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of April 13, 2018. This information is current as Expression Profiling Revealed by Comparative Gene Gallus gallus Existence of Conventional Dendritic Cells in Schwartz-Cornil and Pascale Quéré Le Vern, Bernd Kaspers, Marc Dalod, Isabelle Thien-Phong Vu Manh, Hélène Marty, Pierre Sibille, Yves http://www.jimmunol.org/content/192/10/4510 doi: 10.4049/jimmunol.1303405 April 2014; 2014; 192:4510-4517; Prepublished online 16 J Immunol Material Supplementary 5.DCSupplemental http://www.jimmunol.org/content/suppl/2014/04/16/jimmunol.130340 References http://www.jimmunol.org/content/192/10/4510.full#ref-list-1 , 13 of which you can access for free at: cites 34 articles This article average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive 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 © 2014 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on April 13, 2018 http://www.jimmunol.org/ Downloaded from by guest on April 13, 2018 http://www.jimmunol.org/ Downloaded from

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of April 13, 2018.This information is current as

Expression Profiling Revealed by Comparative GeneGallus gallus

Existence of Conventional Dendritic Cells in

Schwartz-Cornil and Pascale QuéréLe Vern, Bernd Kaspers, Marc Dalod, Isabelle Thien-Phong Vu Manh, Hélène Marty, Pierre Sibille, Yves

http://www.jimmunol.org/content/192/10/4510doi: 10.4049/jimmunol.1303405April 2014;

2014; 192:4510-4517; Prepublished online 16J Immunol 

MaterialSupplementary

5.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/04/16/jimmunol.130340

Referenceshttp://www.jimmunol.org/content/192/10/4510.full#ref-list-1

, 13 of which you can access for free at: cites 34 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive 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 © 2014 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Existence of Conventional Dendritic Cells in Gallus gallusRevealed by Comparative Gene Expression Profiling

Thien-Phong Vu Manh,*,†,‡,1 Helene Marty,†,x,{,1 Pierre Sibille,‖ Yves Le Vern,x,{

Bernd Kaspers,# Marc Dalod,*,†,‡,2 Isabelle Schwartz-Cornil,‖,2 and Pascale Querex,{,2

The existence of conventional dendritic cells (cDCs) has not yet been demonstrated outside mammals. In this article, we identified

bona fide cDCs in chicken spleen. Comparative profiling of global and of immune response gene expression, morphology, and T cell

activation properties show that cDCs and macrophages (MPs) exist as distinct mononuclear phagocytes in the chicken, resembling

their human and mouse cell counterparts. With computational analysis, core gene expression signatures for cDCs, MPs, and Tand

B cells across the chicken, human, and mouse were established, which will facilitate the identification of these subsets in other

vertebrates. Overall, this study, by extending the newly uncovered cDC and MP paradigm to the chicken, suggests that these

two phagocyte lineages were already in place in the common ancestor of reptiles (including birds) and mammals in evolution.

It opens avenues for the design of new vaccines and nutraceuticals that are mandatory for the sustained supply of poultry products

in the expanding human population. The Journal of Immunology, 2014, 192: 4510–4517.

Conventional dendritic cells (cDCs) are functionally char-acterized by their exquisite capacities to present Agsto naive T cells, having a key role in maintenance of

tolerance and induction of immune effectors against invadingpathogens (1). cDCs constitute a unique immune cell lineage, asrecently revealed in mice by genetic cell fate mapping (2) and theprecursor–progeny relationship at the single-cell level (3) and inhumans by comparative gene expression profiling (4). Mouse

cDCs derive from a clonogenic common DC progenitor in thebone marrow and are ontogenetically distinct from other mono-nuclear phagocytes, such as macrophages (MPs), monocyte -de-rived dendritic cells and yolk sac– and fetal liver–derived skinLangerhans cells (LCs) (1, 5–7). Compared with other mononu-clear phagocytes, cDCs are endowed with a much higher efficacyin patrolling virtually all peripheral tissues and in migrating tolymph nodes both at steady state and upon stimulation. Further-more, cDCs and the other types of mononuclear phagocytes aredifferentially involved in setting and tuning specific arms of im-munity, depending on tissue location, inflammatory response, andpathogens (1). Finally, cDCs include two subsets, the CD8a+ andCD11b+ types in the mouse and their homologous BDCA3+

and BDCA1+ types in humans, respectively, that display distinctgene expression programs, surface phenotypes, and functionalspecialization (4, 8, 9). In particular, the mouse CD8a+ type, and,to some extent, the human BDCA3+ type cDCs, are specialized incross-presentation of endocytosed Ags to CD8+ T cells (8, 10–15).The cDCs have been described only in mammals. Nonmamma-

lian vertebrates, such as fishes and birds, have the same basicprinciples of adaptive immunity as mammals, but they presentmany evolutionary particularities, with different repertoires ofgenes, cells, and lymphoid organs, including absence of lymphnodes (16). In these species, cells with dendritic morphology thatseem to be distinct from MPs have been described (16–18). Inchicken in vitro, DC-like cells have been obtained upon differ-entiation of bone marrow cells in GM-CSF and IL-4 cultures (19).In vivo, LCs have been identified in skin (20); in spleen and bursa,which is the primary B cell lymphoid organ in birds, CD83+ cellsand CD205+ cells have been described, the later corresponding toboth putative DCs and MPs (21, 22). A subset of lung phagocytespresenting higher endosomal pH compared with resident MPs hasalso been identified in chicken (23). Although birds seem equippedwith LCs and can develop DC-type cells from bone marrow ex vivo,it is not known whether they dispose of the cDC lineage homolo-gous to that of mammals (23). In addition, whether the biologicaldifferences between cDCs and MPs recently unraveled in mammalspertain to distant vertebrate species such as birds is not known.Gene expression profiling has proved to be a very efficient an-

