of June 3, 2013.This information is current as

Phenotype+ IL-10+γa Nonprotective T-bet+ IFN-

towardDendritic Cells Polarize CD4+ T Cells Leishmania-Infected MHC Class IIhigh

Anabela Cordeiro-da-Silva and Ricardo SilvestreCunha, Bruno Neves, Maria Teresa Cruz, Jérôme Estaquier, Mariana Resende, Diana Moreira, Jorge Augusto, Joana

ol.1203518http://www.jimmunol.org/content/early/2013/05/31/jimmun

published online 31 May 2013J Immunol 

MaterialSupplementary

8.DC1.htmlhttp://www.jimmunol.org/content/suppl/2013/05/31/jimmunol.120351

Subscriptionshttp://jimmunol.org/subscriptions

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2013 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Leishmania-Infected MHC Class IIhigh Dendritic CellsPolarize CD4+ T Cells toward a Nonprotective T-bet+ IFN-g+

IL-10+ Phenotype

Mariana Resende,* Diana Moreira,* Jorge Augusto,* Joana Cunha,*,† Bruno Neves,‡,x

Maria Teresa Cruz,‡ Jerome Estaquier,{,‖ Anabela Cordeiro-da-Silva,*,# and

Ricardo Silvestre*

A differential behavior among infected and bystander dendritic cells (DCs) has been explored in different infection models. We have

analyzed both populations sorted on contact with visceral Leishmania infantum on a susceptible mice model evaluating the

subsequent repercussions on adaptive immune response. Our results demonstrate a clear dichotomy between the immunomod-

ulatory abilities of bystander and infected DCs. The bystander population presents increased levels of IL-12p40 and costimulatory

molecules being capable to induce CD4+ T cell activation with immune protective capabilities. In contrast, infected DCs, which

express lower costimulatory molecules and higher levels of IL-10, promote the development of Leishmania Ag-specific, non-

protective T-bet+IFN-g+IL-10+ CD4+ T cells with an effector phenotype. This specific polarization was found to be dependent

on IL-12p70. Splenic infected DCs recovered from chronic infected animals are similarly capable to polarize ex vivo syngeneic

naive CD4+ T cells toward a T-bet+IFN-g+IL-10+ phenotype. Further analysis revealed that only MHC class IIhigh–infected DCs

were responsible for this polarization. The adoptive transfer of such polarized CD4+ T cells facilitates visceral leishmaniasis in

BALB/c mice in a clear contrast with their counterpart generated with bystander DCs that significantly potentiate protection.

Further, we demonstrated that CD4+ T cells primed by infected DCs in an IL-10 free system, thus deprived of T-bet+IFN-g+IL-10+

population, restore the immune response and reduce parasite load, supporting a deleterious role of IFN-g+IL-10+ T cells in the

maintenance of infection. Overall, our results highlight novel subversion mechanisms by which nonprotective T-bet+IFN-g+

IL-10+ T cells are associated with chronicity and prolonged parasite persistence. The Journal of Immunology, 2013, 191: 000–000.

The intracellular protozoan Leishmania can produce a widespectrum of clinical manifestations, ranging from mild

cutaneous to chronic visceral pathologies, depending on

the species involved. The outcome of these infections is largely

dependent on host immune responses because fatalities often oc-

cur in the absence of treatment. In all cases, a protective response

is associated with the development of IFN-g–producing Th1 im-

munity, which leads to the elimination of the parasites within

infected cells (1). Although IFN-g secretion was shown to be

essential for optimal parasite elimination, high levels of IFN-g are

found during active disease in visceral leishmaniasis (VL) patient’s

plasma, spleen, and bone marrow, which returned to basal levels

after successful treatment (2). Nevertheless, the increased IFN-g

levels were always associated with concomitant accumulation ofIL-10 (2, 3), which play a dominant immunosuppressive role

during VL (4, 5).Among the different cell types that secrete IL-10 (6), recent

evidences have identified CD4+CD252IFN-g+ Th1 cells as the

critical source of IL-10 that suppresses the protective immune

response against Leishmania spp. and control Toxoplasma spp.

or Plasmodium-induced immunopathology (7–10). In an experi-

mental resistant murine model of VL, a CD11chigh dendritic cell

(DC)–driven expansion and/or maintenance of IL-10–producing

Th1 was observed in the passage to chronicity (10, 11). These

IFN-g+IL-10+ double-positive cells were found to arise from IFN-

g–producing Th1 cells and are often observed in both pathogen-

*Parasite Disease Group, Institute for Molecular and Cell Biology, University ofPorto, 4150-180 Porto, Portugal; †Instituto de Ciencias Biomedicas Abel Salazar eFaculdade de Medicina, Universidade do Porto, 4050-313 Porto, Portugal;‡Centre for Neuroscience and Cell Biology, Faculty of Pharmacy, Universidade deCoimbra, 3004-517 Coimbra, Portugal; xDepartamento de Quımica, Centro deEspectrometria de Massa, Quımica Organica, Produtos Naturais e Agro-alimentares,Universidade de Aveiro, 3810-193 Aveiro, Portugal; {Centre National de la RechercheScientifique Formation de Recherche en Evolution 3235, Universite Paris Descartes,75006 Paris, France; ‖Universite Laval, Centre de Recherche en Infectiologie, QuebecG1V 4G2, Canada; and #Departamento de Ciencias Biologicas, Faculdade de Farmacia,Universidade do Porto, 4050-313 Porto, Portugal

Received for publication December 27, 2012. Accepted for publication April 30,2013.

This work was supported by Fundo Europeu de Desenvolvimento Regional (FEDER)funds through the Operational Competitiveness Programme–COMPETE and bynational funds through Fundacao para a Ciencia e a Tecnologia under projectsFCOMP-01-0124-FEDER-011054 (PTDC/SAU-FCF/100749/2008) and FCOMP-01-0124-FEDER-011058 (PTDC/SAU-FCF/101017/2008). M.R. was supported byFCOMP-01-0124-FEDER-011058 (PTDC/SAU-FCF/101017/2008) projects. J.C.and D.M. were supported by SFRH/BD/48626/2008 and SFRH/BD/91543/2012,respectively. R.S. was supported by Programa Ciencia, financed by Programa Oper-

acional Potencial Humano–Quadro de Referencia Estrategica Nacional–Tipologia4.2–Promocao do Emprego Cientıfico, cofunded by Fundo Social Europeu and na-tional funding from the Ministry of Science, Technology and Higher Education. J.E.was supported by an Agence Nationale de la Recherche grant (LEIS-APO, France) andthe Canada Research Chair program.

Address correspondence and reprint requests to Dr. Anabela Cordeiro-da-Silva andDr. Ricardo Silvestre, Parasite Disease Group, Infection and Immunity Unit, Institutode Biologia Molecular e Celular da Universidade do Porto, Rua do Campo Alegre,823, 4150-180 Porto, Portugal. E-mail addresses: cordeiro@ibmc.up.pt (A.C.-d.-S.)and rleal@ibmc.up.pt (R.S.)

The online version of this article contains supplemental material.

Abbreviations used in this article: BMDC, bone marrow–derived dendritic cell;BMM, bone marrow–derived macrophage; CD4 byst, CD4+ T cell polarized withbystander dendritic cells; CD4 CD11c, CD4+ T cell polarized with noninfecteddendritic cells; CD4 inf, CD4+ T cell polarized with infected dendritic cells; DC,dendritic cell; DP, double-producer; IBMC, Instituto de Biologia Molecular e Celu-lar; MHC II, MHC class II; RPMIc, complete RPMI 1640; SLA, soluble LeishmaniaAg; VL, visceral leishmaniasis.

Copyright� 2013 by TheAmerican Association of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203518

Published May 31, 2013, doi:10.4049/jimmunol.1203518 at IN

IST C

NR

S BiblioV

ie on June 3, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

specific and nonspecific T cell clones, which argue in favor ofnonspecific bystander T cell activation (9). Several mechanismshave been proposed to regulate IL-10 production on Th1 cells.Among those, TGF-b, IL-6, IL-21, as well as cytokines belongingto the IL-12 family and ICOS–ICOS ligand interaction wereshown to be involved in the induction of IL-10 (12).DCs are specialized APCs that play a crucial role in driving

adaptive immune responses. Intracellular pathogens are known tocounteract and subvert host immune DCs and effector cell func-tions using distinct strategies that include interference with Agprocessing/presentation, manipulation of costimulatory signals andcytokines, immunosuppression, and increased host cell viability,among others (13). Given that in any chosen model not all of theDCs are infected, one might expect a differential behavior amonginfected and bystander DCs. Carvalho et al. (14) have reporteda functional DC dichotomy after interaction with mucocutaneousLeishmania species, where nonactivated infected DCs producedhigh levels of TNF-a responsible for the increased immunopa-thology, whereas bystander DCs exposed to soluble parasiteproducts were activated promoting T cell responses. Consideringthe substantial differences between the proinflammatory responseobserved in mucocutaneous disease and the IL-10–driven immu-nosuppressive VL, we decided to clarify the functional role of thebystander and infected DC populations during visceral L. infantuminfection on a susceptible mice model evaluating the subsequentrepercussions on adaptive immune response.Our results highlight novel host subversion mechanisms used

by L. infantum. We demonstrate the duality of cytokine expressionpostinfection, indicating that bystander DCs were prone to expressIL-12p40 and IL-6, whereas infected DCs transcribe preferablyIL-10 and TNF-a mRNA. Our data demonstrate the critical role ofinfected DCs to polarize, in an IL-12p70–dependent manner,CD4+ T cells toward a specific nonprotective T-bet+IFN-g+IL-10+

phenotype. Moreover, we prove that only infected MHC classIIhigh (MHC IIhigh) DCs, either developed in vitro or recoveredfrom in vivo infection, were capable to generate the LeishmaniaAg-specific IL-10 Th1 population with effector phenotype. Overall,we identified a cellular mechanism by which nonprotective T-bet+

IFN-g+IL-10+ T cells are associated with chronic parasite per-sistence. Moreover, our data raise a potential dichotomy of IL-27/IL-12p70 in driving protective/pathogenic IFN-g+IL-10+ Th1double-producer (DP) responses.

