Cutting Edge: Expression of XCR1 Defines Mouse Lymphoid-Tissue Resident and Migratory Dendritic Cells of the CD8α+ Type

This information is current as Karine Crozat, Samira Tamoutounour, Thien-Phong Vu of September 23, 2021. Manh, Even Fossum, Hervé Luche, Laurence Ardouin, Martin Guilliams, Hiroaki Azukizawa, Bjarne Bogen, Bernard Malissen, Sandrine Henri and Marc Dalod J Immunol 2011; 187:4411-4415; Prepublished online 23

September 2011; Downloaded from doi: 10.4049/jimmunol.1101717 http://www.jimmunol.org/content/187/9/4411

Supplementary http://www.jimmunol.org/content/suppl/2011/09/23/jimmunol.110171 http://www.jimmunol.org/ Material 7.DC1 References This article cites 19 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/187/9/4411.full#ref-list-1

Why The JI? Submit online. by guest on September 23, 2021 • Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge: Expression of XCR1 Defines Mouse Lymphoid-Tissue Resident and Migratory Dendritic Cells of the CD8a+ Type Karine Crozat,*,†,‡,1 Samira Tamoutounour,*,†,‡,1 Thien-Phong Vu Manh,*,†,‡ x ´ ,†,‡ ,†,‡ ,†,‡ Even Fossum, Herve{ Luche,* Laurencex Ardouin,* Martin Guilliams,* Hiroaki Azukizawa, Bjarne Bogen, Bernard Malissen,*,†,‡ Sandrine Henri,*,†,‡ and Marc Dalod*,†,‡ 2 2 2 Subsets of dendritic cells (DCs) have been described CD11bhiCD24 DCs, CD11b CD24+CD103 DCs, and 2 according to their functions and anatomical locations. CD11b CD24+CD103+ DCs (1), hereafter referred to as + 2 + Conventional DC subsets are defined by reciprocal ex- CD11b DCs, CD103 DCs, and CD103 DCs (Table I). Downloaded from pression of CD11b and CD8a in lymphoid tissues Although LCs and all dermal DC subsets constitutively mi- + (LT), and of CD11b and CD103 in non-LT (NLT). grate from skin to cutaneous LN (CLN), the CD103 DCs a+ + stand out as the most potent subset for presenting keratinocyte- Spleen CD8 and dermal CD103 DCs share a high + efficiency for Ag cross-presentation and a developmen- derived Ags to CD8 T cells in the CLN (1). This capacity tal dependency on specific transcription factors. How- is reminiscent of the high efficiency of lymphoid tissue (LT)- a+ + http://www.jimmunol.org/ ever, it is not known whether all NLT-derived CD103+ resident CD8 DCs for cross-presentation (1). CD103 int- a+ DCs are also found in other anatomical places such as lung DCs and LT-resident CD8 DCs are similar despite + their different anatomical locations. XCR1 was previ- and gut. The development of CD103 int-DCs and LT- resident CD8a+ DCs selectively depends on a common set ously described as exclusively expressed on mouse a+ + of transcription factors (2, 3). Hence, these mouse DC spleen CD8 DCs and human blood BDCA3 DCs. populations may belong to a unique category of CD8a+-type In this article, we showed that LT-resident CD8a+ + DCs (1). DCs and NLT-derived CD103 DCs specifically ex- CD8a+-type DCs exist in human and sheep, where their press XCR1 and are characterized by a unique tran- identification was based on their expression of a unique by guest on September 23, 2021 scriptional fingerprint, irrespective of their tissue of transcriptional fingerprint shared with mouse spleen CD8a+ a+ + origin. Therefore, CD8 DCs and CD103 DCs belong DCs (4, 5) and on their efficiency for Ag cross-presentation to a common DC subset which is unequivocally identi- (5–9). We proposed a universal classification of DCs in five fied by XCR1 expression throughout the body. The major subsets irrespective of tissues and species: monocyte- Journal of Immunology,2011,187:4411–4415. derived inflammatory DCs, LCs, plasmacytoid DCs, CD11b+- type DCs, and CD8a+-type DCs (1). Yet, a discriminating + endritic cells (DCs) exert their functions of immune marker identifying all CD8a -type DCs across tissues sentinels in different anatomical places. The DCs that remained to be determined. Moreover, the relationships be- reside in the parenchyma of nonlymphoid tissues tween LT-resident CD8a+ DCs and CD103+ mig-DCs or D + (NLT) are called interstitial DCs (int-DCs). These DCs shuttle CD103 int-DCs need to be confirmed. The chemokine re- tissue Ags to draining lymph nodes (LNs), where they are ceptor XCR1 is specifically expressed by CD8a+-type DCs in called migratory DCs (mig-DCs). mouse spleen, human blood, and sheep lymph (4–7, 10). The In mouse skin, DCs make up epidermal Langerhans function of XCR1 was first unveiled by the group of cells (LCs) and three major subsets of dermal DCs: R. Kroczek (10), who showed that CD8+ T cell cross-priming

*Centre d’Immunologie Marseille-Luminy, Universite´ de la Me´diterrane´e, 13288 Mar- Recherche sur le Cancer (to K.C.) and the Ministe`re de la Recherche (to S.T.), a Marie seille, France; †INSERM, Unite´ 631, 13288 Marseille, France; ‡Centre National de la Curie fellowship (to M.G.), and the Centre National de la Recherche Scientifique and Recherche Scientifique, Unite´ Mixte de Recherche 6102, 13288 Marseille, France; System biology of T cell activation (SYBILLA) project (to H.L.). x Center for Immune Regulation, Institute of Immunology, University of Oslo and Oslo { Address correspondence and reprint requests to Dr. Marc Dalod or Dr. Karine Crozat, University Hospital Rikshospitalet, 0424 Oslo, Norway; and Course of Integrated Centre d’Immunologie Marseille-Luminy, Case 906, 13288 Marseille Cedex 09, France. Medicine, Department of Dermatology, Osaka University, Suita, Osaka 565-0871, E-mail addresses: [email protected] or [email protected] Japan The online version of this article contains supplemental material. 1K.C. and S.T. contributed equally to this work. Abbreviations used in this article: CLN, cutaneous lymph node; DC, dendritic cell; Received for publication June 14, 2011. Accepted for publication August 25, 2011. bGal, b-galactosidase; int-DC, interstitial dendritic cell; LC, Langerhans cell; LN, This work was supported by institutional funding from the Centre National de la lymph node; LT, lymphoid tissue; medLN, mediastinal LN; mig-DC, migratory den- Recherche Scientifique and INSERM, grants from the Association pour la Recherche dritic cell; MLN, mesenteric LN; NLT, nonlymphoid tissue; TN, triple-negative. sur le Cancer (to M.D.), European Communities Framework Program 7 (MASTER- SWITCH Integrating Project; HEALTH-F2-2008-223404) (to B.M.), the Association Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 Franc¸aise Contre les Myopathies (to B.M.), and the Agence National pour la Recherche (Skin-dendritic cells project) (to S.H.), and by fellowships from the Association pour la www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101717 4412 CUTTING EDGE: XCR1 IS A SPECIFIC MARKER OF MOUSE CD8a+-TYPE DCs depends on their ability to secrete the XCR1 ligand XCL1 in experimental models where either the OVA coupled to an anti-CD205 Ab or OVA-expressing allogeneic pre-B cells are - + (CLN) a + administrated in vivo. XCR1 expression on CD8 DCs was CD4 - 2 + 2 (liver) + (liver) (MLN) +