alytical approach to delineate homologies between mononuclear

*Centre d’Immunologie de Marseille-Luminy, Aix-Marseille University, UM2,13288 Marseille Cedex 9, France; †INSERM, U1104, 13288 Marseille, France;‡Centre National de la Recherche Scientifique, Unite Mixte de Recherche 7280,13288 Marseille, France; xInstitut National de la Recherche Agronomique, UniteMixte de Recherche 1282 Infectiologie et Sante Publique, 37380 Nouzilly, France;{Universite Francois Rabelais de Tours, Unite Mixte de Recherche 1282, 37000Tours, France; ‖Institut National de la Recherche Agronomique, Unite de Recherche892 Virologie et Immunologie Moleculaires, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France; and #Department of Veterinary Sciences, University of Munich,80539 Munich, Germany

1T.-P.V.M. and H.M. contributed equally to this work.

2M.D., I.S.-C., and P.Q. are senior authors.

Received for publication December 23, 2013. Accepted for publication March 14,2014.

This work was supported by institutional funding from the Agence Nationale de laRecherche PhyloGenDC, ANR-09-BLAN-0073-02; the PhyloGenDC ANR grant andthe European Research Council (T.-P.V.M.); and European Research Council FrameProgram 2007-2013 Grant Agreement 281225 for the SystemsDendritic project(M.D.).

I.S.-C. orchestrated the research program; M.D., I.S.-C., and P.Q. designed experi-ments and directed research; I.S.-C. wrote the paper with input from M.D., T.-P.V.M.,H.M., P.S., and P.Q.; H.M., P.S., Y.L.V., I.S.-C., and P.Q. performed experiments andanalyzed data; T.-P.V.M. and M.D. carried out bioinformatics analyses; and B.K.provided reagents and array annotations.

The microarray data presented in this article have been submitted to the Gene Ex-pression Omnibus (http://www.ncbi.nlm.nih.gov/geo/info/) under accession numberGSE55642.

Address correspondence and reprint requests to Dr. Isabelle Schwartz-Cornil, InstitutNational de la Recherche Agronomique, Unite de Recherche 892 Virologie et Im-munologie Moleculaires, Domaine de Vilvert, 78352 Jouy-en-Josas, France. E-mailaddress: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: cDC, conventional dendritic cell; GSEA, gene setenrichment analysis; LC, Langerhans cell; MGG, May–Gr€unwald–Giemsa (stain);MP, macrophage; pDC, plasmacytoid DC; qPCR, qualitative PCR.

Copyright� 2014 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/14/$16.00

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phagocyte subsets of mice, sheep, and humans (4, 24, 25). In thisarticle, a comparative gene expression analysis between chicken(Gallus gallus), human and mouse immune cell subsets shows theexistence in chicken of MPs and of bona fide cDCs that are ca-pable of T cell activation. These data suggest that the establish-ment of the cDC lineage occurred before the common bird/mammalancestor, and is likely to apply to amniotes in general.

Materials and MethodsAnimals

Highly inbred white Leghorn chickens that originate from the GB1 Athensline and are homozygous for the B13 histocompatibility B complex wereused for the cDCs, MPs, and T and B cell characterization. The PA12outbred line was used to isolate CD4+ T cells in the MLR experiments; thePA12 chickens present mainly the B21 MHC haplotype (97%), and a smallproportion of them present the B19 MHC haplotype (3%) (B. Bed’hom,personal). Chickens were 6.5–8.5 wk of age and were raised under specificpathogen–free conditions (Plate-Forme d’Infectiologie Experimentale–Institut National de la Recherche Agronomique, Nouzilly, France). Incompliance with French law, the chickens were euthanized according toprotocols approved by the Animal Ethics Committee (Region Centre,France).

Spleen cell subset isolation

Spleen from four different exsanguinated chickens, indifferently male orfemale, were used for cell isolation. After an incubation of 30 min at 37˚C inRPMI 1640 plus collagenase D (400 U/ml; Roche Applied Science) andDNase (15 mg/ml; Sigma-Aldrich), spleen pieces were crushed on a steelsieve. Isolated splenocytes were washed and centrifuged on a 1.077 densityHistopaque gradient (Sigma-Aldrich) to remove the nucleated erythro-cytes. For B and T cell sorting, splenocytes were labeled with 2 mg/ml PE-conjugated anti–Bu-1 AV20 mAb (26) and PE-conjugated anti-chickenCD3 CT-3 mAb, respectively (Southern Biotech) (27). For MP sorting,splenocytes were labeled with 2 mg/ml PE-conjugated KUL01 mAb (28)and FITC-conjugated anti–MHC-II mAb (Southern Biotech), and theywere sorted as KUL01+ MHC-II+ cells (Southern Biotech). For cDC can-didate cell sorting, splenocytes were first stained with the cell supernatantof the 8F2 hybridoma directed to the putative chicken CD11c (19), fol-lowed by 50 mg/ml Alexa 647–conjugated goat anti-mouse Fab (JacksonImmunoResearch), and subsequently reacted with a mixture of FITC-conjugated mAb anti-chicken MHC-II, PE-conjugated KUL01, anti Bu-1,and anti-CD3 mAbs. The MoFlo Legacy Cell Sorter (Beckman Coulter)was used for the sorting. The labeled cells were analyzed with the FlowJoversion 10 software (TreeStar).

