infected T cells,monocytes, and DCs die by apoptosis (4, 7, 8).

DCs belong to a family of professional APCs responsible for thegeneration of effector CD41 and CD81 T cells (9). They originatefrom CD341 bone marrow progenitors. Immature DCs form a net-work within all epithelia, as Langerhans cells (LCs) in the skin orDCs in the respiratory mucosa. These immature DCs are able tocapture particular Ags via phagocytosis (10) and soluble Ags viamacropinocytosis or receptor-mediated endocytosis (11). They ex-press low levels of MHC class II (MHC-II) molecules at their cellsurface. To become a potent APC, the immature DCs need to beactivated by stimuli that promote their maturation and migration tothe T cell areas of lymphoid tissues. Living bacteria, microbialproducts (LPS), or various cytokines (TNF-a, GM-CSF, IL-1b)stimulate DC maturation. Upon maturation, MHC-II molecules aredelivered to the plasma membrane (12) and the expression of co-stimulatory membrane molecules is increased, thus favoring T cellactivation (13).

When the mature DCs reach secondary lymphoid organs, theyinteract with T cells, receiving signals which induce their terminaldifferentiation into mature effector DCs. CD40-CD40 ligand(CD40L) interaction between DCs and T cells is essential for anoptimal cytokine production. The best-known consequence ofCD40 ligation is the IL-12 production by DCs (14, 15). In human,

*Immunobiologie Fondamentale et Clinique, Institut National de la Sante´ et de laRecherche Medicale U503, Ecole Normale Superieur Lyon, Lyon, France;†Biologiedes Cellules Dendritiques Humaines, Institut National de la Sante et de la RechercheMedicale E 9908, Etablissement de Transfusion Sanguine Strasbourg, Strasbourg,France;‡Immunite et infections virales, Faculte´ de medecine Laennec, IVMC-CentreNational de la Recherche Scientifique-Universite Claude Bernard Lyon Unite Mixtede Recherche 5537, Lyon, France; and§Developpement Normal et Pathologique duSysteme Immunitaire, Institut National de la Sante et de la Recherche Medicale U429,Hopital Necker-Enfants Malades, Paris, France

Received for publication August 9, 1999. Accepted for publication December 2, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by institutional grants from the Institut National de laSante et de la Recherche Medicale, from Ministene de l’Education Nationale et de laRecherche Technologique, from the Agence Francaise du Sang (FORTS 96) and byadditional support from Association pour la Recherche sur le Cancer (CRC 6108 andSM 9501), Ligue Nationale Contre le Cancer, Programme de Recherche Fondamen-tale en Microbiologie et Maladies infectieuses et Parasitaires, and RegionRhone-Alpes.2 Address correspondence and reprint requests to Prof. Chantal Rabourdin-Combe,Institut National de la Sante et de la Recherche Medicale U503, ImmunobiologieFondamentale et Clinique, Ecole Normale Superieure de Lyon, 69364 Lyon cedex 07,France. E-mail address: crabourd@ens-lyon.fr3 Abbreviations used in this paper: MV, measles virus; CD40L, CD40 ligand;CD40L1-L cells, CD40L-transfected L cells; DC, dendritic cell; LC, Langerhans cell;MHC-I/MHC-II, MHC class I/II; MFI, mean fluorescence intensity; NP, MV nucleo-protein; Mo-DC, monocyte-derived DC; UVMV, UV-inactivated MV; rh, recombi-nant human.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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the X-linked immunodeficiency hyper-IgM syndrome has been at-tributed to mutations in the CD40L gene (16). Over the past year,it was recognized that the function of CD40 accounts not only forthe regulation of T-dependent humoral immune responses, but alsofor cellular immune responses (17). Several immune dysfunctionsobserved in CD40L-deficient mice and patients could be explainedby a failure properly to activate APCs (18–20). Recent in vivostudies in mouse demonstrated that CD40 ligation on the DCs canreplace CD41 T cells to prime CD81 cytotoxic responses (21–23).

The mechanisms by which MV infection interferes with thefunctions of DCs remained unknown. In this study, we confirm(24) and further extend that MV replication induces normal mat-uration of immature monocyte-derived DCs and LCs. But, weshow that MV replication leads to an abnormal terminal differen-tiation of CD40L-activated human DCs. Impairment of CD40/CD40L signaling following MV infection was demonstrated byinhibition of tyrosine-phosphorylation level in MV-infected DCsafter CD40 activation. This could explain why DCs display im-paired APC functions and may consequently promote MV-inducedimmunosuppression.