also found critical for the optimal induction of CD8 T cell 2 2 CD4 CD4 CD4 CD207 CD8 +

responses upon Listeria monocytogenes infection (6). We hy- 2 2 (lung) (lung) 2 2 a a a + 2 CD4 pothesized that XCR1 may be the long sought marker de- CD103 CD103 2 lo + b CD8

+ CD8 a

a CD8 CD8 2

fining all CD8 -type DCs throughout the body. In this 2 + CD207 + 2 CD103 2

a CD103

study, to investigate whether LT-resident CD8 DCs and CD8 2 + + CD4 CD11b CD11b

+ + + NLT-derived CD103 DCs belong to the same DC subset + CD11b CD11b CD11b + + + n.a. n.a. n.a. n.a. CD103

and can be identified by a common specific marker across + CD11b CD11b CD11b + + + CD103 CD11c CD11c + mouse tissues, we analyzed their expression of XCR1 and + 2 CD11c CD11c CD11c 2 2 2

compared their gene expression profiles. CD11b « « ImmGen Compendium + « CD11c CD11c CD11c + + 2 CD11b MHC-II MHC-II « + CD3 CD3 CD3 2 + 2 +

Materials and Methods + CD11c Lin Lin hi Mice CD3 + + + MHC-II MHC-II CD11c CD45 tm1Dgen CD45 Xcr1 mice (Xcr1-bGal) (6, 10) generated by Deltagen were bred in hi CD45 CD45 CD45 Centre d’Immunologie Marseille-Luminy animal care facilities. C57BL/6J Downloaded from mice were purchased from Charles River Laboratories (L’Arbresle, France). MHC-II

Studies were performed in accordance with institutional regulations governing MHC-II animal care and use. DC isolation and sorting strategy DCs were isolated from various organs by a combination of enzymatic di-

gestion, mechanical disruption, and gradient density enrichment (11). Sorting http://www.jimmunol.org/ + of CLN DCs was performed as described previously (12). + /+ + 2 2 2 CD24 +

Abs and flow cytometry + 2 /+ 2 2 a 2 CD24 a CD24 CD24 CD24 + + CD24 + 2

Most Abs were purchased from eBioscience or BD Biosciences. Identification + CD8 CD103 CD24 CD24 of mig-DCs was based on their specific pattern of expression of CD11c and CD8 + a 2 + 2 CD24 2 MHC class II (Supplemental Fig. 1). CADM1 staining was performed with + CD103 CD11b CD11b CD11b + 2 CD11b + +

a chicken anti-SynCAM/TSLC1 Ab (clone 3.E.1) revealed with a goat anti- + b CD11b CD11b CD11b CD11b chicken IgG. XCR1 expression was detected using fluorescein di- -D-gal- CD11b + CD11b lo hi hi lo actopyranoside as a fluorogenic substrate for b-galactosidase (bGal) (6). In lo CD11c CD11c CD11b CD11c + CD11c + + lo by guest on September 23, 2021 CLNs, XCR1 expression was also detected using recombinant mouse XCL1 + CD11c CD11c CD11c CD11c covalently coupled to the red fluorescent protein mCherry (E. Fossum and B. CD11c + CD11c hi lo lo hi

Bogen, manuscript in preparation). hi CD11c MHC-II MHC-II MHC-II hi MHC-II 2 2 Flow Cytometric Analyses 2 Microarray analyses and real-time PCR 2 MHC-II Lin Lin MHC-II MHC-II MHC-II Lin MHC-II + Lin 2 + + MHC-II 2 + 2 2 - Quality controls and normalization of a public expression data compendium 2 Lin MHC-II Lin Lin Lin + Lin of mouse DC subsets (ImmGen; Gene Expression Omnibus database 2 CD45.2

GSE15907) were performed as described previously (6). The gene set en- Lin CD45.2 richment analysis (GSEA) method is publicly available at http://www.broad. CD45.2 mit.edu/gsea (13). To test whether GeneSets were enriched in pairwise CD45.2 comparisons between CD8a+ LT-resident DCs or CD103+ NLT-derived DCs versus their CD11b+ counterparts, nominal p values were calculated as well as false-discovery rate (q value), based on 1000 random permutations between GeneSets. Results were considered significant when p , 0.05 and q , 0.25 (13). Real-time PCR experiments were performed as previously described (6) using the Hprt gene for normalization. Primer sequences are available upon request.

Results and Discussion int-DCs int-DCs + mig-DCs mig-DCs + + High level of XCR1 expression is selective for CD103 int-DCs in skin + + int-DCs (skin) Subsets mig-DCs (CLN) LT-resident DCs LT-resident DCs

+ + + and CD103 mig-DCs in CLNs + + int-DCs (skin) a TN DCs (MLN) n.a. CD45 CD103 CD11b CD11b CD103

To investigate which DC subsets express XCR1 in skin and mig-DN DCs (CLN) Lin CD11b CD8 CD11b CLNs, we exploited a reporter mutant mouse model expressing CD11b b-galactosidase (bGal) in place of XCR1. In skin, the int- DC subsets encompass the epidermal LCs and the dermal sub- 2 sets CD103+ DCs, CD103 DCs, CD11b+ DCs, and 2 2 CD11b CD24 DCs (Fig. 1A, Table I). In skin int-DCs, 2 bGal activity was high in CD103+ DCs, low in CD103 2 2 2 DCs, and undetectable in CD11b CD24 DCs, CD11b+ type type CD11b + +

DCs, and LCs (Fig. 1B,1C). In CLNs, only LT-resident 2

+ + a CD8a DCs and CD103 mig-DCs expressed XCR1 (Fig. DC Type Phenotype used for sorting DC subsets by the Immunological Genome consortium (http://www.immgen.org/index_content.html). The consistency between the two phenotyping strategies is shown through underlining of common markers. Phenotype used for DC subset definition in the flow cytometric analyses performed in the study. CD8 CD11b CD24 LCs int (skin) CD45.2 n.a., not applicable. 1D,1F ). The use of fluorescently labeled recombinant mouse a b

XCL1 to stain CLN cells gave a strong and highly specific Table I. Nomenclature and phenotyping for DC subsets as designated in the text The Journal of Immunology 4413 Downloaded from http://www.jimmunol.org/

FIGURE 1.