RNA extraction and hybridization on microarrays

Total RNA from subsets was extracted using the PicoPure RNA IsolationKit (Arcturus Life Technologies) and checked for quality with an Agilent2100 Bioanalyzer using RNA 6000 Nano or Pico Kits (Agilent Technologies).All RNA samples had an RNA integrity number .9. Hybridizations wereperformed at the Platform Biopuces et Sequencage (Institut de Genetiqueet de Biologie Moleculaire et Cellulaire, 67400 Illkirch, France, www.igbmc.fr/grandesstructures/cbi/). When insufficient total RNA amountsfor hydridization were obtained (,50 ng), the RNA from the sorted subsetsof distinct spleen pools was mixed. RNA labeling was performed using theone-color Low Input Quick Amp Labeling Kit (Agilent Technologies),following the manufacturer’s recommendations. Each RNA sample (50 ng)was amplified and cyanin 3 labeled, and subsequently the cRNAwas checkedfor quality on Nanodrop and the Agilent 2100 Bioanalyzer. Subsequently,the cRNA (600 ng) was fragmented and used for hybridization on custom-designed Agilent chicken arrays. Our custom-designed array is based onand includes the entire commercial chicken Agilent technologies array[Gallus gallus oligo microarray (V2), 42 K]. About 1100 new probes weredesigned with the eArray software from Agilent Technologies. The newprobes include gene probes derived from a list of candidates known to beselectively expressed in both mouse and human DC population subsets(4). When direct one-to-one orthologs could be identified between humansand chickens (with genomicus V67.01, www.genomicus.biologie.ens.fr/),the new probes were directly included in the chip. Otherwise, the hu-man and murine candidates were blasted on the Gallus gallus transcriptdatabase to select for potential chicken candidates that were used forthe design of new probes. When no orthologous candidates were found,the human and mouse probes were directly included in the chip (60 Karray).

Bioinformatics analyses

The bioinformatics analyses were adapted from previous reports (4, 24).Raw gene expression data were background corrected using the “normexp”method and quantile normalized with the limma package through Bio-conductor in the R statistical environment (version 2.15.0). Independentbiological triplicates (B cells) and quadruplicates (cDCs, MPs, and T cells)were performed using cells isolated from four different animals. Qualitycontrol of the expression data was assessed by boxplots of raw expressiondata, density plots of normalized data, scatter plots, and calculation of thePearson correlation coefficients between arrays, using the Ringo package.The microarray data have been assigned the Gene Expression Omnibusnumber GSE55642 (www.ncbi.nlm.nih.gov/geo/info/), and they are pub-licly available.

To statistically test whether mouse and human cell transcriptional fin-gerprints were enriched in specific chicken cell types, we performedpairwise comparisons between cell types, using the gene set enrichmentanalysis (GSEA) method from the Massachusetts Institute of Technology(www.broad.mit.edu/gsea).

Themouse and human cell-specific transcriptional fingerprints (SupplementalDataset 2) correspond to genes that were found more highly expressed(.1.5 fold) in the cell population of interest compared with all other cellpopulations considered in the preselected compendium of arrays [see listof public database array IDs taken into account in the human and themouse compendia (Supplemental Dataset 1)]. The cut-off of 1.5-fold hasbeen applied using the stringent “min/max” procedure calculating foldchange as (minimum expression among all replicates for all cell typesselected/maximum expression among all other replicates for all other celltypes), as previously published (29). To perform GSEA on chicken ex-pression data, using fingerprints composed of human or mouse genes, weused annotations provided by the Sigenae pipeline on the orthologs in humanor mouse of the chicken genes.

Regulation of immunity-related gene expression in homologous subsetsacross human, mouse, and chicken species. We established a list of 248immunity-related genes (sensors, cytokine, cytokine receptors, signalingreceptors) from which 116 genes 1) were present on the chicken array, 2)gave signals above background in at least one subset, and 3) were differ-entially expressed ($2-fold) across all cell subsets (cDC, MP, and T andB cells). Of these 116 chicken genes, 94 were found to have an orthologousgene on both human and mouse gene chips (except CLEC2B, for which anorthologous gene was present only on the human chip) as well as havinga differential expression of $2-fold across all murine and human cellsubsets. Whenever several probes were found for a given gene, the mosthighly expressed probe across all cell subsets in each species was selected.Gene expression values of the three species were then cross normalized:Each of the three batches of arrays, corresponding to cell subsets from thethree species, were scaled by setting the mean expression value to 0 andthe variance to 1.