Materials and MethodsAnimals and parasites

BALB/c and IL-10 (BALB/c background)–deficient mice were obtainedfrom Instituto de Biologia Molecular e Celular (IBMC; Porto, Portugal)animal facilities. Animal care was in accordance with institutional guide-lines. All experiments were approved by and conducted in accordance withthe IBMC.INEB Animal Ethics Committee and the Portuguese NationalAuthority for Animal Health (Direccao-Geral de Alimentacao e Veterinaria)guidelines. A cloned line of virulent L. infantum (MHOM/MA/67/ITMAP-263) was maintained by weekly subpassages in complete RPMI 1640 (RPMIc)supplemented with 10% heat-inactivated FBS (FCS; Lonza), 100 U/mlpenicillin, 100 mg/ml streptomycin, and 2 mmol/L HEPES (BioWhittaker,Walkersville, MD). All the experiments were realized with promastigotes atpassage four as previously defined (15).

Abs

Abs used include: PE/Cy7 anti-mouse CD40 Ab, clone 3/23, Isotype ratIgG2a,k; PerCP/Cy5 anti-mouse CD80 Ab, clone 16-10A1, Isotype Ar-menian hamster IgG Brilliant Violet 421 anti-mouse CD86 Ab, cloneGL-1, Isotype rat IgG2a,k; Alexa Fluor 647 anti-mouse I-Ad Ab, clone 39-10-8, Isotype mouse (C3H.SW) IgG3; allophycocyanin anti-mouse I-A/I-E Ab,clone M5/114.15.2, Isotype rat IgG2b,k; Pacific Blue anti-mouse CD1d(CD1.1, Ly-38) Ab, clone 1B1, Isotype rat IgG2b,k; PE/Cy7 anti-mouseCD11c Ab, clone N418, Isotype Armenian hamster IgG; allophycocyanin

anti-mouse CD11c Ab, clone N418, Isotype Armenian. hamster IgG; FITCand PE/Cy7 anti-mouse IFN-g Ab, clone XMG 1.2, Isotype IgG1, k; PEand allophycocyanin anti-mouse IL-10 Ab, clone JES5-16E3, Isotype ratIgG2b, k; PerCP/Cy5.5 anti-mouse TNF-a Ab, clone MP6-XT22, Isotyperat IgG1, k; allophycocyanin anti-mouse IL-12/IL-23 p40 Ab, clone C15.6,Isotype rat IgG1,k; PerCP/Cy5 and purified anti-mouse CD3 Ab, clone17A2, Isotype rat IgG2b, k; LEAF purified anti-mouse TCR b-chain Ab,clone H57-597, Isotype Armenian hamster IgG; PE-anti-mouse CD69 Ab,clone H1.2F3, Isotype Armenian hamster IgG; Brilliant Violet 421 anti-T-bet Ab, clone 4B10, Isotype mouse IgG1, k; V500 rat anti-mouse CD4 Ab,clone RM4-5, Isotype rat IgG2a, k; Alexa Fluor 488 anti-mouse FOXP3Flow Kit, clone MF-14; Isotype rat IgG2b, k; PerCP anti-mouse CD25 Ab,clone PC61, Isotype rat IgG1; PE anti-mouse/human CD44 Ab, clone IM7,Isotype rat IgG2b; allophycocyanin anti-mouse CD62L Ab, clone MEL-14, Isotype rat IgG2a. All Abs were purchased from BioLegend (SanDiego, CA), except for CD4-V500, which was purchased from BD Hori-zon. All isotype controls were obtained from BioLegend.

Generation of bone marrow DCs and naive CD4+ T cellisolation

Bone marrow DCs were derived as described previously (16). In brief, bonemarrow from femurs and tibiae of 10- to 12 wk-old mice were flushed withRPMI 1640, using syringes and 25-gauge needles. The tissue was resus-pended, and bone marrow–derived DCs (BMDCs) were obtained byseeding 5 3 106 bone marrow cells in 25 ml RPMIc supplemented with50 mM 2-ME (Sigma Chemical) and 200 U/ml GM-CSF (DC medium;PeproTech, Rocky Hill, NJ) or 10% of a supernatant containing GM-CSF(J558-GM-CSF). Cells were cultured at 37˚C and 5% CO2 for 3 d, afterwhich the same amount of DC medium was added to each flask. At days 6and 8, half of the culture supernatant was collected and centrifuged, andthe cell pellet was resuspended in the same amount of fresh DC mediumand put back into the original flasks. At day 10, the same procedure wasperformed but with use of only 100 U/ml GM-CSF or 5% of GM-CSF–containing supernatant. The purity of BMDC preparations after 12 d ofculture was always superior to 90%. Mouse naive CD4+ T cells wereisolated from the spleens of naive BALB/c or IL-10 knockout mice andpurified using the CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec)following manufacturer’s recommendations. The purity of naive CD4+

T cell preparations was always superior to 95%.

Leishmania staining with CFSE

Stationary-phase promastigotes from each culture medium at a concentra-tion of 1.2 3 108 promastigotes/ml were used for CFSE (Invitrogen) la-beling. L. infantum promastigotes were washed two times with PBS andlabeled with 5 mM CFSE for 10 min at 37˚C with occasional shackingfollowed by 5-min incubation at 4˚C. The parasites were then washedtwice and resuspended in RPMIc before proceeding to infections.

In vitro infection of DCs

For in vitro infection, 12-d nonadherent BMDCs were seeded at 1 3 106

cells/ml RPMIc in 24- or 6-well plates After an overnight incubation pe-riod, CFSE-labeled L. infantum promastigotes were added to the culture atan infection ratio of 10:1 (parasites/cell). Parallel experiments were alsoperformed using heat-killed (30 min at 56˚C), fixed (10 min in glutaral-dehyde), irradiated (500 Gy; Gammacell 1000 Elite) parasites or 2-mm-diameter FITC-latex beads (Sigma-Aldrich) at similar coculture ratios (10FITC-beads for 1 cell). Noninternalized parasites were removed by gentlywashing after 4 h of infection, and fresh RPMIc was added to the wells.Cells were immediately recovered or maintained for 24 h. BMDCs stimu-lated with LPS (1 mg/ml) were used as a positive control for DC activation/maturation. The percentage of infected BMDCs was determined by flowcytometry evaluation of CFSE+ cells in a FACSCanto II cytometer andanalyzed with FlowJo software (Tree Star, Ashland, OR) (17).

Surface and intracellular staining of BMDCs

For the analysis of surface costimulatory markers, 2 3 105 BMDCs wereincubated for 20 min with saturating concentrations of CD40-PE/Cy7,CD80-PerCP/Cy5.5, CD86- Brilliant Violet 421, MHC II–Alexa Fluor647 CD1d–Pacific Blue, and CD11c-PE/Cy7. Mouse isotype controls wereused when necessary. All of the Abs were obtained from BioLegend. Aftertwo washing steps with PBS/2% FBS, the cells were acquired by flowcytometry in a FACSCanto II cytometer. Cells were selected on the basisof forward scatter/side scatter values; BMDCs were gated on CD11c+. BMDCswere fixed with 2% PFA, washed in PBS containing 2% FCS, and per-meabilized in 50 ml of 0.5% saponin, to detect intracellular cytokines. Cellswere then incubated with TNF-a–PerCP/Cy5.5, IL12p40-allophycocyanin,

2 INFECTED DCs INDUCE IFN-g+ IL-10+ Th1 LYMPHOCYTES

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

or isotype-matched controls (all from BioLegend) for 30 min at roomtemperature in the dark. The cells were then washed twice in PBS andacquired on a FACSCanto II cytometer. All analyses were performed usingFlowJo software.

Cell sorting

Dendritic cells. In some experiments, BMDCs were sorted according theirCD11c+ surface expression in the case of noninfected cells and CD11c+

CFSE2 or CD11c+ CFSE+ expression in the case of bystander and infectedcells, respectively. In other cases, BMDCs were sorted according theirCD11c+/MHC II(high or low) and then infected for posterior analysis. Finally,in other experiments, BMDCs were first infected with CFSE-labeled par-asites and bystander or infected BMDCs were sorted according their MHCII expression (high or low) as demonstrated on gating strategy (Supple-mental Fig. 1A). Six populations were sorted according to their expressionon MHC II (low or high) and CFSE labeling.

CD4+ T cells. After coculture with the different subsets of BMDCs, CD4+

T cells were sorted after surface labeling with anti–CD3-PerCP/Cy5.5and anti–CD4-V500. SYTOX Green Nucleic Acid Stain (Invitrogen) wasadded to exclude dead cells. All sorting experiments were conducted in aFACSAria using FACSDiva software. The purity of the separation wasalways confirmed by flow cytometry and was superior to 95%.

BMDCs and CD4+ T cell coculture

In vitro. Noninfected, infected, or bystander BMDCs (23 104) BALB/c orIL-10 knockout mice were cocultured with naive CD4+ T cells of similarorigins at a responder/stimulator 10:1 cell ratio. CD4+ T activation wasmeasured after 36 h of coculture by labeling CD4+ T cells with anti–CD69-PE. Cocultures to evaluate T cell proliferation (CFSE decay) were measuredafter 72 h in the presence of 10 ng/ml IL-2. The CD4+ T cells intracellularcytokine levels were measured after 5 d of coculture. In some cases,IL12p70, IL-6, IL-21, TNF-a, TGF-b (10 ng/ml), anti–IL-12p40, anti-CD275 (ICOSL; BioLegend), anti–IL-27p28 (R&D Systems) or the re-spective isotype control (all at 10 mg/ml) were added to the cocultures.PMA/Ionomycin plus brefeldin was added during the last 16 h. CD4+ T cellswere surface labeled with CD3-PerCP/Cy5.5 and CD4-V500, and then fixedwith 2% PFA, washed in PBS containing 2% FCS, and permeabilized in 50ml of 0.5% saponin. Cells were intracellularly stained with IFN-g–FITC, t-bet–Brilliant Violet 421 and IL-10–allophycocyanin or isotype-matchedcontrols (all from BioLegend) for 30 min at room temperature in thedark. In some cases, Foxp3-PE was included in the labeling followingmanufacturer’s recommendations. The cells were then washed twice in PBSand analyzed on a FACSCanto II cytometer.