XCR1 expression by DC subsets from nonlymphoid and lymphoid organs. A, Gating strategy defining DC subsets in each organ. B–J, Histograms by guest on September 23, 2021 showing bgal enzymatic activity representative of XCR1 expression in DC subsets of various organs of C57BL/6J (black) and XCR1-bGal (red) mice (except for E where the recombinant XCL1-mCherry was used). B, Epidermis. C, Dermis. D–F, CLNs. F, The percentage of bGal+ cells was calculated by subtracting the percent of bGal+ cells in C57BL/6J mice to the percentage of bGal+ cells in XCR1-bGal mice. G, CADM1 expression in CLN-resident CD11b+ (black) and 2 CD8a+ (red) DCs (left panel) and in CLN-mig CD11b+ (black), CD103 (green), and CD103+ (red) DCs (right panel). H, Liver, lungs, and intestine. I, Mig- DC subsets from MedLNs and MLNs. J, LT-resident DC subsets from spleen, MedLNs and MLNs. Data from intestine and MedLN were obtained from samples pooled from six mice. Representative data are shown from at least two independent experiments (B, C, E, G) and from seven independent experiments (D, F–J ). signal on LT-resident CD8a+ DCs and CD103+ mig-DCs intestine and the CD103+ mig-DCs in MedLN showed sub- from wild-type mice (Fig. 1E ). These data confirmed that the stantial levels of bGal activity in wild-type mice. However, protein XCR1 is specifically expressed on LT-resident CD8a+ a clear increase over that background signal could be detected DCs and CD103+ mig-DCs and validated the use of bGal in the corresponding subsets isolated from XCR1-bGal mice. activity as a faithful reporter of XCR1 expression. Therefore, Within LT-resident DCs, XCR1 expression remained con- in skin and CLNs, a high level of XCR1 expression is selective fined to the CD8a+ subset (Fig. 1J ). 2 for CD8a+-type DCs. In most of the NLT we have examined, CD103 int-DCs 2 expressed intermediate levels of XCR1. CD103 XCR1low a+ XCR1 expression defines CD8 -type DCs in visceral organs and their DCs may be precursors of CD103+XCR1high DCs. In bone 2 2 draining LNs marrow FLT3 ligand cultures, CD11b CD24+CD103 DCs 2 We next analyzed XCR1 expression on DCs residing in dif- represent an immature stage of CD11b CD24+CD103+ ferent tissues. As in skin and CLNs, three main populations DCs not yet endowed with full cross-presentation potential. were defined in the liver, lungs, and the small intestine: In these cultures, the acquisition of cross-presenting compe- 2 CD11b+ DCs, CD103+ DCs, and CD103 DCs (Fig. 1A, tence correlates with the expression of CD103 upon TLR or Table I). In these organs, XCR1 expression was high in cytokine stimulation (14). The acquisition of XCR1 in vivo 2 CD103+ int-DCs, intermediate in CD103 int-DCs, and not might be exploited to follow the development of CD8a+-type detected in CD11b+ int-DCs (Fig. 1H), as observed in the DCs into full-fledged cross-presenting DCs in tissues. skin. In the mig-DCs from mesenteric LNs (MLNs) and a+ + mediastinal LNs (MedLNs) draining, respectively, the in- LT-resident CD8 DCs and NLT-derived CD103 DCs share testine and the lung, XCR1 expression remained highest in a common gene signature the CD103+ subset (Fig. 1I). Despite the use of an inhibitor To investigate the potential similarities between LT-resident of endogeneous Gal activity, the CD103+ int-DCs in the CD8a+ DCs and skin-derived CD103+ mig-DCs, which 4414 CUTTING EDGE: XCR1 IS A SPECIFIC MARKER OF MOUSE CD8a+-TYPE DCs both specifically expressed XCR1, we analyzed the expression family of adaptors critical for signal transduction by ITAM- of several transcripts in DC subsets sorted from CLNs. coupled receptors. CLEC9A contains a hemi-ITAM motif and We focused on Xcr1, Clec9a, Cadm1, Clnk, and Rab7b may signal through CLNK downstream of the Syk tyrosine (5430435G22Rik), because these genes are expressed selectively kinase (15). The small GTPase RAB7B expressed in endosomes in CD8a+-type DCs from mouse spleen, human blood, and may promote assembly of the machinery required for cross- sheep lymph (4, 5). CLEC9A, XCR1, and CADM1 contribute presentation as suggested by the functions of its paralogs to confer their specific functions on spleen CD8a+ DCs by (17). In CLNs, Xcr1, Clnk, Rab7b, Clec9a,andCadm1 were promoting the cross-presentation of dead cell-associated Ags, expressed the highest in LT-resident CD8a+ DCs and CD103+ the priming of CD8+ T cells, and the induction of IL-22 in mig-DCs (Fig. 2A). Tlr3 was expressed identically in all sub- CD8+ T cells (6, 10, 15, 16). CLNK belongs to the SLP-76 sets of mig-DCs. As reported for spleen DC subsets (3), Batf3

Resident Migratory Resident Migratory Resident Migratory Resident Migratory A Xcr1 Clec9a Cadm1 Itgae 0.5 10 0.10 0.3 0.008 6 0.6 1.5 8 0.4 0.08 0.006 0.2 4 1.0 6 0.06 0.4 0.3 0.004 4 0.04 0.2 0.1 2 0.2 0.5 2 0.02 0.002 0.1 Downloaded from Relative expression 0 0.00 0.0 0.000 0 0.0 0.0 0.0 Rab7b Clnk Tlr3 Batf3 4 0.20 0.15 0.010 1.0 0.004 2.0 5 0.8 3 0.15 0.008 0.003 1.5 4 0.10 0.006 0.6 3 2 0.10 0.002 1.0 0.004 0.4 2 0.05 1 0.05 0.002 0.2 0.001 0.5 1 http://www.jimmunol.org/ Relative expression 0 0.00 0.00 0.000 0.0 0.000 0.0 0 + − + + − + + − + + − + + + + + + + + + + + + + + + + + CD8 α CD8 α CD8 α CD8 α CD8 α CD8 α CD8 α CD8 α CD11b CD11b CD11b CD11b CD11b CD11b CD103 CD103 CD11b CD11b CD103 CD103 CD11b CD11b CD103 CD103 CD11b CD11b CD103 CD103 CD24+ CD24+ CD24+ CD24+ CLN Spleen CLN CLN Spleen CLN CLN Spleen CLN CLN Spleen CLN