Real-time PCR

For relative quantitation of gene expression in cellular subsets, RNA wasreverse transcribed using random primers and SuperScript III ReverseTranscriptase (Invitrogen). Real-time qualitative PCR (qPCR) was carriedout with 10 mM primers in a final reaction volume of 15 ml of 2 iQ SYBRGreen Supermix (Bio-Rad). The primers used to amplify the chicken cDNAwere designed with Primer-BLAST (National Center for BiotechnologyInformation), using publicly available GenBank sequences. The primersare as follows: FLT3 forward: 59-CATTCGGACCCAGTACATGTTTAC-39,and reverse: 59-TGAGCCGTAGAAGAGCAGGTATAA-39; ZBTB46 forward:59-CAGGAACGTCATCGAGGTGAT-39, and reverse: 59-GCCTGGACG-ATATCCGTCAT-39; BATF3 forward: 59-GGAGAGAGAAGAACCGAG-TTGCT-39, and reverse: 59-CGTGAAGTTTGTCCGCTTTC-39; MAFBforward: 59-AGGACCGGTTCTCGGATGAC-39, and reverse: 59-CCTCGG-AGGTGCCTGTTG-39; CD14 forward: 59-CATGCTTGGCAGTCTGCAAA-39, and reverse: 59-CAGGAGGACCTCAGGAACCA-39; CADM1 forward:59-GGAGCGTGGACCATGCA-39, and reverse: 59-CAAAGCATGGCAAA-TACAACCA-39; XCR1 forward: 59-CCTTCGGGTGGATTTTTGGT-39, andreverse: 59-CGCTGTAGTAGCCAATGGAGAA-39; BCL11B forward: 59-GCGATGCCAGTGTAAATGCC-39, and reverse: 59-CACATGGTCAGC-CTGTGTGA-39; PLCG1 forward: 59-CACTGCTTCGTGGTCCTCTA-39,and reverse: 59-CATCCTCGGACGTGGCTTG-39; BEND5 forward: 59-TCTCGAGAATGGAAAGAATGTTTGT-39, and reverse: 59-TGACTTT-GGAGAGTCTCTGTGC-39; C5AR1 forward: 59-AACCTTCAACGA-CATCGGCG-39, and reverse: 59-AAGGAAAATGGCGGCGTAGA-39; CD5forward 59-GCGTTACCTCCACCTCTGTC-39, and reverse: 59-TTCGACT-TCTGGCAGACCAC-39; CD79B forward: 59-TACGTGTGCGACAGCAA-GAA-39, and reverse: 59-TTGCTGCGACTCATCACTCT-39; CIITA forward:

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59-TGCCTGAGAAGTGGTACAGC-39, and reverse: 59-TTGCTTCCCT-CCTGAGGTCT-39; CXCR5 forward: 59-CCCTGACCAAGCTAGAAG-CC-39, and reverse: 59-CCAATGGCCTCTGTCACCAT-39; FAM46C for-ward: 59-AGCTGACCGTTTCTTTGGAGA-39, and reverse: 59-AGTTC-AGCACACTGCATGAC-39; INPP4B forward: 59-CACGGTCCATACC-ACTGTCC-39, and reverse: 59-GCCCAGGAAGCTTCTTCCAA-39; KLHL14forward: 59-CTGGCTGTGCAGTCCTTGAT-39, and reverse: 59-GATTT-GTAGGCCCCCATGCT-39; PLEKHA5 forward: 59-CTGTGGGCAGAA-CATCACGA-39, and reverse: 59-ACGTCTGACAACTGGTGCAT-39; TCF7forward: 59-CCGACCTCAAGTCCTCGTTG-39, and reverse: 59-TGGTG-CTTCATACCTGCATCG-39; UBASH3A forward: 59-AGCTCACATCGC-GCAAAAAG-39, and reverse: 59-TTGCAGTGAGAGTGCAACCA-39;VPREB3 forward: 59-AGTGACTTCGGCATCACCTG-39, and reverse:59-GCGCTCCGTGTTGTAGTAGA-39; CD83 forward: 59-CCCTGTGC-AATGTTTGGAGC-39, and reverse: 59-CCAAGACACTGCCTGGTAGG-39;GAPDH forward: 59-GTCCTCTCTGGCAAAGTCCAAG-39, and reverse:59-CCACAACATACTCAGCACCTGC-39; IFNG forward 59-CTCCCGA-TGAACGACTTGAG-39, and reverse 59-CTGAGACTGGCTCCTTTTCC-39.PCR cycling conditions were 95˚C for 5 min, linked to 40 cycles of 95˚Cfor 10 s and 60˚C for 1 min. A melting curve was established using theChromo4 Real-Time Detector (Bio-Rad). The primers were confirmed togive a single hit by primer-BLAST analysis on the whole Gallus RefseqNational Center for Biotechnology Information database, except in thecase of BCL11B, ZBTB46, and BATF3, owing to the existence of variantsof the same gene, and in the case of CADM1, XCR1 CD14, and MAFB, inwhich hits were found on several gene targets for one of the two primers,but with more than three mismatches. In all cases, the melting curve of thePCR product and migration on an agarose gel demonstrated that a singleamplicon was detected, supporting the specificity of all the primer pairsused for all the targeted genes.

Real-time qPCR data were collected by the Opticon Monitor Software(Bio-Rad) and 22DCt calculations for the relative expression of the differentgenes (arbitrary units) were performed using GAPDH for normalization.All qPCR reactions showed .95% efficacy. The qPCR data per gene werenormalized to maximal expression across subsets. In the case of IFN-gqPCR, quantitation of IFN-g and GAPDH transcript numbers was doneusing calibration plasmids.

MLR

After cell sorting, equal numbers of chicken spleen MPs and cDCs (3–5 3104 per well, depending on experiments) were distributed in flat-bottom96-well plates. Flow cytometry–sorted CD4+ T responder cells from anoutbred chicken with a different MHC (PA12 chicken line) were added for24–48 h of culture at 40˚C in DMEM F12 (Fisher) supplemented withpenicillin–streptomycin, nonessential amino acids (Fisher), 50 mM 2-mer-captoethanol, and 2% FCS (Lonza). The ratio of responder to stimulatorcells was 10. Transcription of IFN-g was tested as a readout of the MLR,using qPCR (3 per point, respectively). Cell viability was tested usingtrypan blue exclusion and was .95%.

