Langerin-Expressing Dendritic Cells in Human Tissues Are Related to Cd1c

Article

Langerin-expressing dendritic cells in human tissues are related to CD1c+ dendritic cells and distinct from Langerhans cells and CD141high XCR1+ dendritic cells Venetia Bigley, Naomi McGovern,1 Paul Milne, Rachel Dickinson, Sarah Pagan, Sharon Cookson, Muzlifah Haniffa, and Matthew Collin2 Human Dendritic Cell Laboratory, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom RECEIVED JULY 24, 2014; REVISED OCTOBER 1, 2014; ACCEPTED OCTOBER 16, 2014. DOI: 10.1189/jlb.1HI0714-351R

ABSTRACT mirror the CD1c+ and CD141+ blood DC populations [3]. CD141+ + + Langerin is a C-type lectin expressed at high level by LCs myeloid DCs are homologous to CD8 and CD103 DCs in mice of the epidermis. Langerin is also expressed by CD8+/ that express XCR1 and have enhanced cross-presentation CD103+ XCR1+ cross-presenting DCs of mice but is not capacity. XCR1+ DCs have been referred to as "DC1" and the found on the homologous human CD141high XCR1+ mye- CD1c+ myeloid DC as "DC2" in a recently proposed classification loid DC. Here, we show that langerin is expressed at of mammalian DCs [2]. a low level on DCs isolated from dermis, lung, liver, and + Human LCs are characterized by high expression of langerin, a lymphoid tissue and that langerin DCs are closely C-type lectin, and CD1a. Interstitial tissue DCs also express CD1a related to CD1c+ myeloid DCs. They are distinguishable but at a lower level to that found on LCs [3–9]. Dermal and lung from LCs by the level of expression of CD1a, EpCAM, + + CD11b, CD11c, CD13, and CD33 and are found in CD1a /CD1c DCs (DC2) have been reported previously to be tissues and tissue-draining LNs devoid of LCs. They langerin negative [7, 10], although langerin expression was are unrelated to CD141high XCR1+ myeloid DCs, reported in the lamina propria of human colon [11]. lacking the characteristic expression profile of cross- The selective expression of pattern recognition receptors, presenting DCs, conserved between mammalian spe- including lectins and TLRs, confers specialized functions to cies. Stem cell transplantation and DC deficiency models different populations of DCs. Langerin is able to bind viruses and confirm that dermal langerin+ DCs have an independent other pathogens, is implicated in antigen processing, and is found homeostasis to LCs. Langerin is not expressed by freshly within the Birbeck granule of LCs [12–14]. These observations + + isolated CD1c blood DCs but is rapidly induced on CD1c do not exclude a role for langerin in other DC populations. DCs by serum or TGF-b via an ALK-3-dependent path- In mice, langerin is also expressed by XCR1+ cross- way. These results show that langerin is expressed presenting DCs (DC1), expressing CD103+ in the tissues or CD8+ outside of the LC compartment of humans and highlight – a species difference: langerin is expressed by the XCR1+ in the lymphoid organs [15 20]. In humans, however, the high + "DC1" population of mice but is restricted to the CD1c+ DC1 subset of CD141 XCR1 myeloid DCs is langerin "DC2" population of humans (homologous to CD11b+ DCs negative [3]. in the mouse). J. Leukoc. Biol. 97: 627–634; 2015. In human progenitor cells and monocytes, langerin is induced by a wide range of cytokines in vitro, including GM-CSF, TNF, TGF-b, and BMP-7 [8, 21–24]. These results suggest that langerin Introduction might be expressed by other DCs outside of the LC compartment. In this report, we set out to re-examine the question of Human skin and other nonlymphoid tissues contain several langerin expression by human DCs in situ and found evidence of subsets of DCs and macrophages [1, 2]. In addition to LCs of langerin expression by CD1c+ myeloid DCs. stratified epidermis, the interstitial tissues contain CD1c+ myeloid DCs and a minor population of CD141high myeloid DCs that 1. Current affiliation: Singapore Immunology Network, Agency for Science, Technology and Research, Singapore. Abbreviations: ALK-3 = activin receptor-like kinase 3, APC = allophycocyanin, 2. Correspondence: Institute of Cellular Medicine, Newcastle University, BMP-7 = bone morphogenetic protein 7, CLEC9 = C-type lectin domain Framlington Pl., Newcastle upon Tyne, NE2 4HH, United Kingdom. E-mail: [email protected]; Twitter: http://www.twitter.com/ family 9, DC = dendritic cell, EpCAM = epithelial cell adhesion molecule, FISH = HumanDCLab fluorescence in situ hybridization, HSCT = hematopoietic stem cell This is an Open Access article distributed under the terms of the Creative transplant, LC = Langerhans cell, LCH = Langerhans cell histiocytosis, LN = Commons Attribution 4.0 International (CC BY 4.0) (http://creativecommons. lymph node, NECL2 = nectin-like molecule 2, qPCR = quantitative PCR, org/licenses/by/4.0/) which permits unrestricted use, distribution, and XCR1 = chemokine (C motif) receptor 1 reproduction in any medium, provided the original work is properly cited.