GeneSets B CD8α+DC-specific genes CD11b+DC-specific genes C 6 Xcr1 Clec9a Sirpb1c Ptgs2 5 5 4 4 11b DC (n=92) (n=75) D8α DC ) ) by guest on September 23, 2021 D 2

2 3

3 C 2 2 C 1 1 + Spleen CD8α vs CD11b+ 0 ND 0 -1 -1 α+ + 6 Cadm1 Itgae 5 1810033B17Rik Clec4b1 CLN CD8 vs CD11b 5 4 4 3 3 resident DCs MLN CD8α+ vs CD11b+ 2 2 1 1 0 ND 0 ND ND + + -1 -1 Lung CD103 vs CD11b 6 Clnk Rab7b 5 Ifitm6 Ear2 + +

5 int-DCs Liver CD103 vs CD11b

-like DC] gene expression ratio (Log ratio expression gene -like DC] 4 -like DC] gene expression ratio (Log

4 + + 3 3 8α 1b 2 1

2 D 1 1 CLN CD103+ vs CD11b+ 0 ND 0 ND -1 -1 + 6 Tlr3 Batf3 5 Tmem176a Tmem176b mig-DCs MLN TN vs CD11b

5 C / -like DC + -like DC/ CD -like DC/ 4

+ 4 3 3 2 2 LEGEND: circle diameter = 1

[CD8α 1 normalized enrichment score 0 ND [CD11b 0 -1 -1 3.5 3 enriched in red subset CLN CLN CLN CLN CLN CLN CLN CLN MLN MLN MLN MLN MLN MLN MLN MLN Liver Liver Liver Liver Lung Lung Lung Lung Spleen Spleen Spleen Spleen enriched in green subset s s s s Cs Cs Cs Cs Cs Cs Cs Cs es. es. es. es. r r r r -3 C C C C D D D D <0. 05 D D D D <10 D D D D ig- ig- ig- ig- LT- LT- LT- LT- int- int- int- int- color intensity = FDR q value m m m m FIGURE 2. Gene expression profiling of DC subsets from nonlymphoid and lymphoid organs. A, Relative levels of expression of selected transcripts in DC subsets sorted from spleen and CLNs as assessed by real-time PCR. Graphs represent pooled results from three independent experiments. B, Gene expression ratio in DC subsets isolated from several NLTs and LTs. Results are shown from the analysis of the ImmGen public microarray database for CD8a+ versus CD11b+ DCs purified from spleen, MLN and CLN (LT-resident DCs), CD103+ versus CD11b+ int-DCs purified from lungs and liver, and CD103+/TN versus CD11b+ mig-DCs purified from CLN and MLN. C, Gene set enrichment analysis for comparison of putative CD8a+-type versus CD11b+-type DCs in a number of mouse tissues. CD8a+ DC-specific genes (“CD8a DC” GeneSet encompassing 92 genes) and CD11b+ DC-specific genes (“CD11b DC” GeneSet encompassing 75 genes) were examined for global enrichment of their expression in the ImmGen public microarray data when comparing CD8a+ versus CD11b+ LT-resident DCs from spleen, MLNs and CLNs, or CD103+ versus CD11b+ int-DCs from lungs and liver, or CD103+/TN versus CD11b+ mig-DCs from CLNs and MLNs. The color of circles corresponds to the font color of the DC subset where the GeneSet is enriched. The circle surface area is proportional to the normalized enrichment score, which varies from 1 (no enrichment) to 5 (all genes of the GeneSet are expressed to higher levels in the same DC subset). The color intensity of circles is indicative of the false-discovery rate (FDR) statistical q value, which estimates the likelihood that the enrichment of the GeneSet represents a false- 2 2 2 positive finding. ND, not detectable; TN, triple-negative CD4 CD8a CD11b DCs. The Journal of Immunology 4415 expression was comparable in all CLN DC subsets (Fig. 2A). Acknowledgments As expected, Itgae (CD103) was expressed selectively in We thank Pierre Grenot and Atika Zouine from the Centre d’Immunologie + + CD103 mig-DCs and in CD8a LT-resident DCs. There- Marseille-Luminy Flow Cytometry Core Facility for cell sorting. We thank fore, the common expression of a specific gene signature, Lee Leserman (Centre d’Immunologie Marseille-Luminy) for discussion and including Xcr1, Clec9a, Clnk, Rab7b, and Cadm1, by LT- critical reading of the manuscript. This work benefited from data assembled resident CD8a+ DCs and skin-derived CD103+ mig-DCs by the ImmGen consortium. confirms their belonging to the CD8a+-type DC subset. a+ To determine whether XCR1 expression defined CD8 - Disclosures type DCs across tissues, we exploited public data from the The authors have no financial conflicts of interest. Immunological Genome project, which spans DC subsets from many organs and their draining LNs (18). Xcr1 tran- script was expressed to high levels in all LT-resident CD8a+ + References DCs in lung and liver CD103 int-DCs as well as in MLN 1. Guilliams, M., S. Henri, S. Tamoutounour, L. Ardouin, I. Schwartz-Cornil, 2 2 2 CD4 CD8 CD11b triple-negative (TN) mig-DCs, as M. Dalod, and B. Malissen. 2010. From skin dendritic cells to a simplified classi- + fication of human and mouse dendritic cell subsets. Eur. J. Immunol. 40: 2089– compared with CD11b DCs (Fig. 2B). Xcr1 transcripts were 2094. below the level of detection in CD103+ mig-DCs from CLNs, 2. Ginhoux, F., K. Liu, J. Helft, M. Bogunovic, M. Greter, D. Hashimoto, J. Price, N. Yin, J. Bromberg, S. A. Lira, et al. 2009. The origin and development of likely because of limitations in the sensitivity of the micro- nonlymphoid tissue CD103+ DCs. J. Exp. Med. 206: 3115–3130. array technology. Consistently, Xcr1 expression levels assessed 3. Edelson, B. T., W. Kc, R. Juang, M. Kohyama, L. A. Benoit, P. A. Klekotka, Downloaded from ∼ + C. Moon, J. C. Albring, W. Ise, D. G. Michael, et al. 2010. Peripheral CD103+ by real time-PCR were 100-fold lower in CLN CD103 dendritic cells form a unified subset developmentally related to CD8a+ conventional + mig-DCs as compared with CLN-resident CD8a DCs (Fig. dendritic cells. J. Exp. Med. 207: 823–836. 4. Robbins, S. H., T. Walzer, D. Dembe´le´, C. Thibault, A. Defays, G. Bessou, H. Xu, 2A). A similar reduction was observed for Clec9a, Cadm1, E. Vivier, M. Sellars, P. Pierre, et al. 2008. Novel insights into the relationships Clnk, Itgae, and Rab7b (Fig. 2A,2B). However, XCR1 and between dendritic cell subsets in human and mouse revealed by genome-wide ex- CADM1 were both still highly expressed in these cells at the pression profiling. Genome Biol. 9: R17. 5. Contreras, V., C. Urien, R. Guiton, Y. Alexandre, T. P. Vu Manh, T. Andrieu, protein level (Fig. 1D,1E,1G). Therefore, the transcription K. Crozat, L. Jouneau, N. Bertho, M. Epardaud, et al. 2010. Existence of CD8a- http://www.jimmunol.org/ of some genes may decrease upon maturation of CD103+ int- like dendritic cells with a conserved functional specialization and a common mo- lecular signature in distant mammalian species. J. Immunol. 185: 3313–3325. DCs into mig-DCs, whereas expression of the corresponding 6.Crozat,K.,R.Guiton,V.Contreras,V.Feuillet,C.A.Dutertre,E.Ventre, proteins is maintained as a result of a sufficiently long half life. T. P. Vu Manh, T. Baranek, A. K. Storset, J. Marvel, et al. 2010. The XC che- mokine receptor 1 is a conserved selective marker of mammalian cells homologous To further verify the degree of similarity that exists between to mouse CD8a+ dendritic cells. J. Exp. Med. 207: 1283–1292. DC subsets expressing XCR1, we examined in the conventional 7. Bachem, A., S. Gu¨ttler, E. Hartung, F. Ebstein, M. Schaefer, A. Tannert, A. Salama, K. Movassaghi, C. Opitz, H. W. Mages, et al. 2010. Superior antigen cross- subsets of DCs isolated from the same tissues the enrichment of + + + presentation and XCR1 expression define human CD11c CD141 cells as homo- gene expression signatures (GeneSets) specific of splenic CD8a logues of mouse CD8+ dendritic cells. J. Exp. Med. 207: 1273–1281. + 8. Poulin, L. F., M. Salio, E. Griessinger, F. Anjos-Afonso, L. Craciun, J. L. Chen,