Regulation of immunity-related gene expression in homologoussubsets across human, mouse, and chicken species

We established a list of 116 immunity-related genes (sensors, cytokine,cytokine receptors, signaling receptors) (30), for which the expression inthe cell subsets was normalized across species (Supplemental Fig. 2).

Statistical analyses

Normalized IFN-g transcript values were compared between stimulationconditions, using the nonparametric Mann–Whitney U test.

ResultsIdentification of MP and cDC candidates in chicken spleen

We previously identified DC-like cells in chicken splenocytesthat were morphologically distinct from MPs and that were notlabeled by KUL01, a mAb usually considered to mark MPs (18,28). To gain further insight into the existence of distinct MPand cDC phagocytes in chicken, we followed a flow cytometry sort-ing strategy to isolate candidates from suspensions of inbred SPFLeghorn chicken splenocytes, using a combination of pertinentmarkers among the relatively few number of mAbs available inthis species. We based our reasoning on the fact that in mammals,most cDCs are “lineage”-negative cells that coexpress CD11c and

MHC-II molecules. We used the well-characterized mAbs directedto the CD3 and Bu-1 Ags to exclude T and B cells, respectively,and the KUL01 to exclude MPs. After electronic exclusion gatingof cells positive for CD3, Bu-1, or KUL01, we sorted the re-maining cells in four populations based on their staining patternwith an anti–MHC-II mAb and the 8F2 mAb thought to targetchicken CD11c, which was previously shown to label bone mar-row–derived DCs (Fig. 1A). We sorted, in parallel, MP candidatesas KUL01+ MHC-II+ cells (designated as KUL01+), as well as T(CD3+) and B (Bu-1+) cells (Fig. 1B–D). In an exploratory ap-proach, we tested these cell subset candidates for the expressionof three murine/human cDC-associated genes (FLT3, ZBTB46alias BTBD4, ARGHAP22) (4), and two murine/human MP-associ-ated genes (MAFB, CD14) (4, 31), using qPCR. MAFB and CD14mRNA were found expressed at the highest level in KUL01+ cells

FIGURE 1. Isolation and exploratory analyses of cDC and MP candi-

dates from chicken spleen. (A) cDC candidates were sorted from chicken

splenocytes. FSChi CD32 BU-12 KUL012 cells were first electronically

selected to sort single and double positive cells for MHC-II and CD11c

(putative) expression. (B) B cells were sorted as Bu-1+ cells. (C) T cells

were sorted as CD3+ cells. (D) MP candidates were sorted as KUL01+

MHC class-II+ cells. (E) MGG staining of cDC candidates (MHC-II+

CD11c+). Original magnification 3100. (F) MGG staining of MP candi-

dates (KUL01+). Original magnification 3100. (G) Exploratory qPCR

analysis of cDC- and MP-associated gene expression in the sorted subsets

from two independent experiments (pool of four spleens). The gene ex-

pression was normalized across subsets to maximal expression and is re-

ported as a heat map based on red intensity.

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(Fig. 1G). FLT3 and ZBTB46 mRNA was found expressed at thehighest level in MHC-II+ CD11c+ splenocytes, and at relativelylow levels in MHC-II+ CD11c2 and MHC-II2 CD11c+ spleno-cytes. As expected, comparatively low expression of these cDC-and MP-associated genes was detected in T and B cells. In May–Gr€unwald–Giemsa (MGG) staining (32), the KUL01+ cells resembletypical MPs with a high cytoplasmic/nuclear ratio and vacuolatedcytoplasm, whereas MHC-II+ CD11c+ cells presented large den-drites (Fig. 1E, 1F). The KUL01+ MHC-II+ cells appeared to ofteninclude KUL01hi MHC-IIint and KUL01int MHC-IIhi populations(Fig. 1D) that appeared indistinguishable in the exploratory cDCand MP gene expression analysis and that presented a homoge-neous morphology in the MGG staining (data not shown). Thus,the exploratory qPCR and morphological analyses reassured us inselecting for the next steps the whole KUL01+ and the MHC-II+

CD11c+ populations as the MP* and cDC* candidates, respectively(*marks the candidate status in this article).

Transcriptome mapping of chicken cDC* and MP*

To align the chicken MP* and cDC* with mammalian counter-parts, we devised a scale-up of a global gene expression analyticmethod that was previously demonstrated to be relevant for dis-tinguishing new myeloid human subsets (33, 34) and for charac-terizing sheep cDC subsets (24). The transcriptome of eachchicken subset—that is, B, T, MP*, and cDC* (independent tri- orquadruplicates)—was obtained from mRNA hybridization on cus-tomized chicken gene chips (see Materials and Methods). Frompublicly available expression data (Supplemental Dataset 1), weidentified mouse and human fingerprints for T cells, B cells,plasmacytoid DCs (pDCs), and CD8a+/BDCA3+ cDCs, whichwere established as the list of genes expressed at least 1.5-foldhigher in the index cell population than in a large number of otherimmune cell types (Supplemental Dataset 2). We also identifiedmouse and human “cDC” and “myeloid” fingerprints as genesoverexpressed in all cDCs compared with in other myeloid cells(i.e., monocytes and/or MPs from many different tissues), andreciprocally (Supplemental Dataset 2). We next tested whether theselected mouse and human fingerprints were enriched between thefour chicken subsets by performing pairwise comparisons using

GSEA (35). In all comparisons, the chicken cDCs* were foundenriched for the mouse and human cDCs and CD8a+/BDCA3+