Materials and MethodsReagents

CD1a-PE (BL6), CD3-PE (UCHT1), CD25-PE (B1.49.9), CD32-PE(2E1), CD45RO-PE (UCHL1), CD45RA-PE (ALB11), CD69-PE(TP1.55.3), CD71-FITC (YDJ1.2.2), CD80-FITC (MAB104), CD83-PE (HB15a), CD86-FITC (HA5.2B7), E-cadherin (67A4), and HLA-DR-FITC (B8.12.2) Abs were purchased from Immunotech (Marseille,France); MHC-I (W6/32), CD8-FITC (DK25), and CD4-FITC (MT310)Abs were from DAKO (Glostrup, Denmark); CD40-PE (LOB7/6),CD86 (BU63), and CD11c-FITC (3.9) Abswere from Serotec (Oxford,U.K.); and CD80-PE (L307.4) and MHC-II (L243) were from BectonDickinson Immunocytochemistry Systems (San Jose, CA). A FITC-con-jugated IgG1/PE-conjugated IgG2a irrelevant Ab mixture (Immunotech)was used as isotype controls. Mouse Abs specific for CD46 (20.6; Ref. 25)was produced in our lab. FITC-conjugated, affinity-isolated F(ab9)2 fractionof a sheep anti-mouse Ig Ab (Silenus, Hawthorn, Victoria, Australia) wasused for indirect immunofluorescence labeling procedures. LPS (Esche-richia coli serotype O127:B8) was purchased from Sigma (St. Louis, MO).Recombinant human (rh)GM-CSF and IL-4 were generously provided bythe Schering-Plough Laboratory for Immunological Research (Dardilly,France), whereas rhSCF, rhTNF-a, and purified hTGF-b1 were obtainedfrom R&D Systems (Abingdon, Oxon, U.K.).

Patients

Two patients suffering from X-linked hyper-IgM syndrome were includedin this study. Mutations in the CD40L gene were characterized and led tothe absence of CD40L expression. Informed consent was obtained fromeach patient family for this study.

Cells

Immature LCs were generated in vitro from CD341 progenitors. Positiveselection of CD341 cells was performed as previously described (26).Briefly, PBMC were collected by cytapheresis from myeloma patients whohad received high dose chemotherapy and hematopoietic growth factors(G-CSF or GM-CSF). Informed consent was obtained from all patientsbefore cytapheresis. Hematopoietic progenitors expressing the CD34 Agwere purified (926 2.3%) using the Ceprate LC34-Biotin Kit (CellPro,Bothell, WA) according to the cell separation procedure instructions of themanufacturer. A total of 23 104 purified CD341 cells/ml were cultured in75-ml tissue culture flasks (Falcon 3111, Becton Dickinson Labware,Franklin Lakes, NJ) in serum-free StemPro-34 complete medium (LifeTechnologies, Grand Island, NY) supplemented withL-glutamine (2 mM,Life Technologies), gentamicin (50mg/ml, Life Technologies), rhSCF (20ng/ml), rhTNF-a (0.5 ng/ml), rhGM-CSF (200 ng/ml), and hTGF-b1 (0.5ng/ml). After 7 days of culture the cell suspensions contained 10% CD1a1

cells, with 70–98% of these expressing CD1a and E-cadherin, two markersspecifically found on the immature LCs of the epidermis (13).

Monocyte-derived DCs (Mo-DCs) were generated in vitro, as previ-ously described (4). After 6 days of culture in the presence of 50 ng/mlhrGM-CSF and 500 U/ml hrIL-4,.95% of the cells were DCs as assessedby CD1a labeling. Cultures of the Mo-DCs were performed in 24-well

flat-bottom microtiter plates (Falcon), in a total volume of 1 ml, in RPMI1640 (Life Technologies) supplemented with 10 mM HEPES (Life Tech-nologies), 2 mML-glutamine (Life Technologies), 40mg/ml gentamicin(Life Technologies), and 10% FCS (Boehringer Mannheim, Meylan,France).

PBL were activated with a combination of 10 ng/ml PMA (Sigma) and1 mg/ml ionomycin (Sigma) for 6–12 h. This short-time activation wasprovided to induce CD40L expression by CD41 T cells, but no cytokinesecretion and a limited background proliferation (5,000 cpm) comparedwith DC-dependent allogenic proliferation (80,000 cpm) of PBL. Afteractivation, PBL were washed three times. DCs alone were cultured at 106

cells/ml. In PBL cocultures, 0.53 106 DCs/ml were cultured together with0.5 3 106 activated PBL. In the murine fibroblast cocultures, 106 DCs/mlwere cultured in the presence of 105 irradiated (7000 rads) fibroblasticCD40L or CD32-transfected L cells (both kindly provided by Schering-Plough Laboratory for Immunological Research).

MV infection and detection

Mo-DCs and LCs were infected, at day 6 and 7, respectively, with 1 PFU/cell of Vero cell-derived MV Halle (Halle strain is classified with thevaccine MV strain Edmonston (27)) or pulsed with 1 PFU/cell of MVneutralized by 254 nm UV rays for 30 min (UVMV) or mock-infected.After a 3-h incubation at 37°C, the DCs were washed three times to be freeof unattached virus then put in culture. For CD40 stimulation and detectionof tyrosine phosphorylation, DCs were previously infected with 4 PFU/cell; all of the DCs were infected and 15% were dead by apoptosis 24 hlater when CD40 stimulation was performed. For PFU measurement, viruscontents were quantified by limiting dilution from 10 to 10 until 10210 onconfluent Vero cells. A single plaque in the Vero cells confluent culturerepresents 1 PFU generated by an individual infectious virus. For MVnucleoprotein (NP) staining, after 15 min of permeabilization with 0.33%Saponin (Sigma), cells were stained with anti-NP viral protein mAb (clone25) kindly provided by F. Wild, followed by incubation with PE-labeledanti-mouse Ig (Immunotech). The apoptosis rate differed between cultureconditions according to labeling of fragmented DNA after TUNEL stain-ing: at day 3, 50% of DCs were apoptotic when they are alone or coculturedwith L cells, whereas 80% were apoptotic in DC-PBL cocultures (data notshown).