0741-5400/15/0097-627 © The Author(s) Volume 97, April 2015 Journal of Leukocyte Biology 627 MATERIALS AND METHODS HLA-DR-PerCP-Cy5.5 (L243), langerin-PE (DCGM4; Beckman Coulter, Brea, CA, USA), and EpCAM-APC (EBA-1). Samples and patient data Blood, skin, and surplus biopsy material was obtained from healthy human Microscopy and FISH volunteers and hematopoietic transplant patients under ethical approval from the Newcastle and North Tyneside Research Ethics Committee 1. Skin, tissues, Fluorescence microscopy was performed by use of a Zeiss AxioPlan fl 3 and blood were processed as described previously [3]. 2 microscope EC Plan-Neo uar 40 numerical aperture 0.75 lens and AxioCam running Zeiss AxioVision v2.8 software (Zeiss, Thornwood, NY, USA). Separated epidermal and dermal sheets were fixed in acetone for 20 min and Flow cytometry and cell sorting rehydrated in PBS for 20 min before staining. LCs were stained with CD1a- Cells were stained in aliquots of up to 2 3 106 cells in 50 ml buffer, according FITC (NA1/34; Dako), and langerin+ dermal DCs were visualized with HLA- to standard protocols. Nonviable cells were excluded by staining with DAPI DR FITC (L243; Becton Dickinson), polyclonal rabbit anti-langerin followed (Partec, Sysmex, Görlitz, Germany) or LIVE/DEAD dead cell stain (Invitrogen, by Alexa Fluor 555-conjugated goat anti-rabbit IgG H+L (Invitrogen), and Carlsbad, CA, USA). Flow cytometry was performed on a LSRII cytometer mouse monoclonal anti-CD11c (Becton Dickinson) followed by Alexa Fluor (Becton Dickinson, Franklin Lakes, NJ, USA) and data analyzed with 647 (Invitrogen). Sorted skin DC subsets were spun onto glass slides, fixed in FlowJo (Treestar, Ashland, OR, USA). FACS was performed by use of a BD methanol, and restained for HLA-DR and langerin as above and mounted in FACSAria (100 mm nozzle and 20 pounds/square inch; BD Biosciences, San Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA). For Jose, CA, USA). Sorted cells were collected into Eppendorfs containing RPMI FISH, directly sorted skin cells were fixed in Carnoy’s (methanol: acetic acid with 10% FCS or sorted directly onto glass slides, air dried, and fixed in 3:1). Vysis CEP X SpectrumOrange and CEP X SpectrumGreen DNA probe methanol. Antibodies were from Becton Dickinson unless stated otherwise kit (Abbott Laboratories, Abbott Park, IL, USA) were used, according to the (clone in parentheses). CD1a-FITC (NA1/34; Dako, Glostrup, Denmark), manufacturer’s instructions. CD1a-APC (HI 149), CD1c-PE/APC (AD5-8E7; MACS; Miltenyi Biotec, Bergisch Gladbach, Germany), CD3-FITC (UCHT1), CD11b-APC (ICRF44; BioLegend, San Diego, CA, USA), CD11c-APC/V450 (B-ly6), CD13-APC Langerin Induction on blood DCs (WM15), CD14-FITC/PE/PE-Cy7 (M5E2), QDOT605 (TuK4; Invitrogen), Unfractionated PBMCs (2 3 106) were incubated in 1 ml RPMI with 10% FCS CD16-FITC/PE-Cy7 (3G8), CD19-FITC (SJ25C1), CD20-FITC (L27), CD31- for 18 h, with or without TGF-b (100 ng/ml). In experiments with sorted cells, FITC (WM59), CD34-APC (8G12)/APC-Cy7 (581; BioLegend), CD45-APC- 10,000 cells were cultured in 100 ml. Cells were stained in the same Cy7/V450 (2D1), CD56-FITC (NCAM16.2), CD115-PE (61708; R&D Systems, Eppendorfs for surface expression of langerin and analyzed by flow cytometry Minneapolis, MN, USA), CD123-PE/PerCP-Cy5.5 (9F5), CD135-PE (4G8), initially and after 18 h. Recombinant human BMPR-IA/ALK-3 Fc chimera was CD141-PE (1A4), CD141-APC (AD5-14H12; MACS; Miltenyi Biotec), obtained from R&D Systems (315-BR-100) and used at 2 mg/ml.