Genesets (q values , 0.05 in most cases), whereas the chickenMPs* were found enriched for the mouse and human myeloidGenesets (q values , 0.05) (Fig. 2). In the cDC* versus MP*comparison, the cDC and CD8a+/BDCA3+ signatures were theonly ones to be significantly enriched in a conserved manner.Conversely, in the MP* versus cDC* comparison, the dominantsignature was the myeloid one. As positive controls, we confirmedthat chicken T and B cells were enriched for the respective mouse/human T and B cell Genesets in all comparisons (q values, 0.05).The human and mouse pDC Genesets were not found togetherenriched in most comparisons using our selected populations ofchicken splenocytes, consistent with the fact that our strategy wasnot designed to identify pDCs in chicken that would have requireduse of different cell surface marker combinations.Thus the gene expression profiles show that cDCs* and MPs*

are distinct cell types in the chicken and that they strongly re-semble mouse and human cDCs and myeloid cells, respectively.Furthermore, chicken cDCs* appear to include a significant pro-portion of cells of the CD8a+/BDCA3+ cDC types.

Establishment of core gene expression signatures for T, B,cDC, and MP subsets across mice, humans, and birds

We sought to identify core gene expression signatures for T, B,cDCs, and MPs that would allow distinguishing these subsets fromother immune cell types across chickens, humans, and mice, andthat would likely apply to amniotes.We selected the mRNA transcripts that contribute most to the

enrichment of the mouse T, B, cDC, CD8a+, andMP fingerprints inall the chicken subset pairwise comparisons and that are providedas “leading edge” lists by the GSEA. We repeated the same pro-cedure with the human fingerprints. We identified as core sig-natures the genes found in common in the mouse against chickenand in the human against chicken leading edges lists (Fig. 3). TheMP core signature across chicken, human, and mouse encom-passes the TLR4, CTSB, and CTSD genes. The transspecies cDCcore signature encompasses CIITA, FAM46C, KIT, ZBTB46,FLT3, PLEKHA5 (cDC fingerprint) as well as the XCR1 and

FIGURE 2. Murine and human cDC and myeloid fingerprints are

enriched in the candidate chicken cDC* and MP*, respectively. GSEA

was performed using sets of genes corresponding to the transcriptional

fingerprints of specific murine (left) and human (right) cell types

generated from compendia of expression data from many leukocytes

(see Supplemental Dataset 2). Pairwise comparisons between chicken

cDCs*, MPs*, and B and T cells were performed to assess enrich-

ments of the mouse and human fingerprints. Results are represented as

dots: The red and blue colors of the dots correspond to the color at-

tributed to the subset in which the Geneset was enriched (see boxed

legend within the figure); the circle surface area is proportional to the

absolute value of the normalized enrichment score (NES), which

varies between ~1 (no enrichment) and ~5 (best enrichment possible).

The color intensity of circles is indicative of the false-discovery rate

(FDR) statistical q value.

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CADM1 genes (CD8a+/BDCA3+ cDC type fingerprint). Coresignatures for B cells and T cells across species were also estab-lished (B cells: CXCR5, SWAP70, CD79B, KLHL14, POU2AF1,EBF1, VPREB3, PAX5; T cells: LEF1, CD5, TCF7, UBASH3A,CD28, BCL11B).These lists represent the minimal core signatures that are re-

stricted by the fact that several pertinent genes are probably miss-ing because of misannotated, nonfunctioning, or absent probeson at least one of the three species arrays. To confirm and extendthe transspecies cell subset core signatures, we performed com-plementary qPCR analyses for a selection of genes of the minimalcore signature established above, and we added genes showinga conserved expression pattern between cDCs and MPs in themouse and human for which the signals from the chicken microarrayremained in the noise, with no difference between the forward andreverse probes, suggesting that the forward probes were nonfunc-tioning (Supplemental Fig. 1). This qPCR analysis showed that wecan add ARGHAP22 and BEND5 to the cDC, C5AR1 to the MP,and PCLG1 to the T cell signatures across the three species.

Conserved regulation of immune response–related genes incDCs and MPs in the human, mouse, and chicken

In humans and mice, DC subsets and MPs express specific andrelatively conserved profiles of immune response genes, although

partially overlapping (30). To identify immune response genes thatare differentially regulated in cDCs and monocytes/MPs acrosshumans, mice, and chickens, we analyzed the expression of a se-lection of immune response genes across the arrays used in thisstudy (Supplemental Dataset 1; see Materials and Methods). Agroup of genes clustered together that appeared to be expressedat higher levels in monocytes/MPs and cDCs than in lymphoidcells in several comparisons (Supplemental Fig. 2, pink dendo-gram). Among these genes, some showed a higher expression incDCs than in monocytes/MPs in all three species, that is, FLT3,XCR1, and TLR3 (Fig. 4, right panel). Several immunity mol-ecule mRNAs were overexpressed in chicken cDCs comparedwith MPs, for example, for CD86, ICOSLG, and CSFR2A, whereasit was only the case in one of the two mammals. Conversely,other genes showed a higher expression in monocytes/MPs thanin cDCs in all three species: CD14, TLR2, TLR4, P2RY13, SIRPa,and CSFR1 (Fig. 4, left panel). Finally, inflammatory cytokinegenes (IL6, IL1B) were markedly overexpressed in chicken MPscompared with cDCs and far less clearly so in the correspondingmammalian subsets. The different array data originated from cellsthat were isolated from distinct tissue types with different markersfor subsetting, which may hinder similar regulation of gene ex-pression in homologous immune subsets across species. The ex-pression of CD14, TLR2, and TLR4 was higher in monocytes/MPsthan in cDCs and lymphoid cells, demonstrating a conservedspecialization of MPs in the recognition of bacteria in all threespecies.