Ex vivo. BALB/c mice were infected i.p. with 1 3 108 CFSE-labeled L.infantum promastigotes. Mice were euthanized at 24 h or 28 d postinfectionand the spleens removed. T and B lymphocytes were depleted using CD19+

and CD3ε+ microbeads coupled with LD columns (Miltenyi Biotec). Theremaining cell suspension was labeled with anti–CD11b-PE, anti–CD11c-allophycocyanin, anti–Ly6C-PerCP/Cy5.5, and anti–Ly6G-Pacific Blue,and sorted (in a FACSAria using the FACSDiva software) according to thesurface expression CD11b+CD11chighLy6C2Ly6G2, and surface MHC IIexpression gating on infected (CFSE+CD11b+CD11chighLy6C2Ly6G2) orbystander (CFSE- CD11b+CD11chighLy6C2Ly6G2) splenic DCs. For allexperiments, CD11b+CD11chighLy6C2Ly6G2 cells from the spleen ofnoninfected mice were sorted as a control. In other experiments, totalsplenic DCs of infected or noninfected mice were sorted following a similarapproach. Noninfected, infected, or bystander splenic DCs were coculturedwith naive CD4 T cells of similar origins at a responder/stimulator 10:1 cellratio in the presence of IL-2 (10 ng/ml) for 120 h. In some experiments,a gradient of responder/stimulator ratios was used (20:1; 10:1; 4:1, and 2:1).The intracellular levels of IFN-g and IL-10 on CD4+ T cells were measuredas depicted earlier.

TCR specificity

The Ag-specific proliferative capacity of the in vitro polarized CD4+ T cellpopulations was performed through Leishmania Ag presentation by APCsas previously described (18). In brief, irradiated (3000 cGy) J774 macro-phages (104) were used as APCs. These were cultured overnight with heat-killed Leishmania infantum promastigotes at a 10:1 ratio (parasite:J774;56˚C, 30 min) and irradiated. A total of 105 sorted CFSE-labeled CD4+

T cells (CD4+ T cells polarized with infected dendritic cells [CD4 inf],CD4+ T cells polarized with bystander DCs [CD4 byst], or CD4+ T cellspolarized with noninfected DCs [CD4 CD11c]), which had been polarizedwith the distinct BMDCs, were plated with the above and incubated in thepresence of IL-2 (5 ng/ml) for 72 h at 37˚C 5% CO2. CFSE decay wasmeasured by flow cytometry.

RNA extraction and real-time RT-PCR

Total RNA was isolated from cells with TRIzol reagent (Invitrogen, Bar-celona, Spain) or RNeasy micro kit (Qiagen), according to the manu-facturer’s instructions. The RNA concentration was determined by OD260measurement using a NanoDrop spectrophotometer (Thermo Scientific,Wilmington, DE), and quality was inspected for the absence of degradationor genomic DNA contamination, using Experion RNA StdSens Chips inthe Experion automated microfluidic electrophoresis system (Bio-Rad,Hercules, CA). RNA was stored in RNA Storage Solution (Ambion, Fos-ter City, CA) at 280˚C until use. Real-time RT-PCRs were run in triplicatefor each sample on a Bio-Rad MyCycler iQ5. Primers were designed usingBeacon Designer software (version 7.2; PREMIER Biosoft International,Palo Alto, CA) and thoroughly tested. In brief, 200 ng total RNA wasreverse-transcribed using the iScript Select cDNA Synthesis Kit (Bio-Rad).Real-time PCR was performed as described previously (17). After am-plification, a threshold was set for each gene and Ct values were calculatedfor all samples. Gene expression changes were analyzed using the built-iniQ5 Optical system software (version 2). The results were normalizedusing two reference genes, HPRT-1 and GAPDH, determined with Genexsoftware (MultiD Analyses AB, Goteberg, Sweden) as the most stable forthe conditions used.

Adoptive transfer and parasite burden

BALB/c mice were infected i.p. with 1 3 108 CFSE-labeled L. infantumpromastigotes suspended in sterile PBS. Fifteen days after, 1 3 106 CD4+

cells were adoptively transferred by i.v. injection. Equal volume of PBSwas injected in some groups as a control. Two weeks after receiving theCD4+ T cells, the animals were euthanized. The parasite burden in thespleen and liver was determined by limiting dilution as previously de-scribed (19).

Cytokine ELISA

Spleen cells were cultured in the presence or absence of soluble LeishmaniaAg (SLA; 25 mg/ml) at 37˚C under 5% CO2. The supernatants were col-lected after 72 h of culture for cytokine quantification by ELISA (IFN-gand IL-10), using commercial sandwich immunoassay kits (BioLegend andBD, San Diego, CA) and following the manufacturer’s recommendations.The supernatants of sorted BMDCs were collected after 24 h of culture,and supernatants of BMDC–CD4+ T cell cocultures were collected after72 h of culture for cytokine quantification by ELISA (IL-12p70 and IL-27),using commercial sandwich immunoassay kits (BioLegend and BD) andfollowing the manufacturer’s recommendations.

Immunofluorescence

BMDCs were labeled with MHC II–allophycocyanin for cell sorting. Aftersorting, each BMDC subset was fixed in PFA 2%, and ∼20,000 cells weremounted on slides with Vectashield mounting medium with DAPI (H-1200, Vector Laboratories) and imaged at room temperature using a Axi-oImager Z1 (Carl Zeiss, Germany) with the 633/1.40 Plan-Apochromatoil objective and AxioCam MR version 3.0 (Carl Zeiss, Germany). Thefilter set used included an excitation filter of 640/30 nm and an emissionfilter of 690/50 nm. The settings for contrast, brightness, pinhole, acqui-sition mode, and scanning time were maintained throughout the work.All images were treated using open source ImageJ (National Institutes ofHealth, Bethesda, MD).

Generation of bone marrow–derived macrophages and CD4+

T cell bone marrow–derived macrophage coculture

Bone marrow–derived macrophages (BMMs) were generated from thebone marrow recovered from femurs and tibiae of 10- to 12 wk-old femalemice flushed with RPMI 1640, using syringes coupled with 25-gaugeneedles. The tissue was resuspended, and BMM were obtained by seeding4 3 105 bone marrow cells in 400 ml DMEM supplemented with 10% ofL929 supernatant as source of M-CSF for 10 d. At days 4 and 8, 10% ofL929 supernatant was added to cultures. At day 9 of culture, naive CD4+

T cells were added to BMMs at a responder/stimulator 10:1 cell ratio in thepresence of IL-2 (10 ng/ml) for 120 h. The cytokine intracellular levels ofCD4+ T cells were measured after PMA/Ionomycin plus brefeldin Astimulation as before.

Statistical analysis

The results are presented as means 6 SD, and the statistical differencebetween two groups was determined by the two-sided unpaired Studentt test. For multiple group comparisons, the one-way ANOVA test with

The Journal of Immunology 3

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

a Bonferroni multiple-comparison posttest was used. The tests were per-formed using GraphPad Prism (version 5.02; GraphPad Software, SanDiego, CA). Statistically significant values are as follows: *p , 0.05,**p , 0.01, ***p , 0.001.

ResultsMaturation status and cytokine expression profile of bystanderand L. infantum–infected BDMCs impact differently CD4T cell activation and proliferation

We used an in vitro system where BMDCs are targeted by CFSE-labeled parasites. The infection rate was confirmed by flowcytometry analysis of CFSE+ BMDCs (Fig. 1A). Although we didnot directly address promastigote internalization, we assumed theCFSE+ population as comprised in its majority by DCs containingintracellular parasites. In support of this assumption, we failed todetect surface-associated promastigotes in CFSE+ sorted DCs bymicroscopy, and similar percentages of CFSE+ DCs were detectedin experiments using the crystal violet as a surface fluorescencequencher (20) (data not shown). Regarding the maturation status,the bystander BMDC population (CFSE2) expressed significantlyhigher levels of CD40, CD86, and MHC II molecules whencompared with infected BMDCs (CFSE+) or cells not exposed toparasites. In opposition, the CD1d marker was specifically loweron the bystander population (Fig. 1A). This phenotype was BMDCspecific, because bystander and Leishmania-infected BMMs dis-played similar levels of cell-surface markers (data not shown). Wefurther evaluated the cytokine profile expressed by each one ofthese subpopulations by quantitative PCR. Among all tested cyto-kines, we detected significantly higher levels of Il10 and Tnfatranscripts exclusively on infected BMDCs (Fig. 1B) and a trendfor IL-27p28 (although not significant) compared with uninfectedand bystander (only for Il10) BDMC cells. In contrast, bystanderpopulation produces preferably Il12p40 and Il6 transcripts (Fig.1B). Flow cytometry analyses confirmed that IL-12p40 is prefer-ably detected on bystander population (gating specifically on eachsubpopulation, IL-12p40–producing bystander cells are 26.1 62.8% compared with 16.9 6 2.2% in infected cells; Fig. 1C, 1D).In opposition, 1.2 6 0.2% of bystander BMDCs versus 5.9 60.7% of infected cells expressed TNF-a (Fig. 1C, 1D).Because T cell activation depends on the nature of APCs, we