Figure 1. Langerin expression by cutaneous DCs, distinct from LCs. (A) Identification of langerin+ DCs among the CD45+,DR+, low autofluorescnce (AF) fraction of collagenase-digested skin. LCs are seen in the skin as CD1ahigh langerinhigh cells (red gate); a second CD1a+ langerin+ population is apparent (blue gate). Representative example of 22 experiments. SSC-A, Side-scatter-area. (B) Compar- ison of whole skin, dermis, and epidermis prepara- tions, showing that langerin+ DCs are found in whole skin or dermis but not epidermis and may be separated from LCs by lower expression of CD1a and EpCAM or by higher expression of CD13, CD33, CD11c, and CD11b. Percentage of langerin+ DC (blue) and LCs are indicated (red). A representative example of 5 experiments shown. (C) Heat map summary of mean fluorescence intensity of key surface antigens of langerin+ DCs, CD1a+ dermal DCs, and LCs (mean of 4 experiments).

628 Journal of Leukocyte Biology Volume 97, April 2015 www.jleukbio.org Bigley et al. Langerin+ human dendritic cells

Figure 2. Gene-expression proﬁles of langerin+ DCs compared with other dermal DCs and LCs. (A) Heat map of qPCR for 4 characteristic transcripts of CD141+ cross-presenting DCs. Mean of 3 experiments. (B) Relative expression of growth factor receptors. Mean 6 SEM of 3 experiments. (C) Relative expression of TLRs. Mean + SEM of 3 experiments

qPCR (Invitrogen) with deoxyribonucleotide triphosphates (Roche, Indianapolis, IN, USA), random hexamers (Pharmacia, Uppsala, Sweden), and RNasin RNA was extracted from sorted cell populations or LCH lesions by use of the (Promega, Madison, WI, USA). qPCR reactions were performed with TaqMan Qiagen Micro kit (Qiagen, Valencia, CA, USA), according to the manufac- Gene Expression Assays (Applied Biosciences, Life Technologies, Carlsbad, turer’s instructions. Single-stranded cDNA was generated by use of an CA, USA) by use of the 7900HT Fast Real-Time PCR system (Applied Moloney Murine Leukemai Virus Reverse Trnascriptase (M-MLV RT) kit Biosciences, Life Technologies). GAPDH was used to calculate DCT.