Phenotypic analysis

All immunostaining were performed in 1% BSA and 3% human serum-PBS. Direct immunostainings were performed using 2mg/ml of FITC-conjugated or PE-conjugated Abs. Indirect immunostainings were per-formed using 2mg/ml of the first mouse mAb and revealed with 2mg/mlof the FITC-conjugated, affinity-isolated F(ab9)2 fraction of a sheep anti-mouse Ig Ab. Viable DCs were gated according to negative staining withpropidium iodide.

RNase protection assays

RNA was extracted from 107 treated Mo-DCs using RNA NOW-TC re-agent (Biogentex, Seabrook, TX). The RNase protection was performedusing 4mg of RNA with the RiboQuant multiprobe RNase assay system(PharMingen, San Diego, CA), following the manufacturer’s specification.In brief, RNA was hybridized overnight with the in vitro-translated32P-labeled probe (hCK-2 kits, PharMingen). Following hybridization, sampleswere treated with RNase A1T1 and proteinase K, phenol-chloroform ex-tracted, and ethanol precipitated. The protected fragments were resolved byelectrophoresis on a 5% acrylamide/urea gel and exposed on a PhosphorScreen (Molecular Dynamics, Sunnyvale, CA) for 12 h to quantify theintensity of the bands with ImageQuant (Molecular Dynamics).

Cell counts

CD41 and CD81-labeled T cells in DC-PBL cocultures were numbered bya time-monitored FACS analysis during 2 min at high speed (1ml/s). Asthe CD8 percentages differed in PBL used (34% in healthy donors, 8% and20% in CD40L-deficient patients), results of Fig. 2 were calculated for 53104 CD81 T cells put in culture at day 0.

CD40 stimulation and detection of tyrosine phosphorylation

DCs were stimulated with 10mg/ml of monoclonal anti-CD40 (mAb 89)generously provided by the Schering-Plough Laboratory for Immunologi-cal Research or irrelevant IgG1 control Abs (Sigma) for 10 min at 37°C.Stimulation was terminated by lysis in RIPA buffer (150 mM NaCl, 50 mMTris-HCl (pH 8.0), 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS)containing 5 mM EGTA, 1 mM Na vanadate, and a mixture of proteaseinhibitors (complete, Boehringer Mannheim) for 15 min at 4°C. Insoluble

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material was removed by centrifugation at 10,0003 g for 10 min. Proteinsfrom cell lysates were separated by SDS-PAGE under reducing conditionsand transferred to Immobilon-P membranes (Millipore, Bedford, MA).Membranes were blocked using 5% nonfat dried milk in TBS-T (20 mMTris (pH 7.6), 130 mM NaCl, 0.1% Tween 20) and incubated for 1 h withthe anti-P-Tyr Ab 4G10 (Upstate Biotechnology, Lake Placid, NY) inTBS-T. Immunoreactive bands were visualized by using secondary horse-radish peroxidase-conjugated Abs (Promega, Madison, WI) and chemilu-minescence (ECL, Amersham. Little Chalfont, U.K.).

ResultsMV replication in immature DCs6 PBL

To show that MV replicates in immature DCs, FACS analysis ofNP staining and measurement of infectious virus particles wereperformed. NP is the earliest MV protein transcribed during viralcell cycle and its amount, measured by mean fluorescence intensity(MFI), reflects the intensity of viral replication in infected cells.Immature LCs or Mo-DCs were MV-infected. Mo-DCs were cul-tured for 5 days alone or with CD40L1-L cells or normal PBL orCD40L-deficient PBL. At day 3 of culture, 12% of LCs and around50% of DCs were NP1, but normal PBL or CD40L1-L cells en-hanced MFI of NP1 DCs (Table I, columns 1 and 2). At day 5,

PFU were measured in supernatants of culture. We have previ-ously shown that CD40L1 T cells enhance viral production byDCs (4). In MV DC-CD40L-deficient PBL cocultures, the absenceof CD40L decreased PFU measured in supernatant. High MV pro-duction was restored by addition of CD40L1-L cells (Table I,column 3). Thus, high MV replication in immature DCs correlateswith CD40 triggering.