examined the impact of each DC subpopulation on the activationlevel of naive CD4+ T cells by quantifying the expression ofa T cell activation marker, CD69 (Fig. 1E). Our results showedthat bystander BMDCs induced higher levels of cell activation(8.10 6 2.3%) compared with infected or noninfected BMDCs(5.25 6 0.3 and 5.12 6 0.8%, respectively; Fig. 1E). Experimentsperformed evaluating the proliferative capacity of CFSE-labeledCD4+ T cell (Fig. 1F, 1G) corroborated the differences observed atthe activation level (Fig. 1E). BMDCs exhibit a heterogeneouspopulation based on the surface expression of MHC II (21, 22).On the basis of MHC II surface expression, we observed thatMHC IIlow BMDCs acting as highly phagocytic cells uptake morethan double the number of L. infantum promastigotes than MHCIIhigh cells do either in the absence of cell sorting (SupplementalFig. 1A, 1B) or after cell sorting (Supplemental Fig. 1C–E). Ac-cordingly, T cell activation was measured upon coculture withMHC IIlow or MHC IIhigh sorted noninfected, bystander, and in-fected BMDCs (Supplemental Fig. 1F). The profile of bystanderMHC IIhigh revealed increased Il12p40 and Il6 transcripts (Sup-plemental Fig. 1G), which was not surprising because the majorityof bystander cells displayed a matured phenotype (Fig. 1A),whereas only MHC IIhigh infected BMDCs showed significant in-creased levels of Il10 and Tnfa (Supplemental Fig. 1G, 1H). Ourdata demonstrate that the differential activation and proliferative

phenotype were not a direct result of disparate levels of MHC IIat the cell surface of bystander in comparison with infectedcells. Indeed, infected BMDCs were always unable to induceCD4+ T cell activation (Supplemental Fig. 1F). Moreover, evenin the presence of high levels of MHC II, infected BMDCs wereunable to induce similar levels of CD4+ T proliferation (CFSEdecay) than bystander BMDCs (Supplemental Fig. 1I–J). Inter-estingly, sorted MHC IIhigh BMDCs exposed to L. infantum in-fection displayed a downregulation of MHC II surface levelsspecifically on the infected population, suggesting a new sub-version mechanism of host immune responses (SupplementalFig. 1C).

Polarization of naive T cells toward a T-bet+IFN-g+IL-10+

phenotype depends on infected BDMCs

We then evaluated the profile of CD4+ T cells induced in thoseconditions. IL-4, IL-10, IL-17, and IFN-g, initially used for dis-criminating Th2, Tr1, Th17, and Th1 cells, respectively, were an-alyzed. Whereas bystander BMDCs primed CD4+ T cells to IFN-gsecretion, infected BMDCs polarize CD4+ T cells toward an IFN-g+IL-10+ DP phenotype (the percentage of DP varied from 2.8 to13.4% in independent experiments; Fig. 2A, 2B). Few cells werepositive for IL-4 and IL-17, and we did not find any significantdifferences on the IL-21, IL-22, or TNF-a level among the differentcocultures. The absence of contact between BDMCs and CD4+

T cells abrogate any IFN-g and/or IL-10 production (data notshown). Interestingly, the CD4+ polarization was restricted to theDC population because DP cells were never generated when in-fected BMMs were used as APCs (Supplemental Fig. 2). Studieson Leishmania spp. (7, 23, 24) and Toxoplasma gondii (9) infec-tion classified the DP cells arising during infection as effectorTh1 lymphocytes. Accordingly, the DP population generated withinfected DCs expressed the highest levels of the Th1 master tran-scription factor T-bet, but not the classical regulatory T cell markerFoxp3 (Fig. 2C). Similarly, this population was found negative forthe CD25 marker displaying an effector phenotype (CD62L2

CD44+; Fig. 2D). To gain insight about the specificity of CD4+

T cells generated in vitro after 5 d of coculture, we restimulatedCD4+ T cells with APCs loaded with Leishmania Ags. CD4 infproliferate in response to SLA, whereas those cultured with by-stander cells failed to proliferate (Fig. 2E, 2F). Nevertheless, we donot discard the possibility that CD4 byst may recognize specificLeishmania secreted products, which are poorly presented by J774macrophages in our experimental settings regarding the initial dif-ferentiation mediated by bystander and infected DCs, and mayreflect distinct Ag specificity of CD4+ T cells. Thus, although initiallyless potent to proliferate in the presence of infected DCs, the CD4inf cells acquired specific Ag response.

IL-10 controls the activation of CD4+ T cells polarized byinfected BMDCs playing a crucial role in their effectorfunctions

Considering the IL-10 inhibitory role for Ag-specific activationand proliferation of CD4+ T cells (25) and because infectedBMDCs presented increased IL-10 production (Fig. 1B), com-plementary experiments were performed with BMDCs recoveredfrom IL-10 knockout mice cocultured with syngeneic BALB/cnaive CD4+ T cells. Although the level of CD4+ T cell activa-tion remained unchanged when noninfected or bystander BMDCswere used as APCs, the absence of IL-10 from infected BMDCsrestored the CD4+ T activation to similar levels as bystandercocultures (Fig. 3A, 3B). Similarly, the absence of IL-10 frommyeloid origin rescued the CD4+ T proliferation upon coculturewith infected BMDCs to comparable levels as bystander cocul-

4 INFECTED DCs INDUCE IFN-g+ IL-10+ Th1 LYMPHOCYTES

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

tures (Fig. 3C, 3D). Overall, these data demonstrate that IL-10secreted by infected BMDCs is responsible, at some extent, forinhibiting the activation and proliferation of Ag-specific CD4+

T cells.Cytokine quantification on coculture supernatant showed in-

creasing quantities of IL-10 concomitantly to a decrease of IFN-glevels when CD4 inf cells were used in comparison with CD4 bystcoculture conditions (Fig. 4A, 4B). Given the major immuno-suppressive role of IL-10, we repeated the experiment in thepresence of anti–IL-10 mAb. Remarkably, the blockage of IL-10

restored the IFN-g levels in coculture with CD4 inf to similarlevels of CD4 byst cocultures (Fig. 4C).

Ex vivo infected DCs polarize naive T cells toward an IFN-g+

IL-10+ DPs CD4+ T cell phenotype

To confirm the impact of bystander and infected DCs, we infectedBALB/c mice with CFSE-labeled L. infantum, and 24 h later weisolated splenic CD11c+ cells. Thereafter, we sorted infected andbystander DCs, and accessed syngeneic naive CD4+ T cell polari-zation upon coculture. As a control, noninfected DCs from naive

FIGURE 1. L. infantum shapes the maturation and cytokine expression profile of bystander and infected BDMCs resulting in distinct levels of T cell

activation and proliferation. The surface costimulatory molecules were analyzed in bystander (blue) and infected gated populations (red) of BALB/c

BMDCs infected with CFSE-labeled L. infantum. For comparison, the histograms display the same markers on noninfected BMDCs (black) (A). Four hours

postinfection, CD11c+ bystander and infected BMDCs were sorted, and the transcript levels of the indicated cytokines were determined by qPCR. Values

are normalized for noninfected CD11c+ sorted cells (B). The intracellular levels of IL-12p40 and TNF-a were accessed by flow cytometry 18 h postin-

fection (C). The graphic depicts the relative percentage of noninfected, bystander, or infected cells producing IL-12p40 and TNF-a as detected by flow

cytometry (D). Sorted CD11c+ bystander, infected, or noninfected BMDCs from BALB/c mice were cocultured with syngeneic naive CD4+ T cells. Thirty-

six hours later, the activation of CD4+ T lymphocytes was measured through CD69 expression (E). Seventy-two hours later, the proliferation of CD4+ T

lymphocytes was measured through CFSE decay in the presence of 10 ng/ml IL-2 (F). The mean 6 SD from one representative experiment out of five is

shown (G). *p , 0.05, ** p , 0.01, *** p , 0.001.

The Journal of Immunology 5

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

mice were used. The DP CD4+ T cell population was exclusivelydetected when CD4+ T cells were in contact with infected DCs(Fig. 5A) demonstrating that only infected splenic BMDCs arecapable to polarize naive CD4+ T cell toward an DP phenotype.

To ascertain the true relevance of this DP phenotype on CD4+

T cells, we evaluated their presence during different time points ofinfection in our in vivo susceptible model (BALB/c). In this study,the frequency of splenic DP CD4+ T cells was found to increase

FIGURE 2. Infected DCs induce Leishmania

Ag-specific effector Th1 CD4+ T cells with

IFN-g+IL-10+ DP phenotype. Sorted CD11c+

bystander, infected, or noninfected BMDCs

from BALB/c mice were cocultured with syn-

geneic naive CD4+ T cells. After 5 d of co-

culture, the IFN-g and IL-10 intracellular levels

were determined on CD4+ T cells (A). The

mean6 SD from one representative experiment

out of eight is shown (B). T-bet and Foxp3

expression were measured on CD4+ T cells

cocultured with infected BMDCs on each of the

indicated gates (C). CD44, CD62L, and CD25

surface markers were measured on the IFN-g+

IL-10+ DP CD4+ T cells (black). Isotype con-

trol is represented (gray) (D). After 5 d of co-

culture, the CD4+ T lymphocytes of each one of

the cultures (CD4 byst, CD4 Inf, CD4 CD11c

corresponding to the source of DCs that were

used in the coculture, bystander, infected, or

noninfected, respectively) were sorted and fur-

ther cultured with APCs presenting Leishmania

Ags. The Leishmania-specific proliferative ca-

pacity of each CD4+ T cell was quantified after

96 h by CFSE decay (E). The mean 6 SD from

one representative experiment out of five is

shown (F). *p, 0.05, **p, 0.01, ***p, 0.001.

FIGURE 3. IL-10 controls the activation and proliferation of CD4+ polarized by infected BMDCs. Sorted CD11c+ bystander, infected, or noninfected

BMDCs from IL-10ko mice were cocultured with syngeneic naive WT CD4+ T cells. Thirty-six hours later, the activation of CD4+ T lymphocytes was

measured through CD69 expression (A). The mean 6 SD from one representative experiment out of five with BMDCs from BALB/c or IL-10ko origin is

shown (B). Upon seventy-two hours of coculture, the proliferation of WT CD4+ T lymphocytes was measured through CFSE decay in the presence of 10 ng/

ml IL-2 (C). The mean 6 SD from one representative experiment out of three is shown (D). *p , 0.05, **p , 0.01, ***p , 0.001.