DCs or of splenic CD11b DCs (Fig. 2C). In all pairwise by guest on September 23, 2021 a+ A.M.Keller,O.Joffre,S.Zelenay,E.Nye, et al. 2010. Characterization of comparisons, we observed that the CD8 DC-specific Gen- human DNGR-1+BDCA3+ leukocytes as putative equivalents of mouse CD8a+ eSet was significantly enriched in LT-resident CD8a+ DCs, dendritic cells. J. Exp. Med. 207: 1261–1271. + + 9. Jongbloed, S. L., A. J. Kassianos, K. J. McDonald, G. J. Clark, X. Ju, C. E. Angel, CD103 int-DCs, CD103 mig-DCs, or TN mig-DCs (Fig. C. J. Chen, P. R. Dunbar, R. B. Wadley, V. Jeet, et al. 2010. Human + 2C). Reciprocally, the CD11b DC-specific GeneSet was sig- CD141+(BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset nificantly enriched in CD11b+ DCs from all tissues. Nineteen that cross-presents necrotic cell antigens. J. Exp. Med. 207: 1247–1260. + 10. Dorner, B. G., M. B. Dorner, X. Zhou, C. Opitz, A. Mora, S. Gu¨ttler, A. Hutloff, genes from the CD8a DC GeneSet were consistently ex- H. W. Mages, K. Ranke, M. Schaefer, et al. 2009. Selective expression of the a+ chemokine receptor XCR1 on cross-presenting dendritic cells determines co- pressed to higher levels in all CD8 -type DCs as compared + + operation with CD8 T cells. Immunity 31: 823–833. with their CD11b -type DC counterparts (Supplemental Fig. 11. Guilliams, M., K. Crozat, S. Henri, S. Tamoutounour, P. Grenot, E. Devilard, a+ B. de Bovis, L. Alexopoulou, M. Dalod, and B. Malissen. 2010. Skin-draining 2). Some genes from the CD8 DC GeneSet were not ex- – a+ lymph nodes contain dermis-derived CD103 dendritic cells that constitutively pressed on all CD8 -type DCs. This observation pinpoints produce retinoic acid and induce Foxp3+ regulatory T cells. Blood 115: 1958–1968. the necessity to assess the relationships between two cell sub- 12. Henri, S., L. F. Poulin, S. Tamoutounour, L. Ardouin, M. Guilliams, B. de Bovis, E. Devilard, C. Viret, H. Azukizawa, A. Kissenpfennig, and B. Malissen. 2010. sets, based on their comparative expression of many markers. CD207+CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens + Although highly expressed by all CD8a -type DCs, Clec9a irrespective of the presence of Langerhans cells. J. Exp. Med. 207: 189–206. and Cadm1 are also expressed by cell subsets other than 13. Subramanian, A., H. Kuehn, J. Gould, P. Tamayo, and J. P. Mesirov. 2007. + GSEA-P: a desktop application for Gene Set Enrichment Analysis. Bioinformatics CD8a -type DCs in mice and humans, contrary to Xcr1 (6). 23: 3251–3253. This genome-wide expression profiling analysis confirmed the 14. Sathe, P., J. Pooley, D. Vremec, J. Mintern, J. O. Jin, L. Wu, J. Y. Kwak, a+ J. A. Villadangos, and K. Shortman. 2011. The acquisition of antigen cross- existence of CD8 -type DCs characterized by XCR1 ex- presentation function by newly formed dendritic cells. J. Immunol. 186: 5184– pression across all examined tissues in mice. 5192. 15. Sancho, D., O. P. Joffre, A. M. Keller, N. C. Rogers, D. Martı´nez, P. Hernanz- In conclusion, we identified for the first time to our Falco´n, I. Rosewell, and C. Reis e Sousa. 2009. Identification of a dendritic cell knowledge a robust and specific marker defining mouse receptor that couples sensing of necrosis to immunity. Nature 458: 899–903. a+ 16. Galibert, L., G. S. Diemer, Z. Liu, R. S. Johnson, J. L. Smith, T. Walzer, CD8 -type DCs in all the LT and NLT examined. This M. R. Comeau, C. T. Rauch, M. F. Wolfson, R. A. Sorensen, et al. 2005. Nectin- marker, the chemokine receptor XCR1, is also specifically like protein 2 defines a subset of T-cell zone dendritic cells and is a ligand for class-I- expressed by human blood BDCA3+ DCs and sheep lymph restricted T-cell–associated molecule. J. Biol. Chem. 280: 21955–21964. + 17. Zou, L., J. Zhou, J. Zhang, J. Li, N. Liu, L. Chai, N. Li, T. Liu, L. Li, Z. Xie, et al. CD26 DCs and conserved during evolution (5, 6). Thus, the 2009. The GTPase Rab3b/3c-positive recycling vesicles are involved in cross- community of the researchers working on DCs may consider presentation in dendritic cells. Proc. Natl. Acad. Sci. USA 106: 15801–15806. + a+ 18. Heng, T. S., M. W. Painter, and Immunological Genome Project Consortium. using the term “XCR1 DCs” to designate CD8 -type DCs 2008. The Immunological Genome Project: networks of gene expression in immune in a consistent manner across all mammalian species and cells. Nat. Immunol. 9: 1091–1094. 19. Ziegler-Heitbrock, L., P. Ancuta, S. Crowe, M. Dalod, V. Grau, D. N. Hart, tissues to further implement the definition of a universal P. J. Leenen, Y. J. Liu, G. MacPherson, G. J. Randolph, et al. 2010. Nomenclature nomenclature for mouse and human DC subsets (19). of monocytes and dendritic cells in blood. Blood 116: e74–e80. A.