Phenotypic maturation of LCs and monocyte-derived DCs isinduced by MV replication

Physiologically, MV may encounter immature DCs at its entry sitein the respiratory mucosa. As previously described (24), immatureDCs isolated from peripheral blood up-regulated MHC-II, CD83,and CD86. LCs can be used as a model for epithelial immatureDCs functionally close to the respiratory tract DCs (28). ImmatureLCs were MV-infected, then cultured for three days (Table II, leftcolumns 1–3). At day 3, immature LCs were positive for E-cad-herin, MHC-II, CD1a, and CD80, and negative for CD86 andCD83. In contrast, after MV replication, E-cadherin and CD1awere down-regulated, MHC-II and CD80 were up-regulated,

Table I. MV replication in immature DCs6 PBLa

% NP1 DC MFI of NP1 DC PFU/Million of DCs

LC 126 1.6 986 10 NDMo-DC 506 3.2 1036 7 2,6006 230Mo-DC 1 CD40L1-L cells 556 4.2 5086 43 53,2006 4,200Mo-DC 1 PBL 506 5.6 4326 44 49,4006 5,100Mo-DC 1 CD40L-deficient PBL 496 6.1 1156 15 10,1006 1,600Mo-DC 1 CD40L-deficient PBL1

CD40L1-L cells516 4.9 4526 38 50,3006 4,700

a Columns 1 and 2: At day 3 of culture, LC or Mo-DC were double stained with FITC-anti-CD1a/PE-anti-NP or FITC-MHCII/PE-anti-NP, respectively. Percentages and mean fluorescence intensity (MFI) of NP1 DCs are shown. Column 3: At day 5 ofculture, MV productions in the culture supernatant were measured by PFU on Vero cell layer. Mean6 SD of three separateexperiments. ND, not done.

Table II. Phenotype of MV-infected immature DCs in absence or in presence of CD40L signala

Absence of CD40Lb Presence of CD40Lc

Langerhans cells Mo-DC Mo-DC

Alone TNF-a MV Alone LPS MV CD40LLPS 1CD40L

MV 1CD40L

E-cadherin* 111 111 6MHC-I* 11 111 111 MHC-I* 11 11 11MHC-II* 1 111 11 11 111 111 MHC-II* 111 111 111CD1a 111 11 6 11 2 2 CD1a 2 2 2CD25 2 11 111 CD25 11 11 2CD69 2 1 11 CD69 111 111 2CD71 2 11 6 CD71 11 11 2CD11c* 11 1 1 CD11c* 1 1 1CD32 6 2 2 CD32 2 2 2CD40* 1 11 111 CD40* 11 11 6CD80* 1 111 111 1 111 111 CD80* 111 111 6CD86 2 1 111 2 11 111 CD86 11 11 6CD83 2 6 11 2 11 11 CD83 11 11 2CD46* 11 11 1 CD46* 11 11 1CD4 1 1 1 CD4 1 1 1

a 2, 1, 11, 111 are used to gradually notify the percentage of cells from 0 to 100% expression of the designed marker analyzed by FACS;6 is used when the phenotypepresented two subpopulations; *, notifies that 100% of cells were positive for staining; in that case,2, 1, 11, 111 reflects the MFI from low to high expression.

b Enclosed signs are mature phenotype of DCs. Immature LCs were uninfected or infected with MV, then cultured for 3 days. LCs cultured in the presence of TNF-a (50ng/ml) were used as maturation control. Double stainings on CD80 or CD1a were performed. The expressions of E-cadherin, MHC-II HLA-DR, CD1a, CD86, CD83, on gatedCD80-positive viable LCs, and the expression of CD80 on gated CD1a-positive viable LCs are shown. Data shown are from one representative experiment out of six performedon six different donors; SD were below 20%. Immature Mo-DCs were uninfected or infected with MV, then cultured for 3 days. DCs cultured in the presence of LPS (1mg/ml)were used as maturation control. Data shown are from one representative experiment out of eight performed on eight different donors; SD were below 15%.

c Enclosed signs are abnormal phenotype of mature DCs. Immature Mo-DCs were uninfected or LPS-activated or MV-infected, then CD40-activated and cultured for 3 days.Data shown are mean of six experiments performed on six different donors; SD were below 15%.

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whereas CD86 and CD83 were induced. Mock supernatant andUVMV did not induce phenotypic maturation (data not shown).Regarding CD1a, CD86, and CD83 expressions, MV replicationinduced stronger LC maturation than TNF-a.

Similar experiments were repeated using Mo-DCs as a source ofimmature DCs. By comparison with immature Mo-DCs phenotypeat day 3 (Table II, left columns 4–6), MV replication down-reg-ulated CD1a, CD11c, and CD32 expression; up-regulated MHC-I,MHC-II, CD80, and CD40 expression; and induced CD25, CD69,CD71, CD86, and CD83 expression in Mo-DCs. CD46 were alsodown-regulated as a function of MV production in agreement withthe fact that CD46 is a receptor for MV (29, 30). Throughout the14 markers that were studied, a similar maturation profile wasobserved after MV infection compared with LPS activation. Mocksupernatant and UVMV did not induce Mo-DC maturation (datanot shown). On the basis of this phenotypic study, we confirm andfurther extend to the LCs and Mo-DCs that MV replication inducesmaturation of immature DCs.