6 INFECTED DCs INDUCE IFN-g+ IL-10+ Th1 LYMPHOCYTES

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

during the acute phase, reaching its peak at 28 d postinfection(0.10 6 0.04% in naive mice; 2.40 6 0.60% at day 28 postin-fection), diminishing in percentage and total numbers duringchronic phase (1.10 6 0.25%; Fig. 5B, 5C). Thus, an �25-fold

expansion in the number of CD4+ T cells capable of IFN-g and IL-10 simultaneous production was found at day 28 postinfection(2.446 1.023 104 in naive mice versus 6.006 1.253 105 at day28 postinfection). Importantly, at day 84 postinfection, ∼81.1 6

FIGURE 4. IL-10 secreted from CD4+ polarized by

infected BMDCs plays a crucial role in their effector

functions. Naive CFSE-labeled CD4+ T (2.5 3 104)

cells were activated with anti-CD3 (1 mg/ml) and

cultured with in vitro polarized and sorted CD4 byst,

CD4 Inf, or CD4 CD11c (2.53 104) in the presence of

irradiated APCs. After 5 d, the levels of IL-10 (A) and

IFN-g (B) were measured in the culture supernatant.

Similarly, the levels of IFN-g were quantified when

naive CD4+ T cells (2.5 3 104) were cultured with

in vitro polarized CD4+ T cells (1.0 3 104) in the

presence of anti–IL-10 (10 mg/ml) or the respective

isotype control (IgG1; C). The mean 6 SD from one

representative experiment out of three is shown. *p ,0.05, **p , 0.01.

FIGURE 5. Ex vivo infected DCs induce the polarization of CD4+ T cells with IFN-g+IL-10+ DP phenotype. BALB/c mice were infected i.p. with 1 3108 CFSE-labeled L. infantum promastigotes. Twenty-four hours later, the spleens were removed and the bystander and infected DCs sorted. As a control,

DCs were sorted from the spleen of noninfected mice. Each DC population was cocultured with syngeneic naive CD4+ T cells for 5 d, and the IFN-g and

IL-10 intracellular levels were determined by flow cytometry (A). The percentage (B) and total number (C) of splenic CD4+ T cells with Leishmania Ag-

specific IFN-g+IL-10+ DP phenotype were quantified in noninfected mice (naive) or after 14, 28, or 84 d of infection (d14, d28, or d84, respectively). The

spleens of noninfected (naive) or d28 infected mice (infected) were recovered and the CD11c+ MHC IIhigh DC population purified by cell sorting. Each DC

population was cocultured with syngeneic naive CD4+ T cells for 5 d at the displayed conditions, and the IFN-g and IL-10 intracellular levels were

determined by flow cytometry (D). Noninfected, infected, or bystander BMDCs sorted according their MHC II expression (high or low) were cocultured

with naive CD4+ T cells. After 5 d of coculture, the IFN-g and IL-10 intracellular levels were determined on CD4+ T cells (E).

The Journal of Immunology 7

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

4.1 Leishmania Ag-specific IL-10+–producing cells and 32.2 613.4 IFN-g+ were characterized as DPs.We then assessed whether splenic DCs from chronically infected

micewere capable of generating in vitro DP CD4 T cells from naivecells. We isolated total splenic DCs defined as CD11c+ MHC IIhigh

from naive or 28 d postinfection mice and accessed syngeneicCD4+ T cell polarization upon coculture. Again, the DP CD4+

T cell population was exclusively detected when CD4+ T cellswere in contact with DCs recovered from infected mice in adose-dependent manner (Fig. 5D). Because we were unable todiscriminate between bystander and infected spleen DCs usingCFSE-labeled parasites after 28 d of infection, the cocultures wereperformed with increased quantities of DCs. This was a necessarystep because at chronic phase, the percentage of actually infectedDCs should be reduced.Because splenic DCs are in their majority MHC IIhigh, we

performed in vitro complementary experiments to evaluate theimpact of surface MHC II in CD4+ polarization. The DP CD4+

T cells were found only when these were polarized by MHC IIhigh–infected BMDCs, whereas MHC IIlow polarizes only IFN-g+ single-producer CD4+ T cells (Fig. 5E), thus demonstrating the differentimpact of high and low MHC II–infected BMDCs on CD4+

T cell polarization. As expected, bystander or noninfected cellswere always unable to generate DP CD4+ T cells independentlyof their MHC II intensity. Altogether, our data demonstrate thatMHC IIhigh–infected DCs have a direct impact on naive T cellpolarization toward the development of Leishmania Ag-specificeffector CD25–T-bet+IFN-g+IL-10+ T cells.

Critical role for IL-12p70 in the development of IFN-g+IL-10+

DP CD4+ T cells

Several cytokines have been proposed to induce DP CD4+ T cells.Therefore, we performed complementary experiments to explorewhich cytokines could be involved in the polarization of DP cellsby infected DCs. As expected, the addition of IL-12p70 increased

the secretion of high levels of IFN-g (Fig. 6A, 6B). In infected Ag-presenting BMDCs, IL-12p70 increased the percentage of DPCD4+ T cells. In contrary, the addition of IL-12p70 had no impacton DP-producing cells from bystander cells. By blocking IL-12p70 activity, we were able to impede the emergence of DPCD4+ T cells (Fig. 6A, 6B). As expected, the addition of IL-12potentially increased the expression of single IFN-g producers,whereas its neutralization by the use of a mAb, but not with mAbisotype control, abrogated IFN-g production. Among all othercytokine cocktails tested, we have found no differences in thepercentage of DP CD4+ T cells induced by infected BMDCs in thepresence of rIL-6, rIL-21, rTNF-a, or rTGF-b, or after ICOS–ICOS ligand blockage (Supplemental Fig. 3A, 3B). IL-27 added atthe beginning of the culture did not impact on DP but abrogatedthe production of IL-10 single-producer cells (Supplemental Fig.3C, 3D). Moreover, the blockage of IL-27 by a specific Ab did notalter the polarization of CD4+ T cells cocultured with infectedBMDCs toward a DP phenotype (Supplemental Fig. 3A, 3B). Wethen quantified by ELISA the presence of endogenous IL-12p70and IL-27 in the coculture experiments. Indeed, a significant in-crease of IL-12p70 levels was found only on cocultures involvingthe infected BMDCs (Fig. 6C), whereas IL-27 levels were increasedat equivalent levels on both bystander and infected BMDCs/CD4+

T cell cocultures (Fig. 6C). These data suggest the main role ofIL-12p70 in triggering the DP CD4+ populations.

CD4+ T cells primed by infected DCs but deprived of T-bet+

IFN-g+IL-10+ DP population restore the immune response andreduce parasite load

The functional in vivo significance of the CD4+ T lymphocytepopulations derived from in vitro cultures with infected or by-stander BMDCs was then evaluated by adoptive transfer in BALB/cmice. Groups of eight age-matched BALB/c mice at the peak ofthe acute phase of infection were i.v. injected with 1 3 106 viableCD4+ T cells polarized with bystander (CD4 byst) or infected

FIGURE 6. Critical role for IL-

12p70 in the development of IFN-g+

IL-10+ DP CD4+ T cells. Sorted

CD11c+ bystander, infected, or non-

infected BMDCs from BALB/c mice

were cocultured with syngeneic naive

BALB/c CD4+ T cells in the presence

or absence of rIL-12p70 (10 ng/ml),

anti–IL-12p40, or the correspondent

isotype control (10 mg/ml), and the

intracellular levels of IFN-g and IL-

10 on CD4+ T cells were measured

after PMA/Ionomycin stimulation (A).

The mean 6 SD from one represen-

tative experiment out of four is shown.

*p, 0.05, **p, 0.01, ***p, 0.001

in comparison with isotype control or

mock treated (None) (B). The levels of

IL-12p70 and IL-27 were quantified

on the supernatant of sorted CD11c+

bystander, infected, or noninfected

BMDCs cultured in the absence

(2CD4+ T cells) or presence (+CD4+

T cells) of naive CD4+ T cells (C). The

mean 6 SD from one representative

experiment out of three is shown.

*p, 0.05, **p, 0.01, ***p, 0.001.

8 INFECTED DCs INDUCE IFN-g+ IL-10+ Th1 LYMPHOCYTES

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

BMDCs (CD4 inf). As controls, we transferred equal numbers ofCD4 CD11c or treated the mice with an equivalent volume of PBS(Fig. 7A). Subsequent assessments of parasite burden in the spleenand liver of all groups of mice were made at 15 d after adoptivetransfer. Remarkably, the transfer of CD4+ T cells primed bybystander DCs induced a significant decrease in liver and spleenparasite burden (1.54 and 1.31 log, respectively) when comparedwith mice that received PBS or CD4 CD11c (Fig. 7A). Moreimportantly, this protection was lost when CD4+ T cells werepolarized with infected DCs. Indeed, the transfer of a CD4+

population containing T-bet+IFN-g+IL-10+ T cells not only failedto induce any protection in the liver (6.24 6 0.16 for CD4 infagainst 5.84 6 0.23 and 5.56 6 0.22 for PBS and CD4 CD11c,respectively), but it could even increase splenic parasite burden(6.71 6 0.15 for CD4 inf against 5.91 6 0.46 and 5.72 6 0.22 forPBS and CD4 CD11c, respectively; Fig. 7A). The levels of IL-10and IFN-g produced by splenocytes after stimulation with SLA(25 mg/ml) demonstrated an increase in Leishmania-specific IFN-g secretion in the group receiving CD4 byst cells, whereas spleencells from mice that were adoptively transferred with CD4 inf(containing the T-bet+IFN-g+IL-10+ population) displayed a pref-erential secretion of IL-10 (Fig. 7B). This led to a disequilibriumof the IFN-g/IL-10 ratio, a known indicator of the infection out-come (19), contributing to an impaired effective immune responseand prolonged parasite persistence.To ensure that the observed effect was related to IL-10 from

lymphoid origin, we performed the same assay in the context of anIL-10–free system therefore absent of T-bet+IFN-g+IL-10+ T cells.