TRITC+ CD11c CD11c

TRITC MHC-II

B. CCR7+/+ CCR7-/- CD11c CD11c

MHC-II MHC-II

Supplemental figure 1 Spleen CD8+ DC GeneSet Spleen CD11b+ DC GeneSet 4 5 3 2 9 7 11 6 12 10 14 13 1 8 Spleen Spleen mig-CLN mig-MLN Res-CLN Res-CLN Res-MLN Res-Liver mig-CLN Res-Lung mig-MLN

PROBE DESCRIPTION PROBE DESCRIPTION Res-MLN Res-Liver Res-Lung 10500710 BC037703 6.8 7.5 3.8 2.5 2.2 5.3 9.2 10350516 Ptgs2 5.4 7.3 18.1 20.2 19.5 4.1 2.5 10359870 Pbx1 4.6 4.3 3.8 5.0 4.5 2.6 2.1 10569877 1810033B17Rik 4.4 5.0 3.6 4.5 5.7 3.9 2.4 10438753 Leprel1 27.5 15.2 12.2 6.9 9.3 1.5 5.5 10497356 Sirpb1c 34.7 24.7 13.5 19.0 6.4 1.7 2.7 10506968 Ttc39a 6.6 4.6 3.7 5.7 7.8 5.0 2.1 10423363 Ank 3.2 4.8 4.4 3.0 2.7 2.9 1.6 10542181 Clec9a 34.5 14.2 20.5 42.4 8.1 1.9 2.9 10392560 Abca9 5.2 14.1 4.6 10.9 5.6 1.4 1.9 10585048 Cadm1 10.8 3.0 7.2 4.6 4.3 2.6 1.7 10419156 Ear10 2.9 4.1 2.3 3.3 1.2 4.3 2.1 10423593 Laptm4b 6.2 1.8 2.0 3.0 1.8 4.1 2.2 10414262 Ear2 3.0 3.2 2.5 3.1 1.1 7.6 2.3 10432540 Lima1 6.9 3.6 2.2 2.6 2.2 1.6 1.8 10497079 Ptger3 5.9 2.6 1.9 2.5 0.9 2.0 1.2 10509441 Ece1 3.8 5.5 3.8 3.3 2.9 1.6 1.7 10569020 Ifitm6 5.9 4.7 8.0 29.0 4.7 1.2 2.0 10509127 Fuca1 3.8 3.7 4.4 1.4 2.0 1.5 1.4 10432682 Krt80 3.6 2.8 2.7 2.3 1.7 1.1 1.9 10439218 Pdia5 15.6 8.7 5.9 3.1 5.0 1.5 1.5 10538150 Tmem176a 2.2 3.6 3.5 8.3 5.2 1.2 1.7 10578477 Fam149a 6.4 4.8 4.7 4.7 7.1 1.3 1.4 10544596 Tmem176b 2.6 3.0 3.7 8.2 6.6 1.2 2.0 10409464 Dbn1 3.5 3.1 3.6 5.5 4.6 1.3 1.3 10541587 Clec4a2 4.2 2.5 2.7 4.0 3.1 1.2 1.3 10405539 Txndc15 2.7 2.2 2.5 2.4 2.1 1.3 1.3 10457733 B4galt6 5.3 3.1 5.8 4.2 1.6 1.1 1.2 10408616 Slc22a23 3.1 1.9 1.9 4.5 3.2 1.1 2.1 10441195 Dscam 17.9 3.8 13.5 0.9 0.8 2.7 1.6 10411958 Rnf180 3.2 2.1 1.9 2.8 2.1 1.8 1.3 10504582 1300002K09Rik 2.8 1.8 4.6 1.2 1.5 1.3 2.7 10504891 Tmeff1 2.8 2.2 2.2 2.5 2.4 1.7 1.3 10447275 1700106N22Rik 4.1 1.3 1.8 1.1 1.2 2.0 1.5 10386909 Cenpv 3.3 2.1 1.8 2.5 2.1 1.2 1.1 10435009 Bex6 12.3 3.0 5.6 1.9 1.7 1.0 1.7 10527936 Fzd1 4.6 2.9 2.8 9.8 5.1 1.5 1.2 10478973 Cass4 3.4 4.5 4.5 1.0 2.4 1.1 1.3 10604656 Xlr 2.3 0.9 2.2 4.5 5.1 2.0 1.4 10463224 Marveld1 2.9 2.1 3.0 1.6 1.6 1.3 1.3 10407211 Ppap2a 15.6 7.7 7.0 12.2 1.6 2.2 2.9 10603051 Ap1s2 3.3 1.8 2.0 1.4 2.0 1.1 1.6 10476443 Plcb4 2.6 2.1 1.5 2.6 1.6 2.4 1.8 10360349 Cadm3 4.0 3.3 4.3 1.5 1.8 5.1 1.1 10562181 Lsr 2.0 2.0 2.1 1.5 1.3 1.6 1.4 10542414 Ptpro 1.5 2.5 2.0 1.4 1.6 1.6 1.1 10407982 A530099J19Rik 48.9 28.0 14.7 23.1 60.4 0.9 2.5 10541581 Clec4b1 5.7 4.6 3.3 8.6 2.5 1.2 1.1 10597996 Xcr1 28.4 22.0 18.5 35.8 40.0 1.0 1.9 10426689 Spats2 3.3 1.4 2.2 0.7 1.1 1.4 1.2 10542917 Hepacam2 18.2 10.5 12.5 1.7 10.1 1.1 2.7 10444016 Pram1 3.1 1.1 2.3 1.8 1.5 1.0 1.4 10536949 Fam40b 5.6 3.1 3.5 6.2 5.4 0.9 1.9 10584317 Esam 2.5 1.0 1.6 0.8 1.5 0.4 1.3 10349661 5430435G22Rik (Rab7b) 33.1 21.5 14.0 16.5 4.9 1.1 1.2 10367746 Sash1 4.3 1.1 1.8 0.8 1.4 1.0 1.1 10436024 Gcet2 26.4 13.9 13.3 21.1 19.8 1.2 1.3 10455873 Slc12a2 5.3 0.8 2.4 4.7 0.7 1.4 1.3 10507784 Ppt1 7.9 6.6 5.6 5.1 4.2 0.9 1.2 10551736 Ppp1r14a 5.1 2.0 3.8 1.2 0.7 1.0 1.8 10594582 Snx22 6.3 6.2 6.6 5.6 7.8 1.1 1.5 10562117 Ffar2 4.1 2.4 4.1 1.0 1.1 0.9 1.2 10603059 Tmem27 3.3 2.1 2.0 1.3 2.6 1.1 1.4 10443876 Gm9705 5.9 2.0 5.2 0.9 1.2 0.8 1.2 10578493 Tlr3 16.1 12.4 10.1 19.5 25.4 1.0 1.5 10492306 Sucnr1 5.5 1.6 2.3 0.9 1.0 1.8 1.1 10462922 Plce1 4.4 2.6 3.4 9.4 6.1 0.9 1.1 10435048 Tctex1d2 3.0 1.5 2.0 1.4 1.4 1.5 1.1 10423556 Pgcp 5.0 2.5 4.1 1.6 1.9 0.9 1.2 10443869 Cyp4f16 6.8 4.9 9.4 1.8 1.9 0.9 1.2 10404965 Rnf144b 4.0 4.0 3.3 1.9 2.9 2.4 1.2 10358879 Npl 3.9 1.6 3.2 0.9 1.4 0.8 0.9 10406334 Mctp1 2.6 4.5 3.0 2.0 5.4 2.4 1.0 10437483 Rogdi 1.8 1.2 1.7 0.7 0.6 1.0 1.4 10534102 Gusb 6.3 4.3 4.3 1.7 1.9 1.8 1.3 10377938 Eno3 1.5 0.6 1.5 2.1 0.7 0.9 1.3 10385883 Slc22a21 3.6 2.5 2.6 1.6 2.5 1.6 0.9 10583847 Bbs9 1.7 1.0 1.5 0.9 0.8 1.0 1.2 10425726 Sept3 3.2 2.6 2.0 12.0 4.7 1.3 1.2 10521337 Rgs12 3.1 0.7 