CD40-induced DC maturation is impaired by MV infection

Although MV-induced DC maturation was similar to LPS-inducedDC maturation, MV-infected DCs are deficient in APC functions(4, 5) in contrast to LPS-activated DCs. The CD40L expressed byactivated T cells is a potent signal to induce terminal differentiationof DCs in mature professional APCs. We compared the conse-quences of LPS activation vs MV infection of DCs for the inte-gration of CD40L signal. Effective CD40 activation of B cells (31)and DCs (4) were previously shown by using CD40L-transfectedmouse fibroblasts (CD40L1-L cells). Mo-DCs were either LPS-activated or MV-infected for 3 h, then cocultured with CD40L1-Lcells for 3 days (Table II, right). As compared with immature DCs(Table II, left), CD40 ligation alone or CD40 ligation of LPS-activated DCs showed a typical mature phenotype with highMHC-I, MHC-II, CD25, CD69, CD71, CD40, CD80, CD86, andCD83 expression. In contrast, CD40 ligation of MV-infected DCsinhibited induction of CD25, CD69, CD71, CD86, and CD83 andup-regulation of CD40 and CD80 expression. MV infection alsodown-regulated expression of the CD46/MV receptor. Impaired

phenotype was also observed when CD40 was ligated 48 h beforeinfection, and 24 or 48 h after infection (data not shown). Thus,CD40-dependent maturation of Mo-DCs is inhibited by MVreplication.

CD40-induced cytokine pattern in DCs is modified by MVinfection

We then compared cytokine mRNA productions of uninfected,LPS-activated, MV-infected, CD40L-activated, and MV-infected1 CD40L-activated DCs. Mo-DCs were treated for 3 h, and thencultured for 24 h. RNase protection assay was performed by usingspecific probes to quantify the level of eight cytokine mRNAs(Fig. 1). In immature DCs, only lowlevels of IL-1b and IL-1RAmRNAs were detected. LPS activation induced IL-12p35, IL-12p40,IL-10, IL-1a, and IL-6 mRNAs, whereas IL-1b and IL-1RA mRNAswere enhanced. MV infection weakly induced IL-12p35, IL-12p40,IL-1a, and IL-6 mRNAs, whereasIL-1b and IL-1RA mRNAs wereup-regulated. CD40L signal strongly induced IL-12p40; inducedIL-12p35, IL-1a, and IL-6 mRNAs; and up-regulated IL-1b andIL-1RA mRNAs in immature DCs. MV replication modified theCD40L-induced cytokine pattern: IL-10 mRNA was induced,whereas neither CD40L-activation nor MV infection alone in-duced IL-10 gene transcription. Furthermore, IL-12p35, IL-12p40,and IL-1a/b mRNAs were reduced in MV1CD40L conditioncompared with CD40L activation alone. Thus, MV replication al-ters the cytokine pattern induced by CD40L activation of DCs.

MV infection prevents CD40L-dependent CD81 T lymphocyteproliferation

Our data indicate that MV replication could modify the signaltransduced by CD40L in DCs. To investigate this point, PBL fromhealthy donors or from CD40L-deficient patients were used. Nor-mal T lymphocyte numbers were detected in these patients, but Tcell subpopulations were profoundly affected (Table III), as patientno. 1 had only 8% CD81 T cells and 4% of CD41/CD45RO1

lymphocytes, whereas patient no. 2 presented normal percentagesof CD41 and CD81, but the number of CD45RO1 T cells wasabnormally low.

FIGURE 1. Effect of MV infection on CD40L-induced mRNA cytokine pattern in DCs. Immature Mo-DCs have been uninfected, LPS-activated,MV-infected, CD40L-activated with CD40L1-L cells, or MV-infected and CD40L-activated. After 24 h of culture, RNAs were extracted and used forRNase protection using the hCK-2 probe kit and developed by the PhosphorImager system after 6-h exposition. Local background has been subtracted fromeach signal. The levels of mRNAs were quantified by densitometry and scanning comparison with control probes (GAPDH and L32). The tables are shownfor IL-12p40, IL-10, and IL-1b. Data shown are from one representative experiment (exp 3) of three.

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We first assessed whether, as has been reported in the mouse(21–23), CD40-CD40L interaction was required for maturation ofeffector DCs able to generate CD81 T cells. Preactivated PBLoriginating either from healthy CD40L1 donors or from CD40L-deficient patients were used as sources of T cells. In DC-PBLcocultures, allogeneic DCs induced CD81 T cell proliferation(314.6 at day 5) (Fig. 2A). In contrast, no viable CD81 T cellscould be detected at day 5 when DCs were cocultured withCD40L-deficient PBL. Detection of CD81 T cells at day 5 wasdependent on CD40L because bystander CD40L activation of theDCs by CD40L1-L cells permitted us to recover a high number ofCD81 T cells (331.6 at day 5) in CD40L-deficient PBL. Higherproliferation rate obtained with CD40L1-L cells was attributed tothe higher CD40L expression in transfected L cells than in acti-vated T cells (data not shown). The absence of CD81 T cells in theDC-CD40L-deficient PBL cocultures did not result from an overallinhibition of T cell proliferation. Indeed, PBL from CD40L-defi-cient patients incorporated thymidine and allogeneic CD41 T cellswere detected in increased number (38.9 at day 5; data notshown). Under the culture conditions performed with a short pre-activation of PBL by PMA and ionomycine, CD81 T cell prolif-eration in DC-PBL cocultures was dependent on CD40-activationof DCs by CD40L1 CD41-activated T cells.