IL-10ko CD4+ T cells were polarized with noninfected, bystander,or infected IL-10ko BMDCs. No significant differences werefound on the levels of IFN-g produced by the polarized CD4+

T cells (Supplemental Fig. 4A, 4B). Once more, the adoptivetransfer of CD4+ T cells polarized with bystander BMDCs in-duced a significant decrease in liver and spleen parasite burden(1.45 and 1.40 log, respectively; Fig. 7C). Importantly, CD4+

T cells primed by infected DCs in an IL-10–free system reducedsignificantly the parasite burden in both the liver and spleens ofinfected mice. These results clearly demonstrated the critical roleof T-bet+IFN-g+IL-10+ cells in the maintenance of infection.

DiscussionDCs are specialized APCs that play a crucial role in drivingadaptive immune responses. However, most of the analyses per-formed on DC functions upon protozoan infections are generallyperformed without discriminating between the infected and thebystander (exposed but noninfected) populations. Exploring theCFSE-labeled L. infantum-BMDCs model allowed us to underlinethe existence of two major distinct DC subsets with opposite rolesfor T cell activation and polarization. Hence when facing a ma-tured DC, Leishmania induces the secretion of myeloid IL-10 thatlimits T cell activation and proliferation. On the other side, thebystander population, which contacted with the parasite or withparasite-secreted products, presents increased transcription levelsof inflammatory cytokines being capable to induce CD4+ T cellactivation and proliferation with immune protective capabilities.Our results demonstrated that in vitro or ex vivo only infected DCs

FIGURE 7. Adoptive transfer of T-

bet+IFN-g+IL-10+ T cells is associated

with incapacity to mount an effective

immune response. Sorted bystander,

infected, or noninfected BMDCs from

BALB/c mice were cocultured with

syngeneic naive BALB/c CD4+ T cells

(A). After 5 d of coculture, CD4+ T

lymphocytes of each one of the cul-

tures (CD4 byst, CD4 Inf, CD4 CD11c

corresponding to the source of DCs

that were used in the coculture, by-

stander, infected, or noninfected, re-

spectively) were sorted and injected

i.v. in previously infected BALB/c

mice. Two weeks later, the spleens and

livers were recovered and parasite loads

determined in all groups. The levels of

IFN-g and IL-10 were measured by

ELISA in the supernatant of spleno-

cytes cultured in the presence or ab-

sence of SLAs for 3 d (B). A similar

experiment as shown in (A) was per-

formed with BMDC and CD4+ T cells

from IL-10ko origin (C). Each value

represents the mean 6 SD from at

least eight mice per group (*p , 0.05,

**p , 0.01, ***p , 0.001).

The Journal of Immunology 9

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

induced the polarization of naive CD4+ T cells toward an IFN-g+

IL-10+ DP phenotype. This phenotype of DP cells correspondsphenotypically and functionally to the IL-10–producing Th1identified in cutaneous or VL lesions (7, 11, 23, 26). Most im-portantly, we demonstrate that the adoptive transfer of DP cellsfavors disease progression and highlights the importance of IL-10as a major factor inhibiting parasite elimination. In contrary, theadoptive transfer of CD4+ T cells polarized in the presence ofbystander DCs displayed a striking phenotype with reducedsplenic burden and enhanced IFN-g production. Therefore, ourresults contrast with other infectious models such as malaria orToxoplasma, where the generation of DP cells is protective (8, 27).The general consensus for an effective response toward all

forms of leishmaniasis is the preferential development of Th1-mediated immune response. Nevertheless, patients with activeVL disease present high levels of IFN-g and IL-12p70 that areconcomitantly detected with elevated IL-10 production (2, 23, 28).Among its cellular sources, IL-10 of myeloid origin has been longassociated with disease progression (29–31). In this study, wedemonstrate that the secreted IL-10 from infected DCs impaired tosome extent the development of an adaptive response by de-creasing the activation and proliferation of Ag-specific CD4+

T cells. More importantly, recent data indicated that IL-10–pro-ducing Th1 cells, which are activated early in a strong inflam-matory setting, are the critical mediators of immune suppressionin a chronic cutaneous or VL (7, 23). The detailed characterizationof this population indicated that the DP cells are T-bet+, Foxp32,and CD252 while maintaining an effector phenotype as previ-ously described (CD62L2 CD44+) (7, 9, 24). Moreover, the im-pact of the adoptive transfer of IFN-g+IL-10+ DP cells on diseaseprogression highlights the importance of lymphoid IL-10 asa major factor inhibiting parasite elimination because the adoptivetransfer of T cells deficient for IL-10 cultured in the same con-ditions do not exacerbate disease progression.Our data also demonstrated the dynamics of DP CD4+ T cells

during acute and chronic infection. A clear increase of this pop-ulation was observed upon infection, reaching its peak in thetransition to chronic phase (day 28). Although the observed con-traction phase of DP cells is in late chronic phase, their relativeabundance among single IFN-g+– or IL-10+–producing cells issustained at high levels. This suggests a modification of thesplenic microenvironment toward a more parasite-permissive cy-tokine enrichment. Importantly, we demonstrated that splenic DCsrecovered at day 28 postinfection were very efficient in the in-duction of DP cells. Thus, our data demonstrate a previouslyproposed association between the frequency of IFN-g+IL-10+ DPCD4+ T cells and susceptibility in leishmaniasis (7, 11, 32). Arecent study underlined, in a resistant model (C57BL/6 mice) ofvisceral L. donovani infection, also the role of splenic CD11chigh

DCs in the development of IL-10–producing Th1 cells and diseaseprogression (10). Nevertheless, the differential behavior amonginfected and bystander DCs and the cytokines involved in CD4T cell polarization were not explored.It is well-known that the balance of IL-10 and IL-12 is central

in determining T cell activation (33). Our results indicated thatinfected DCs control the level of activation of naive T cellsthrough their capacity to secrete IL-10 because once this cyto-kine is blocked, the amount of IFN-g–producing CD4+ T cellsinduced is increased to levels similar to the ones induced by by-stander or noninfected DCs. However, we found that these DPcells acquire a striking capacity to proliferate secondary to a re-exposure to Leishmania Ag presentation. Most interestingly, wedemonstrated that IL-12p70 play a role in the polarization ofIFN-g+IL-10+ DP CD4+ T in our experimental settings. Thus, the

blockage of IL-12p70 abrogated the generation of such pop-ulation. In symptomatic VL, patients present significantly higherlevels of IL-12 than asymptomatic or healthy individuals in thesame endemic area (28, 34). Although infected DCs did not up-regulate IL-12p40 or IL-12p35 mRNA transcription upon L.infantum infection, we detect higher levels of IL-12 in thesupernatants coculture. This apparent paradox may be explainedby the requirement of DC–T cell surface interactions for the se-cretion of large amounts of IL-12p70 (35). In addition, membrane-associated IL-12p70 stores were shown to be released by humanand murine DCs after in vitro or in vivo contact with visceralLeishmania species (36). Overall, our data demonstrate that IL-12is needed to prime CD4+ T cells toward a DP phenotype.Other stimuli such as IL-27 (8, 37), IL-21 (38), TGF-b (39),

ICOS, and the transcription factor c-maf (40) have also beenshown to drive IL-10 expression. IL-27 was shown in differentinfectious models to be crucial in the development of IFN-g+IL-10+ DP CD4+ T cells (8, 41, 42). More recently, a correlativepresence of IL-27–producing splenic DCs and DP CD4+ T po-larization in vivo was shown, although a direct link was not proved(10). In our DC–L. infantum experimental model, we failed toconfirm this link. Not only did IL-27 fail to induce DP cells, butwe did not detect any increment on the transcription or the se-cretion levels of IL-27p28, EBI3, or IL-27 upon infection. Instead,in this study, we found that the addition of IL-27 inhibits theexpression of IL-10.An ongoing debate discusses the DC subtypes responsible for

the induction of protective immunity against Leishmania infection.Leishmania parasites in different steps of the infection processwill interact with DCs presenting distinct maturation degrees. Inthe course to visceralization, Leishmania parasites interact withboth MHC IIhigh dermal DC immigrants and epidermal Langer-hans cells (43). In addition, MHC IIint dermal monocyte-derivedDCs (44) or even resident MHC IIlow lymphoid tissue DCs (45)were suggested to play a preponderant role in the development ofT cell immunity against pathogens. Once in the spleen, Leish-mania will contact with DCs expressing high levels of cell-surfaceMHC II molecules (10, 46). In this article, we demonstrated thatonly infected MHC IIhigh DCs, both of in vitro or ex vivo origin,were capable of inducing the polarization of CD4+ T cells towarda DP phenotype. Moreover, only infected MHC IIhigh BMDCsshowed significant increased levels of Il10 and Tnfa, whereasinfected MHC IIlow did not modify the expression of any of thetested cytokines. Finally, the profile of bystander MHC IIhigh

revealed increased Il12p40 and Il6 transcripts, which was notsurprising because the majority of bystander cells displayeda matured phenotype. Although high levels of MHC II surfaceexpression on DCs can be viewed as essential for Ag presentationand DP polarization, our results revealed a profound difference incytokine expression, which probably represents the main deter-minant in DP polarization that we observed. Thus, MHC IIlow didnot express IL-10. Altogether, our results highlight a new level ofcomplexity in Leishmania–DCs interaction and suggest that futurestudies should dissect DC immune responses in view of MHC IIexpression.Our results highlight novel subversion mechanisms used by L.

infantum parasites. We demonstrate a clear dichotomy betweenbystander and infected BDMCs revealing opposite roles on T cellactivation and polarization. First, IL-10 secreted from MHC IIhigh

DCs is capable to restrain to a certain amount the activation andproliferation of CD4+ T cells. Second, our work identified for thefirst time, to our knowledge, an infectious model where IL-12p70–driven IFN-g+IL-10+ DP CD4+ Th1 cells play a critical role in themaintenance of protozoan infection, underlying the role of lym-

10 INFECTED DCs INDUCE IFN-g+ IL-10+ Th1 LYMPHOCYTES

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

phoid IL-10 as judged by the adoptive transfer of IL-10ko CD4+

T cells contrasting the beneficial role of this subset (IFN-g+IL-10+

DP cells) in T. gondii or Plasmodium spp. infections. Our obser-vation that IL-12p70 favors nonprotective T-bet+IFN-g+IL-10+

T cells without any involvement of IL-27 unravel a potentialdichotomy of IL-27/IL12p70 in driving protective/pathogenic IFN-g+IL-10+ DP Th1 responses. Thus, the identification of the mech-anisms by which IFN-g+IL-10+ DP CD4+ T populations are inducedin the context of Leishmania infection could represent a newstrategic therapeutic target.