Chicken cDCs are efficient at CD4+ T cell activation

In mammals, cDCs are potent APCs that can activate naive Tcells with optimal efficacy. We thus tested whether chicken cDCspresented the capacity to activate allogeneic CD4+ T cells. In fiveindependent experiments, we observed that chicken spleen cDCs,without exogenous activation, were efficient at stimulating allo-geneic CD4+ T cells for IFN-g (p , 0.05), whereas splenic MPsisolated in parallel were not (Fig. 5). Thus, as in mammals, cDCsare more potent in activating allogeneic T cells than are MPs.

DiscussionIn this article, we show that MP and cDC subsets exist in thechicken as two distinct immune cell lineages that display geneexpression profiles sharing substantial homology with their mam-malian counterparts. To our knowledge, this is the first time thatcDCs have been identified outside mammals, and it shows that thecDC/MP paradigm extended beyond mammals, to vertebrates thatdiverged .300 million years ago from the common ancestor ofreptiles, birds, and mammals. Birds also present other phagocyticcells that mammals do not—including heterophils (the chickenfunctional equivalent of mammalian neutrophils) and thrombocytes(homologous in function to mammalian platelets, which are absentin the chicken)—for which their role as APCs is not established.Comparative gene expression profiling allowed us to determine

similarities between immune cell subsets across the chicken, hu-man, and mouse. Whereas this approach avoids biases of a priorihypotheses, it unravels similarities between subsets, but not exactequivalences, for different reasons. First, the MHC-II+ CD11c+

subset that was sorted with our strategy, corresponding to ∼1% ofsplenocytes, was clearly enriched for bona fide cDCs, but it ispossible that non-cDCs, such as NK and innate lymphoid cells,were included. In addition, our selection process of human andmurine cell subset fingerprints was stringent and eliminated manygenes that were not found in common in the whole range of thedatasets corresponding to a population type (MP, DC), owing totissue or species exception, absence of functional probes, or mis-

FIGURE 3. Establishment of a core signature of cDCs, MPs, and B and

T cells across chicken, human, and mouse species. Bar chart representing

the chicken gene chip normalized expression values of signature genes for

chicken cDCs (red), MPs (grey), B cells (blue), and T cells (green). Genes

were selected as 1) being part of both the mouse and human transcriptomic

fingerprints (T, MP, CD8a/BDCA3+-type cDC, cDC), and as 2) found

enriched in the equivalent chicken cell type according to the GSEA “lead-

ing edge” definition. Each bar corresponds to the average value of three

to four replicates for the four different chicken cell types. Error bars rep-

resent the SD for the replicates. Normalized expression values were scaled

to the highest expression value for each gene.

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annotated probes. An example is the absence of CD14 and MAFBfrom the mouse myeloid signature because of their low expressionin MPs of some tissues. Consequently, CD14 and MAFB, which

are highly expressed in chicken MPs, do not fall in the myeloidcore signature across the chicken, human, and mouse. To avoidthis problem, the human and mouse fingerprints should have been

FIGURE 4. Comparative expression of immune response–re-

lated genes in cDCs, MPs, and T and B cells across chicken,

human, and mouse species. Bar chart representing the relative

expression (mean 6 SD) of a selection of monocyte/MP-specific

(top) or cDC-specific (bottom) immune response–related genes

in chicken (left), mouse (middle), and human (right) cDC and

monocyte/MP populations (cDC in red/orange and MP in black/

gray in the legend, respectively). These populations include, for

chicken: MPs and cDCs; for human: monocyte (mono)–derived

MPs, PBMC-derived MPs, nonclassical blood monocytes, clas-

sical monocytes, BDCA3+ cDCs, and BDCA1+ cDCs; for mouse:

peritoneal cavity MPs, lung MPs, nonclassical monocytes, clas-

sical monocytes, splenic CD8a+ cDCs, cutaneous lymph node

CD8a+ cDCs, splenic CD11b+ cDCs, and cutaneous lymph node

CD11b+ cDCs. Values were computed from two to five inde-

pendent replicates for each cell population, except for human

mono-derived MP (n = 1). Normalized gene expression values

were scaled for each gene and species to the highest expression

value across all cell types.