We then investigated whether MV replication could impair thisCD40L-dependent CD81 T cell proliferation. Experiments per-formed in Fig. 2Awere repeated with MV-infected DCs (Fig. 2B).When the DCs were infected by MV, CD81 T cell proliferationwas abolished in allogeneic DC-PBL cocultures. Furthermore, theCD81 T cell proliferation obtained by adding CD40L1-L cells in

coculture of DC with CD40L-deficient PBL was inhibited by MVinfection of the DCs.

In conclusion, we have demonstrated that 1) CD40L activationof DCs is required to sustain human CD81 T cell proliferation, invitro, and 2) MV infection of DCs prevents this CD40L-dependentCD81 T cell proliferation.

MV replication impairs CD40 signaling in DCs

To determine whether MV could modify CD40 signaling into theMo-DCs, we studied the expression of membrane Ags that wereinduced or up-regulated by CD40L activation in DCs. DC-PBLcocultures were performed using allogeneic PBL either fromhealthy donors or from CD40L-deficient patients. In the absence ofMV infection (Fig. 3A), DCs cocultured with normal PBL exhib-ited a normal mature phenotype (CD861, CD80high, MHC-IIhigh),whereas DCs cocultured with CD40L-deficient PBL looked like anintermediate stage of maturation with immature-type expression ofCD86. CD86 expression was induced when CD40L1-L cells wereadded to the CD40L-deficient PBL, thus demonstrating that aCD40L signal was required to increase CD86 expression. By con-trast, the CD40L signal, which by itself enhanced CD80 andMHC-II expression (Table II, right), could be replaced by other(s)T cell signal(s) that increased CD80 and MHC-II expressions onDCs (Fig. 3A). When DCs were MV-infected (Fig. 3B), inhibitionof CD86 expression was observed. This latter event occurred evenin the presence of CD40L1-L cells, suggesting a blockade inCD40 signaling. CD80 expression was inhibited only when MV-infected DCs were CD40L activated either with CD40L1-PBL orwith CD40L1-L cells. Thus, even in the presence of other(s) T cellsignal(s) able to up-regulate CD80 expression, CD40 triggering ofMV-infected DCs did not up-regulate CD80 expression. There-fore, both MV replication and CD40 triggering of DCs wereneeded for inhibition of CD80 and CD86 expression. Although thenature of the CD40 signaling pathway in DCs has not been eluci-dated, CD40 signaling in monocytes and B cells has been shownto involve protein tyrosine kinase activity (32). Since we demon-strated that MV infection impairs CD40-induced maturation ofDCs, we investigated whether MV infection influences anti-CD40-induced tyrosine phosphorylation (Fig. 4). The effect of CD40stimulation on overall levels of tyrosine phosphorylation in mock-treated or MV-infected DCs was examined by Western blot anal-ysis of total protein using anti-phosphotyrosine Abs. The enhancedtyrosine phosphorylation was evident in mock-treated DCs after 10min of stimulation with anti-CD40. But MV infection stronglyinhibited anti-CD40 enhanced tyrosine phosphorylation.

DiscussionThis study was realized with MV Halle strain classified with thevaccine MV strain Edmonston. In this paper we show that MVreplication in immature DCs induces phenotypic maturation. Bycontrast, in the presence of CD40L activation, MV replication in

Table III. Subpopulations of T lymphocytes in CD40L-deficient patient, in vivoa

Patient 1 Patient 2 Normal Range

CD3 CD45RA CD45RO CD3 CD45RA CD45RO CD3 CD45RA CD45RO

CD4 77 93 4 58 82 13 35–55 40–60 40–60CD8 8 52 40 20 45 24 15–35 40–60 40–60

a Peripheral blood lymphocytes were double stained for CD3 and CD4 or CD8; for CD4 and CD45RA or CD45RO; and for CD8 and CD45RA orCD45RO. SD of FACS analysis was 1%,n 5 2. The lymphocytes/mm3 for patient 1 are 2800, for patient 2 are 4800, and the normal range is 2000–5000.The % CD3 for patient 1 is 85%, for patient 2 is 78%, and the normal range is 60–80%.

FIGURE 2. Effect of MV replication on CD40L-dependent CD81 Tlymphocytes proliferation, in vitro. Uninfected (A) or MV-infected (B)DCs were cocultured with either normal allogeneic activated PBL, or al-logeneic activated PBL from CD40L-deficient patients. CD40L1-L cellswere used to restore bystander CD40L activation of the DCs. CD32L1-Lcells were used as a control for CD40L1-L cells. Viable CD81 T cellnumber was quantified by FACS at different time points. Results are meansof triplicate experiments with normal PBL (n5 4) or with PBL comingfrom two different CD40L-deficient patients (n5 2).