AcknowledgmentsWe thank Catarina Leitao (IBMC, Porto University, Portugal) and Sofia

Lamas (IBMC) for all the technical help.

DisclosuresThe authors have no financial conflicts of interest.

References1. Kaye, P., and P. Scott. 2011. Leishmaniasis: complexity at the host-pathogen

interface. Nat. Rev. Microbiol. 9: 604–615.2. Karp, C. L., S. H. el-Safi, T. A. Wynn, M. M. Satti, A. M. Kordofani,

F. A. Hashim, M. Hag-Ali, F. A. Neva, T. B. Nutman, and D. L. Sacks. 1993.In vivo cytokine profiles in patients with kala-azar. Marked elevation of bothinterleukin-10 and interferon-gamma. J. Clin. Invest. 91: 1644–1648.

3. Ghalib, H. W., M. R. Piuvezam, Y. A. Skeiky, M. Siddig, F. A. Hashim, A. M. el-Hassan, D. M. Russo, and S. G. Reed. 1993. Interleukin 10 production correlateswith pathology in human Leishmania donovani infections. J. Clin. Invest. 92:324–329.

4. Murray, H. W., C. M. Lu, S. Mauze, S. Freeman, A. L. Moreira, G. Kaplan, andR. L. Coffman. 2002. Interleukin-10 (IL-10) in experimental visceral leish-maniasis and IL-10 receptor blockade as immunotherapy. Infect. Immun. 70:6284–6293.

5. Murray, H. W., A. L. Moreira, C. M. Lu, J. L. DeVecchio, M. Matsuhashi, X. Ma,and F. P. Heinzel. 2003. Determinants of response to interleukin-10 receptorblockade immunotherapy in experimental visceral leishmaniasis. J. Infect. Dis.188: 458–464.

6. O’Garra, A., and P. Vieira. 2007. T(H)1 cells control themselves by producinginterleukin-10. Nat. Rev. Immunol. 7: 425–428.

7. Anderson, C. F., M. Oukka, V. J. Kuchroo, and D. Sacks. 2007. CD4(+)CD25(-)Foxp3(-) Th1 cells are the source of IL-10-mediated immune suppression inchronic cutaneous leishmaniasis. J. Exp. Med. 204: 285–297.

8. Freitas do Rosario, A. P., T. Lamb, P. Spence, R. Stephens, A. Lang, A. Roers,W. Muller, A. O’Garra, and J. Langhorne. 2012. IL-27 promotes IL-10 pro-duction by effector Th1 CD4+ T cells: a critical mechanism for protection fromsevere immunopathology during malaria infection. J. Immunol. 188: 1178–1190.

9. Jankovic, D., M. C. Kullberg, C. G. Feng, R. S. Goldszmid, C. M. Collazo,M. Wilson, T. A. Wynn, M. Kamanaka, R. A. Flavell, and A. Sher. 2007.Conventional T-bet(+)Foxp3(-) Th1 cells are the major source of host-protectiveregulatory IL-10 during intracellular protozoan infection. J. Exp. Med. 204: 273–283.

10. Owens, B. M., L. Beattie, J. W. Moore, N. Brown, J. L. Mann, J. E. Dalton,A. Maroof, and P. M. Kaye. 2012. IL-10-producing Th1 cells and disease pro-gression are regulated by distinct CD11c⁺ cell populations during visceralleishmaniasis. PLoS Pathog. 8: e1002827.

11. Stager, S., A. Maroof, S. Zubairi, S. L. Sanos, M. Kopf, and P. M. Kaye. 2006.Distinct roles for IL-6 and IL-12p40 in mediating protection against Leishmaniadonovani and the expansion of IL-10+ CD4+ T cells. Eur. J. Immunol. 36: 1764–1771.

12. Jankovic, D., D. G. Kugler, and A. Sher. 2010. IL-10 production by CD4+ ef-fector T cells: a mechanism for self-regulation. Mucosal Immunol. 3: 239–246.

13. Khan, N., U. Gowthaman, S. Pahari, and J. N. Agrewala. 2012. Manipulation ofcostimulatory molecules by intracellular pathogens: veni, vidi, vici!! PLoSPathog. 8: e1002676.

14. Carvalho, L. P., E. J. Pearce, and P. Scott. 2008. Functional dichotomy of den-dritic cells following interaction with Leishmania braziliensis: infected cellsproduce high levels of TNF-alpha, whereas bystander dendritic cells are acti-vated to promote T cell responses. J. Immunol. 181: 6473–6480.

15. Moreira, D., N. Santarem, I. Loureiro, J. Tavares, A. M. Silva, A. M. Amorim,A. Ouaissi, A. Cordeiro-da-Silva, and R. Silvestre. 2012. Impact of continuousaxenic cultivation in Leishmania infantum virulence. PLoS Negl. Trop. Dis. 6:e1469.

16. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, andG. Schuler. 1999. An advanced culture method for generating large quantities ofhighly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:77–92.

17. Neves, B. M., R. Silvestre, M. Resende, A. Ouaissi, J. Cunha, J. Tavares,I. Loureiro, N. Santarem, A. M. Silva, M. C. Lopes, et al. 2010. Activation of

phosphatidylinositol 3-kinase/Akt and impairment of nuclear factor-kappaB:molecular mechanisms behind the arrested maturation/activation state ofLeishmania infantum-infected dendritic cells. Am. J. Pathol. 177: 2898–2911.

18. Kalupahana, R. S., P. Mastroeni, D. Maskell, and B. A. Blacklaws. 2005. Ac-tivation of murine dendritic cells and macrophages induced by Salmonellaenterica serovar Typhimurium. Immunology 115: 462–472.

19. Silvestre, R., A. Cordeiro-Da-Silva, N. Santarem, B. Vergnes, D. Sereno, andA. Ouaissi. 2007. SIR2-deficient Leishmania infantum induces a defined IFN-gamma/IL-10 pattern that correlates with protection. J. Immunol. 179: 3161–3170.

20. Mathew, S., Y. C. Lim, and A. Kishen. 2008. Quenching of fluorescence bycrystal violet and its use to differentiate between surface-bound and internalizedbacteria. Proc. SPIE 6791, Saratov Fall Meeting 2007: Optical Technologies inBiophysics and Medicine IX, 67910C.

21. Masurier, C., C. Pioche-Durieu, B. M. Colombo, R. Lacave, F. M. Lemoine,D. Klatzmann, and M. Guigon. 1999. Immunophenotypical and functional het-erogeneity of dendritic cells generated from murine bone marrow cultured withdifferent cytokine combinations: implications for anti-tumoral cell therapy. Im-munology 96: 569–577.

22. Menges, M., T. Baumeister, S. Rossner, P. Stoitzner, N. Romani, A. Gessner, andM. B. Lutz. 2005. IL-4 supports the generation of a dendritic cell subset frommurine bone marrow with altered endocytosis capacity. J. Leukoc. Biol. 77: 535–543.

23. Nylen, S., R. Maurya, L. Eidsmo, K. D. Manandhar, S. Sundar, and D. Sacks.2007. Splenic accumulation of IL-10 mRNA in T cells distinct from CD4+CD25+ (Foxp3) regulatory T cells in human visceral leishmaniasis. J. Exp. Med. 204:805–817.

24. Pagan, A. J., N. C. Peters, A. Debrabant, F. Ribeiro-Gomes, M. Pepper, C. L.Karp, M. K. Jenkins, and D. L. Sacks. 2013. Tracking antigen-specific CD4(+)T cells throughout the course of chronic Leishmania major infection in resistantmice. Eur. J. Immunol. 43: 427–38.

25. Taga, K., H. Mostowski, and G. Tosato. 1993. Human interleukin-10 can directlyinhibit T-cell growth. Blood 81: 2964–2971.

26. Ranatunga, D., C. M. Hedrich, F. Wang, D. W. McVicar, N. Nowak, T. Joshi,L. Feigenbaum, L. R. Grant, S. Stager, and J. H. Bream. 2009. A human IL10BAC transgene reveals tissue-specific control of IL-10 expression and altersdisease outcome. Proc. Natl. Acad. Sci. USA 106: 17123–17128.

27. Hall, A. O., D. P. Beiting, C. Tato, B. John, G. Oldenhove, C. G. Lombana,G. H. Pritchard, J. S. Silver, N. Bouladoux, J. S. Stumhofer, et al. 2012. Thecytokines interleukin 27 and interferon-g promote distinct Treg cell populationsrequired to limit infection-induced pathology. Immunity 37: 511–523.

28. Milano, S., G. Di Bella, P. D’Agostino, C. Barbera, R. Caruso, M. La Rosa,V. Ferlazzo, G. Vitale, C. La Russa, G. Gambino, et al. 2002. IL-15 in humanvisceral leishmaniasis caused by Leishmania infantum. Clin. Exp. Immunol. 127:360–365.