1.6 1.1 0.7 1.0 1.1 10604633 Cxx1b 1.8 2.1 1.7 5.4 7.0 1.3 1.0 10419151 Ear1 1.7 1.6 1.5 0.2 1.0 1.1 1.1 10531370 Naaa 11.8 5.1 6.1 2.7 2.6 0.6 1.1 10417759 Ube2e2 4.4 1.1 1.7 1.2 0.8 2.0 0.7 10531348 Ppef2 7.2 5.6 5.6 3.0 4.5 0.9 1.2 10400926 Rtn1 3.1 1.7 2.6 0.9 0.6 1.3 1.1 10427369 Pde1b 4.7 4.0 3.5 2.6 3.2 0.4 0.9 10587226 Lysmd2 3.6 1.9 3.6 1.0 0.8 1.0 1.0 10430834 Naga 3.2 2.8 2.7 2.4 3.3 1.3 1.0 10519527 Abcb1a 8.4 1.5 3.5 1.2 1.2 1.0 1.2 10599654 Cxx1c 2.5 3.7 2.6 5.0 13.6 1.0 0.7 10567343 2310008H09Rik 1.9 1.5 1.5 1.0 0.9 1.4 1.2 10467182 Htr7 4.9 2.4 3.0 5.5 2.7 0.9 1.0 10547540 Mical3 1.4 1.7 1.7 0.9 1.0 1.1 1.1 10444352 Notch4 2.9 3.6 2.2 5.2 2.4 1.1 1.0 10513164 Ptpn3 3.2 1.2 3.5 1.0 0.5 0.4 0.8 10443506 Fgd2 2.1 2.8 2.4 2.1 2.4 0.8 0.9 10579468 Haus8 2.7 1.2 3.7 1.2 1.2 1.2 1.1 10468762 4930506M07Rik 2.2 2.3 2.2 2.5 3.2 0.4 1.0 10597935 Cdcp1 2.5 1.4 2.7 0.9 0.9 1.4 1.1 10363773 Rhobtb1 2.9 2.2 2.4 2.7 2.7 1.2 1.2 10560575 Relb 1.9 1.2 2.2 1.0 1.3 1.0 1.1 10362294 Arhgap18 2.5 2.6 2.7 2.0 3.1 1.0 1.1 10467739 Avpi1 1.9 1.3 1.7 1.0 0.9 0.8 0.9 10568001 Sult1a1 2.5 2.1 2.9 3.3 3.4 1.2 0.9 10541318 Slc6a13 1.0 0.7 1.0 0.7 1.0 0.8 1.7 10410092 Zfp367 2.0 2.0 2.0 1.9 2.1 1.1 1.0 10457731 Rpl35 0.7 1.0 0.8 1.0 1.0 1.6 1.2 10443007 Neurl1B 2.3 1.9 1.8 2.7 1.4 1.0 1.2 10485840 Ryr3 1.2 1.5 1.5 0.9 0.8 1.1 1.2 10350003 Cyb5r1 2.8 2.1 3.4 1.6 1.1 1.3 0.8 10353117 Slco5a1 1.3 1.2 1.6 0.9 0.8 1.1 1.3 10450484 Aif1 6.0 3.3 3.7 1.3 1.0 1.2 0.6 10480090 Itga8 9.0 3.8 7.2 4.2 1.5 1.0 1.6 10500276 BC028528 6.3 2.6 4.4 2.2 1.2 0.8 1.8 LEGEND: 10385872 Slc22a5 2.5 1.9 1.9 1.3 2.0 1.2 1.1 >2-fold higher in CD8+ or CD103+ DCs 10370339 Trpm2 3.6 5.7 4.2 1.0 2.7 1.0 1.6 <2-fold higher in CD8+ or CD103+ DCs 10545742 Cd207 2.3 3.0 1.1 13.0 1.1 1.6 1.3 not in leading edge from GSEA analysis 10567010 Dkk3 2.6 1.6 1.5 4.7 1.8 1.5 0.9 around or below detection threshold 10440206 Arl6 1.8 1.5 1.5 1.2 1.5 1.4 1.3 <2-fold higher in CD11b+ DCs 10487011 Gatm 4.6 5.4 6.9 3.4 1.1 0.9 1.2 >2-fold higher in CD11b+ DCs 10546104 Eefsec 4.2 3.4 2.6 1.2 2.6 0.9 1.4 10382300 Map2k6 2.4 1.8 2.1 1.2 2.2 0.9 1.0 Gene expression ratio calculation: 10531407 Cxcl9 16.5 6.6 12.7 1.1 0.6 0.7 1.5 Spleen1: spleen CD8+ DCs / spleen CD11b+ DCs 10593628 Rab39 1.8 1.8 1.8 2.0 1.8 0.9 1.0 Res-CLN2: CLN CD8+ res-DCs / CLN CD11b+ res-DCs 10537921 Zfp282 1.7 1.6 1.7 1.5 1.7 1.0 1.0 Res-MLN3: MLN CD8+ res-DCs / MLN CD11b+ res-DCs 10388430 Serpinf1 2.1 1.5 1.6 1.5 1.5 1.1 1.1 Res-Lung4: Lung CD103+ int-DCs / lung CD11b+ int-DCs 10599680 3830403N18Rik 1.2 1.0 0.9 3.1 1.4 1.3 1.4 Res-Liver5: Liver CD103+ int-DCs / Liver CD11b+ intDCs 10436978 Cbr3 2.2 1.0 1.3 1.1 1.5 1.1 0.9 mig-CLN6: CLN CD103+ mig-DCs / CLN CD11b+ mig-DCs 10517689 Pqlc2 1.9 1.9 1.5 1.2 1.6 1.0 1.0 mig-MLN7: MLN TN mig-DCs / MLN CD11b+ mig-DCs 10470529 Olfm1 3.6 1.1 3.0 0.9 2.0 0.9 0.9 Spleen8: Spleen CD11b+ DCs / spleen CD8+ DCs 10430186 Apol7c 3.6 0.9 1.0 9.6 3.9 0.8 0.8 Res-CLN9: CLN CD11b+ res-DCs / CLN CD8+ res-DCs 10496023 Casp6 2.8 1.5 2.3 0.8 1.3 0.8 1.8 Res-MLN10: MLN CD11b+ res-DCs / MLN CD8+ res-DCs 10535807 Flt1 3.4 1.0 2.1 1.2 0.3 1.1 1.7 Res-Lung11: Lung CD11b+ int-DCs / lung CD103+ int-DCs 10413951 Arhgap22 1.8 1.5 1.2 0.7 1.2 2.0 0.9 Res-Liver12: Liver CD11b+ intDCs / liver CD103+ int-DCs 10466530 Pcsk5 1.5 1.0 0.9 1.3 1.0 1.4 1.0 mig-CLN13: CLN CD11b+ mig-DCs / CLN CD103+ mig-DCs 10489878 Ptgis 1.5 0.8 1.2 1.2 1.4 1.0 1.0 mig-MLN14: MLN CD11b+ mig-DCs / MLN TN mig-DCs 10489701 4833422F24Rik 2.1 1.0 1.5 1.1 0.8 1.1 0.8 10567366 Gp2 4.4 1.0 0.8 1.1 0.8 1.0 1.1 10454881 0610010O12Rik 1.5 1.0 1.1 1.7 1.4 1.2 1.1 10541910 Vwf 1.2 1.0 1.0 1.4 0.8 1.0 0.9 10392056 Cyb561 1.1 1.4 1.1 1.1 1.3 1.2 1.2 10430166 Apol7a 1.2 1.0 1.1 0.9 1.1 1.2 1.1 10595990 Armc8 1.1 1.1 0.9 0.9 1.1 0.8 0.8 10523255 Stbd1 1.3 1.1 1.3 1.2 1.0 0.7 0.9 10459930 Ctdp1 0.9 1.0 0.9 0.7 0.8 1.0 1.1 Supplemental figure legends