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derived from the same tissue (= spleen) and with similar gatingstrategies as the one used for chicken, but such transcriptomic dataare at present not available. Finally, the commercial chicken genechips, although improved by us, were not as optimal as the com-mercial human and mouse ones, owing to misannotations, missingprobes, missing known orthologs to mammals, and nonfunctioningprobes. These handicaps resulted in a reduced number of analyzedprobes that probably led to an underestimation of the GSEA and toincomplete core gene expression lists. Specifically, as mentionedbefore, no probes were identified on the chicken microarrays asbeing specific for PLCG1, C5AR1, BEND5, and ARGHAPP22, butwe designed qRT-PCR for these genes to complement knowledgeon the core lists featuring genes with a conserved expressionpattern between human, mouse, and chicken immune cells. Othergenes with a conserved expression pattern across cell types inhumans and mice, for which no probes were identified on thechicken microarrays but for which an ortholog exists in thisspecies according to Ensembl, included HLA-DOB and ETV3for the cDC fingerprint; TCF4 (E2-2) for the pDC fingerprint;BANK1, CD22, FCRL1, and FCRL5 for the B cell fingerprint;CD3G, CD6, and DGKA for the T cell fingerprint; and MRC1(CD206), which is selectively expressed in monocyte-derivedDCs and some MPs in both the mouse and human. In the 12thAvian Immunology Research Group Conference (in 2012), theKUL01 mAb was reported to be specific to CD206 (36). Thus,in consistency with what was observed in the mouse and human,CD206 seems selectively expressed in the monocyte/MP and notin the cDC lineage in the chicken. No orthologs are yet reportedin the chicken for CLEC9A for the signature of XCR1+ DC,CD19 and CD79A for the B cell signature, as well as for SELPLG(CD162) and TNFSF18 (GITRL). For CD162, CD19, and CD79A,orthologs have been identified in birds other than the chicken, inreptiles, and in fishes, strongly suggesting that orthologs exist inthe chicken but still await identification. For CLEC9A and TNFSF18,no orthologs have been identified outside mammals, strongly sug-gesting that these genes do not exist in other vertebrates, includingbirds. In consistency with this, in the case of CLEC9A, we includedprobes on the gene chip corresponding to the mouse and humangene, but they did not give any signal. LY75 (CD205) is selectively,although not specifically, expressed on XCR1+ DCs in the humanand mouse. Two corresponding probes were present on the chickenarray and gave an ∼2-fold higher signal in cDCs and MPs than in T

and B lymphocytes, with no difference between cDCs and MPs.In agreement with this finding, a newly generated anti-chickenCD205 mAb was found to label both putative DCs (CD83+) andMPs (KUL01+) in chicken spleen (22). Thus, in the chicken,CD205 may not discriminate cDCs from MPs.The core gene expression profiles we identified in this study

allow proposal of a unifying molecular definition of T, B, MP, andcDC subsets across the chicken, human, and mouse, which shouldbe valid across amniotes. These core signatures can be used tosearch for homologous subsets in other model and applicationspecies, including distant vertebrates such as fishes. Most genes ofthe transspecies MP and cDC core signatures are included in thecore gene lists of MP and cDCs across mouse tissues identified inan independent study by the Immgen group (31). Several of theCD8a+ cDC-type genes, such as CADM1 and XCR1, were foundin the transspecies cDC core signature, strongly indicating thatchicken are equipped in CD8a+-type cDCs and that their cDClineage is likely composed of the two subsets previously identifiedin mammals, CD8a+-type and CD11b+-type cDCs. Whereas sev-eral genes of the cDC signature have already been studied for theirbiological implication in cDC biology, nothing is known regardingPLEKHA5 and FAM46C. Similarly, the molecules of the T andB cell core signature are well known for their function, except forKLHL14. As these genes are conserved in the gene expressionprogram of specific immune cell subsets across evolution, theyshould be studied for their role in the specific function or devel-opment of these subsets.Our results underline some degree of conservation of functions

for cDCs and MPs across amniotes. Several immunity-relatedgenes were similarly regulated between cDCs and MPs acrossthe three species under study. The conserved selective expressionof sensors that are considered more viral (TLR3) and bacterialoriented (TLR2, TLR4, CD14) in cDCs and MPs, respectively,suggest that these two lineages are specialized in distinct pathogensensing in amniotes. Furthermore, CTSB and CTSD are membersof the MP core signature, further pointing to the strong lysosomalactivity in MPs for degradation purposes. In addition, the strongercapacity for CD4 T cell activation, as well as the higher expres-sion of the CD86 and ICOSLG genes in chicken cDCs comparedwith MPs, shows that in all species, cDCs developed as special-ized in T cell activation.Efficient strategies to prevent disease outbreak in chicken farms

is mandatory to supply the growing world demand for poultryproducts that is expected to increase at a 2.5% rate per year until2030. The poultry sector strongly relies on vaccination for main-tenance of health and production status, with birds receiving .20vaccines during their short life (16). Our identification and mo-lecular characterization of cDCs in chickens will help in designingbetter vaccine strategies and in developing new immunomodula-tors as alternatives to antibiotics.

AcknowledgmentsWe thank Christelle Thibault-Carpentier from the Plate-forme Biopuces

(Strasbourg, France) for performing the microarray experiments (www-

microarrays.u-strasbg.fr); Mickael Bourge (Imagif, Gif-sur-Yvette, France)

for the pilot flow cytometry sorting experiments; the Plate-Forme d’Infec-

tiologie Experimentale, Nouzilly (France) for breeding and providing

high standard SPF chickens; Bertrand Bed’hom (Institut National de la

Recherche Agronomique, France) for the genotyping of the MHC of the

PA12 chicken line; Pierre Boudinot for helpful and enlightening discus-

sions; and Luc Jouneau for guidance on chicken microarray improvements.

DisclosuresThe authors have no financial conflicts of interest.

FIGURE 5. Chicken cDCs are efficient at allogeneic CD4+ T cell

stimulation. cDCs and MPs were sorted from pools of four chicken spleens

(inbred B13 MHC line), and they were plated at a 1:10 ratio with sorted

allogeneic splenic CD4+ T cells (outbred PA12 chicken). After cocul-

ture, the RNA was extracted and subjected to qPCR for IFN transcript

quantitation, using GAPDH for normalization. The relative IFN-g mRNA

copy numbers from five independent experiments (shown as distinct

symbols) are reported with means and SEM. Significant statistical

differences were given by the nonparametric Mann–Whitney U test.

*p , 0.05.

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