29. Kane, M. M., and D. M. Mosser. 2001. The role of IL-10 in promoting diseaseprogression in leishmaniasis. J. Immunol. 166: 1141–1147.

30. Nandan, D., C. Camargo de Oliveira, A. Moeenrezakhanlou, M. Lopez,J. M. Silverman, J. Subek, and N. E. Reiner. 2012. Myeloid cell IL-10 productionin response to leishmania involves inactivation of glycogen synthase kinase-3bdownstream of phosphatidylinositol-3 kinase. J. Immunol. 188: 367–378.

31. Padigel, U. M., and J. P. Farrell. 2005. Control of infection with Leishmaniamajor in susceptible BALB/c mice lacking the common gamma-chain for FcR isassociated with reduced production of IL-10 and TGF-beta by parasitized cells.J. Immunol. 174: 6340–6345.

32. Kemp, K., M. Kemp, A. Kharazmi, A. Ismail, J. A. Kurtzhals, L. Hviid, andT. G. Theander. 1999. Leishmania-specific T cells expressing interferon-gamma(IFN-gamma) and IL-10 upon activation are expanded in individuals cured ofvisceral leishmaniasis. Clin. Exp. Immunol. 116: 500–504.

33. O’Garra, A., and K. M. Murphy. 2009. From IL-10 to IL-12: how pathogens andtheir products stimulate APCs to induce T(H)1 development. Nat. Immunol. 10:929–932.

34. Hailu, A., T. van der Poll, N. Berhe, and P. A. Kager. 2004. Elevated plasmalevels of interferon (IFN)-gamma, IFN-gamma inducing cytokines, and IFN-gamma inducible CXC chemokines in visceral leishmaniasis. Am. J. Trop.Med. Hyg. 71: 561–567.

35. Marovich, M. A., M. A. McDowell, E. K. Thomas, and T. B. Nutman. 2000. IL-12p70 production by Leishmania major-harboring human dendritic cells isa CD40/CD40 ligand-dependent process. J. Immunol. 164: 5858–5865.

36. Quinones, M., S. K. Ahuja, P. C. Melby, L. Pate, R. L. Reddick, and S. S. Ahuja.2000. Preformed membrane-associated stores of interleukin (IL)-12 are a previ-ously unrecognized source of bioactive IL-12 that is mobilized within minutes ofcontact with an intracellular parasite. J. Exp. Med. 192: 507–516.

37. Stumhofer, J. S., J. S. Silver, A. Laurence, P. M. Porrett, T. H. Harris,L. A. Turka, M. Ernst, C. J. Saris, J. J. O’Shea, and C. A. Hunter. 2007. Inter-leukins 27 and 6 induce STAT3-mediated T cell production of interleukin 10.Nat. Immunol. 8: 1363–1371.

38. Spolski, R., H. P. Kim, W. Zhu, D. E. Levy, and W. J. Leonard. 2009. IL-21mediates suppressive effects via its induction of IL-10. J. Immunol. 182: 2859–2867.

39. Huss, D. J., R. C. Winger, H. Peng, Y. Yang, M. K. Racke, and A. E. Lovett-Racke. 2010. TGF-beta enhances effector Th1 cell activation but promotes self-regulation via IL-10. J. Immunol. 184: 5628–5636.

40. Pot, C., H. Jin, A. Awasthi, S. M. Liu, C. Y. Lai, R. Madan, A. H. Sharpe,C. L. Karp, S. C. Miaw, I. C. Ho, and V. K. Kuchroo. 2009. Cutting edge: IL-27induces the transcription factor c-Maf, cytokine IL-21, and the costimulatory

The Journal of Immunology 11

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

receptor ICOS that coordinately act together to promote differentiation of IL-10-producing Tr1 cells. J. Immunol. 183: 797–801.

41. Ansari, N. A., R. Kumar, S. Gautam, S. Nylen, O. P. Singh, S. Sundar, andD. Sacks. 2011. IL-27 and IL-21 are associated with T cell IL-10 responses inhuman visceral leishmaniasis. J. Immunol. 186: 3977–3985.

42. Batten, M., J. Li, S. Yi, N. M. Kljavin, D. M. Danilenko, S. Lucas, J. Lee, F. J. deSauvage, and N. Ghilardi. 2006. Interleukin 27 limits autoimmune encephalo-myelitis by suppressing the development of interleukin 17-producing T cells.Nat. Immunol. 7: 929–936.

43. Brewig, N., A. Kissenpfennig, B. Malissen, A. Veit, T. Bickert, B. Fleischer,S. Mostbock, and U. Ritter. 2009. Priming of CD8+ and CD4+ T cells in ex-

perimental leishmaniasis is initiated by different dendritic cell subtypes. J.Immunol. 182: 774–783.

44. Leon, B., M. Lopez-Bravo, and C. Ardavın. 2007. Monocyte-derived dendriticcells formed at the infection site control the induction of protective T helper 1responses against Leishmania. Immunity 26: 519–531.

45. Iezzi, G., A. Frohlich, B. Ernst, F. Ampenberger, S. Saeland, N. Glaichenhaus,and M. Kopf. 2006. Lymph node resident rather than skin-derived dendritic cellsinitiate specific T cell responses after Leishmania major infection. J. Immunol.177: 1250–1256.

46. Prasad, S. J., and C. C. Goodnow. 2002. Cell-intrinsic effects of non-MHC NODgenes on dendritic cell generation in vivo. Int. Immunol. 14: 677–684.

12 INFECTED DCs INDUCE IFN-g+ IL-10+ Th1 LYMPHOCYTES

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

1

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

2

S1. Maturation status and survival of bystander and L. infantum infected BDMCs. The

percentage of infected cells in relation to their surface MHCII expression was performed

accordingly to the depicted gating strategy (A). The percentage of infected MHCIIlow or high

is given by the percentage of the respective infected population over the corresponding

bystander cells (B). BMDCs were sorted according to their surface MHCII expression (high

or low) and infected with CFSE-labeled parasites. After 24 hours, the MHCII intensity on

bystander and infected cells as well as the percentage of infection on MHCIIlow and

MHCIIhigh populations were determined by flow cytometry (C). Sorted MHCII (high or

low) BMDCs were infected with CFSE-labeled L. infantum or cultured with irradiated

CFSE-parasites or FITC-Beads and their uptake analyzed by flow cytometry. The

percentage of uptake in each case is shown (D). Representative immunofluorescence

pictures are depicted for each gated population (a, b, c and d) according the gate strategy

shown on figure S2C. The scale bar represent 10 μm (E). Sorted MHCIIhigh or low bystander,

infected or non-infected BMDCs from Balb/c mice were co-cultured with syngeneic naïve

Balb/c CD4+ T cells. Thirty-six hours later, the activation of CD4+ T lymphocytes were

measured through CD69 expression (F). RNA levels of the indicated cytokines were

determined by qPCR for MHCIIhigh (G) or MHCIIlow (H) infected and bystander BMDCs

sorted as depicted on Fig. S1A. Values are normalized for non-infected cells MHCIIhigh or

low, respectively (A-B). Sorted MHCIIhigh bystander, infected or non-infected BMDCs from

BALB/c mice were co-cultured with syngeneic naïve BALB/c CD4+ T cells. Seventy-two

hours later the proliferation of CD4+ T lymphocytes were measured through CFSE decay in

the presence of 10 ng/ml of IL-2 (I). The mean ± S.D. from one representative experiment

out of three is shown (J). Each value represents the mean ± S.D. from at least four

independent experiments. (*p<0.05; ** p<0.01; *** p<0.001).

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

3

S2. Infected bone marrow-derived macrophages (BMM) are unable to induce Th1

CD4+ T cells with IFN- +IL-10+ double producer phenotype. Sorted bystander, infected

or non-infected BMM from BALB/c mice were co-cultured with naïve BALB/c CD4+ T

cells. After 5 days of co-culture, the IFN- and IL-10 intracellular levels were determined

on CD4+ T cells. The results are from a representative experiment of three carried out

independently.

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

4

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

5

S3. The addition or blockage of distinct anti or pro-inflammatory cytokines does not

modifies the IFN- + IL-10+ double producer phenotype induced by infected DCs.

Sorted bystander, infected or non-infected BMDCs from BALB/c mice were co-cultured

with naïve BALB/c CD4+ T cells in the presence of distinct cytokines (IL-6, IL-21, TNF- ,

TGF- ; added at a final concentration of 10 ng/ml) or in the presence of mAb against

CD275 (ICOSL), IL-27p28 or the correspondent isotype controls (all mAb were added at a

final concentration of 10 μg/ml). The IFN- and IL-10 intracellular levels were determined

on CD4+ T cells after 5 days of co-culture (A). The mean ± S.D. from one representative

experiment out of three is shown (B). Similarly, sorted bystander, infected or non-infected

BMDCs from BALB/c mice were co-cultured with naïve BALB/c CD4+ T cells in the

presence of IL-27 or IL-27 plus IL-12p70 (all cytokines added at a final concentration of 10

ng/ml). The IFN- and IL-10 intracellular levels were determined on CD4+ T cells after 5

days of co-culture (C). The mean ± S.D. from one representative experiment out of three is

shown (D) (*p<0.05).

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from

6

S4. Profile of naïve CD4+ T cells from IL-10 knockout mice co-cultured with

bystander, infected or non-infected BMDCs from IL-10 knockout mice. Sorted

bystander, infected or non-infected BMDCs from IL-10ko mice were co-cultured with

syngeneic naïve IL-10ko CD4+ T cells. After 5 days of co-culture, the IFN- and IL-10

intracellular levels were determined on CD4+ T cells (A). The mean ± S.D. from one

representative experiment out of three is shown (B) (*p<0.05).

at INIST

CN

RS B

iblioVie on June 3, 2013

http://ww

w.jim

munol.org/

Dow

nloaded from