Supplemental Figure 1: Phenotypic characterization of migratory DCs in CLNs. A)

The gates defining mig-DCs in CLNs were based on phenotypic characteristics of tetramethyl rhodamine isothiocyanate (TRITC)-positive DCs that have migrated from the skin to the CLNs 48 hours after TRITC was applied to the dorsal and ventral sides of each ear. In CLNs, mig-DCs are CD11c+MHC-IIhigh. B) CD11c+MHC-IIhigh DCs are absent from the CLNs of CCR7-deficient mice confirming that these cells are mig-DCs. Dot plots are representative of at least 3 independent experiments.

Supplemental Figure 2: Microarray analysis of CD8α+ DC-specific and CD11b+

DC-specific genes in LT-resident CD8α+ DCs, CD103+ int-DCs, CD103+ mig-DCs or

TN mig-DCs versus their CD11b+ counterparts purified from several lymphoid and non-lymphoid organs. One representative ProbeSet of each gene is shown. Red represents higher expression in CD8α+-type DCs, green represents higher expression in CD11b+-type DCs, yellow signifies lack of strong preferential expression between

DC subsets as determined by the fact that the corresponding gene is not included in the leading edge of the GSEA analysis, and gray signifies that gene expression is around or below the detection threshold of the microarray experiments in both

CD8α+-type and CD11b+-type DC subsets. The median of the relative expression ratios between CD8α+-type DCs and CD11b+-type DCs are indicated in each cell for each gene and pair-wise comparison.