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Mo-DCs induce an abnormal DC phenotype consisting of a de-creased expression of coactivation membrane molecules (CD40,CD80, CD86) and a lack of activation (CD25, CD69, CD71) andmaturation (CD83) marker expressions. Furthermore, CD40-in-duced cytokine pattern in DCs was modified by MV replicationbecause IL-12 and IL-1a/b mRNA were decreased, whereas IL-10mRNA was induced. Using PBL originating from CD40L-defi-cient patients and CD40L1-L cells, we demonstrate that CD40Lactivation of DCs is required to induce CD81 T cell proliferationin vitro and that MV replication inhibits this CD40L-dependentCD81 T cell proliferation. Finally, the inhibition of CD80 andCD86 expression could be related to the impairment of CD40 sig-naling, which is demonstrated by the inhibition of tyrosine-phos-phorylation level in MV-infected CD40-activated DCs. Therefore,the CD40 triggering of MV-infected DCs could play a pivotal rolein MV-induced immunosuppression.

Networks of DCs are found in tissues that come into closestcontact with the external environment. According to the studies ofHolt et al. (28) in rats, lung wall DCs share some properties of

LCs: they can effectively bind inhaled Ags in situ, at the entry siteof MV, but require additional maturation/activation signals beforethey can efficiently present the Ag to T cells. At the present time,we do not have formal proof that DCs are infected in vivo duringmeasles, but five types of immature DCs are susceptible to MVinfection and actively suppress T cell proliferation in vitro: Mo-DCs (4), CD341-derived DCs (5) that contains two subpopulationsof immature DCs (13), freshly isolated LCs contrary to surround-ing keratinocytes (6), and now CD341-derived LCs. So far, thebest-characterized peripheral DCs are the epidermal LCs. To be-come potent APCs, these immature DCs must undergo a terminaldifferentiation step during the migration from the skin to draininglymph nodes that is induced by various stimuli such as LPS, cy-tokines, or pathogens. Phenotypic DC maturation induced by bac-teria (reviewed in Ref. 33) and parasites (34) is documented. Con-cerning virus-induced DC maturation, poor information isavailable; blood-derived DCs up-regulate MHC-I, MHC-II, CD38,CD83, and CD86 after infection with influenza virus (35). Our dataconfirm previous work (24) and further extend to the LCs andMo-DCs that DC maturation can be obtained with MV. MV in-duces down-regulation of E-cadherin in LCs; this loss of E-cad-herin expression may be of particular relevance for effective mi-gration, because E-cadherin participates in the homophilicadhesion between LCs and keratinocytes in the epidermis (36).Moreover, MV infection of Mo-DCs weakly induces IL-12 andIL-6 mRNAs. Weak IL-12 secretion by MV-infected human bloodDC precursors has been already described (24).

When DCs were infected before CD40-mediated signal, MVreplication decreased expression of coactivation (CD40, CD80,CD86), activation (CD25, CD69, CD71), and maturation (CD83)marker molecules, whereas MHC-I and MHC-II expressions re-mained high. Abnormal phenotype was also observed when Mo-DCs were infected 48 h after CD40 activation (data not shown). Insuch culture conditions, DCs showed normal mature phenotypebefore they were infected. Thus, MV infection is able to revertnormal CD40-induced mature phenotype in DCs. By contrast, MVinfection did not modified LPS-induced mature phenotype in DCs(data not shown), suggesting that MV infection specifically blocksCD40L-mediated DC maturation. Thus, abnormal phenotype in-duced by MV infection seems to be dependent on CD40 triggeringof DCs rather than their maturation stage. Functionally, the highCD40L-induced mRNA synthesis of IL-12 and IL-1a/b were in-hibited by MV infection, whereas IL-10 mRNA synthesis was in-duced. Thus MV infection inhibits the CD40-dependent IL-12 se-cretion by DCs (4) at the transcriptional level. Consequently, the

FIGURE 3. Effect of MV replication onCD40 signaling. Immature Mo-DCs were un-infected (A) or MV-infected (B), then cocul-tured for 3 days with either normal allogeneicactivated PBL or allogeneic activated PBLcoming from CD40L-deficient patients.CD40L1-L cells were used to restore by-stander CD40L-activation of the DCs. FACSanalysis on gated viable DCs was performed.Results are means of two triplicate experi-ments with PBL coming from two differentCD40L-deficient patients.

FIGURE 4. CD40-induced tyrosine phosphorylation of cellular proteinsin DCs and down-regulation by MV infection. DCs were either mock-treated or MV-infected with 4 PFU/cell. After 24 h of culture, DCs werewashed then stimulated by anti-CD40 or control IgG1 Ab (10mg/ml) for10 min. Protein extracts were separated by 10% SDS-PAGE and then trans-ferred to a polyvinylidene difluoride membrane and blotted with either aspecific anti-phosphotyrosine Ab or an anti-b-tubulin Ab as a control ofloaded proteins. Molecular mass standards in kilodaltons are indicated onthe right. Data shown are from one representative experiment of three.

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Th1-type response mediated by IL-12 secretion of CD40-activatedDCs may be inhibited. IL-10 has been involved in the inhibition ofDC maturation and their APC functions (37). IL-10 mRNA syn-thesis is also induced by LPS activation of DCs whether the DCsare CD40 activated or not (data not shown). Yet, LPS bacterialendotoxin is a potent mediator of DC maturation. Therefore thepresence of IL-10 mRNA, induced by CD40 ligation of MV-in-fected DCs, may participate to immunosuppression but cannot ac-count for the various inhibitory effects observed. At a first sight,DC apoptosis induced by MV infection could explain that MV-infected DCs are deficient in APC functions, but several featuresdemonstrate that other mechanisms are involved: 1) cytokinemRNA synthesis was modified in infected DCs as soon as 24 hafter infection DC, whereas massive apoptosis occurred at day 3 ofculture; 2) when DCs were pulsed with UVMV, no apoptosis wasobserved, yet IL-12 secretion by UVMV-pulsed DCs was 30%decreased and T cell proliferation was 30% inhibited; 3) thoughDC apoptosis was delayed 3 days after MV infection, the inhibi-tion of T cell proliferation occurred at the beginning of the culture(4); and 4) abnormal DC phenotype in MV-infected CD40-acti-vated DCs was observed on viable DCs. Thus, MV-induced DCapoptosis certainly reinforces the inability of DCs to maintain theirAPC function, but cannot explain the discrepancies of the APCproperties between normal DCs and MV-infected DCs.

Whatever signal(s) the DCs received to initiate their maturation,the CD40L signal may be needed to achieve their complete mat-uration and thus generate mature effector DCs. CD40L signalingcan be provided by activated T cells in secondary lymphoid or-gans. Hyper-IgM patients, who are CD40L deficient, display clin-ical symptoms suggestive of immune deficiency not limited to Bcells. In particular, the frequent occurrence ofPneumocystis care-nii andCryptosporidiuminfections indicate impaired T cell acti-vation (18). As reported in mouse models (21–23), the lack ofCD40 ligation on DCs prevents the generation of CD81 cytotoxicT cells for certain pathogens. Furthermore, the CD40L signal hasbeen involved in the generation of memory CD81 CTL (38). Thepresent study demonstrates that 1) the lack of CD40L signal, invivo, in two hyper-IgM patients, is not strictly correlated with aCD81 deficiency, but rather with a defective number ofCD45RO1 T cells reported as memory T cells. This is confirmedby a recent study where reduced Ag-primed population has beenobserved in both CD41 and CD81 populations, as determined byCD45RO expression (39). 2) CD40L-deficient activated PBL fromthese patients induce an intermediate stage (CD862 CD801) ofDC phenotypic maturation, in vitro. 3) CD40L activation of DCsis required to activate CD81 T cells proliferation, in vitro. In thecase of MV infection, instead of getting mature effector humanDCs able to activate CD81 T cells proliferation, CD40 triggeringof MV-infected DCs prevents CD81 T cell proliferation.

In DC-PBL cocultures, both CD86 and CD80 expressions wereinhibited by the CD40 triggering of MV-infected DCs. CD86 ex-pression was only up-regulated by CD40L1 T cells. In contrast,CD80 up-regulation was also observed using CD40L-deficient Tcells. Thus, in the absence of MV infection, other unidentified Tcell signals, termed “X,” different from CD40L, can up-regulateCD80 expression on DCs. As MV replication abrogated the Xpathway when DCs are CD40 triggered, we propose that the neg-ative effect induced by CD40 triggering of MV-infected DCs isdominant and abrogated the X pathway. As a whole, these datasuggest that CD40 triggering of MV-infected DCs leads to an in-hibitory signal. Indeed, tyrosine-phosphorylation level induced byCD40 activation in DCs is inhibited by MV infection. However,CD40 pathway into DCs have not yet been described. It would beuseful to analyze CD40 signaling in DCs and then the relationship

between MV infection doses, DC differentiation, DC death, andmodification of CD40-signaling in DCs.

The location of DCs identifies them as one of the cell populationmost likely to have the earliest contact with viruses during infec-tion. DCs have been involved in primary antiviral immune reac-tion, but also in the propagation of viral infection (reviewed in Ref.40). Modification of CD40 signaling by MV infection may be themajor mechanism which induces MV immunosuppression be-cause: 1) Terminal differentiation of DCs in mature effector DCs isprevented. 2) DCs are used by at least two immunosuppressiveviruses as a reservoir that is activated by CD40L and upon inter-action with T cells. Indeed, MV replication in DCs is one logenhanced after CD40 activation (4). The same observation wasrealized for HIV replication (41). Thus the spreading of these im-munosuppressive viruses, in vivo, may be linked to CD40 activa-tion of DCs. 3) The requirement of CD40L probably locates theinitiation of immunosuppression in T cell area of secondary lym-phoid organs where immune response is organized. 4) The impair-ment of CD40 signaling could also occur in other cell types asmacrophages or B cells.

Beyond virus infection, modifying CD40 signaling in DCscould be a powerful tool to modulate immune response.

AcknowledgmentsWe thank Dr. A. Bohbot for providing CD341 cells. We also thank Drs.J. Marvel, A. Astier, V. Lotteau, and H. Valentin for critical reading of themanuscript; M. Perret for technical assistance; and A. Thomas and S.Mouradian for FACS settings.

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