Figure 3. Langerin+ DCs in other tissues and draining LNs. (A) Langerin+ DCs identiﬁed within CD1c+ DCs of lung, liver, and tonsil, showing variable coexpression of CD1a. As in the dermis, langerin+ DCs were found in CD45+ HLA-DR+ 2 2 lineage and autoﬂuorescence negative (Lin /AF ) 2 CD14 fraction. Data represent 3, 3, and 7 samples of lung, liver, and tonsil, respectively. (B) Detailed phenotype of langerin+ cells isolated from lung and lung draining LN (DLN). Skin is shown for comparison. Both skin and skin draining nodes contain langerin+ DCs (blue gate) and CD11clow CD1ahigh LCs (red gate). The langerin+ populations of lung and lung draining LNs contain only langerin+ DCsbutnoLCs.Thesebothhaveasimilarphenotype 2 to dermal langerin+ DCs and are EpCAM CD11blow CD31+ CD11c+.

www.jleukbio.org Volume 97, April 2015 Journal of Leukocyte Biology 629 Statistical analysis in langerin+ DCs, contrasting with the intense perinuclear Golgi + Graphs were plotted with Prism Version 5.0a (GraphPad Software, La Jolla, staining of LCs. Langerin DCs were smaller than LCs and resembled + CA, USA). Mean and SD calculation, paired t-tests, and linear-regression CD1a DCs in appearance (Fig. 4A). With the use of CD11c analysis were performed within the graphing software. All P values were two expression to separate langerin+ DCs from LCs, it was possible to tailed. Heat maps of median ﬂuorescence intensity were generated by use of detect occasional langerin+ DCs in situ in the apical dermis. These MultiExperiment Viewer (http://www.tm4.org/index.html; TM4 Microarray also showed a similar diffuse pattern of langerin staining in contrast Software Suite). to the bright langerin expression of migrating LCs (Fig. 4B). Several attempts were made to detect Birbeck granules in sorted populations RESULTS of langerin+ DCs, but none was found (not shown).

+ Langerin expression on a fraction of CD1a/c DCs in + normal human tissues The homeostasis of langerin dermal DCs is distinct from that of LCs Collagenase-digested whole skin was analyzed by flow cytometry + + fi Maturing LCs down-regulate langerin expression and may for langerin expression. From live CD45 HLA-DR cells, xed + macrophages and monocyte-derived cells were excluded by acquire the characteristics of myeloid DCs. To verify that langerin gating out autofluorescent and CD14+ cells. Two populations of dermal DCs were not derived from LCs, we examined 2 in vivo langerin+ cells were observed within the remaining population; one with high langerin and CD1a and the other with in- termediate langerin and CD1a (Fig. 1A). Neither CD14+ cells nor the CD141high subset of DCs expressed langerin, as described previously [3]. This observation suggested that in addition to langerinhigh CD1ahigh LCs originating from the epidermis, there was a lower level of langerin expression by CD1a+ dermal DCs. To define this further, dermis and epidermis were examined separately for langerin expression together with CD1a and EpCAM to distinguish LCs (CD1ahigh EpCAM+) from dermal DCs (CD1alow 2 EpCAM ). This consistently demonstrated langerin+ dermal DCs (Fig. 1B). Additional antigens CD13, CD31, CD11c, and CD11b were able to dissociate LCs from langerin+ DCs (Fig. 1B). Comparison of all surface antigens analyzed suggested that langerin+ DCs were closely related to CD1a+ dermal DCs and distinct from LCs. All subsets expressed CD1c (Fig. 1C). The phenotype of langerin+ DCs was explored further with qPCR. First, comparison with the signature of CD141high XCR1+ DCs showed that dermal langerin+ DCs did not express the characteristic markers of cross-presenting DCs: XCR1, NECL2, and CLEC9 (Fig. 2A). Growth factor receptor expression profiles showed that langerin+ DCs had a high Flt-3/low M-CSFR signature in common with CD1a+ dermal DCs and distinct from LCs and CD14+ monocyte-derived cells (Fig. 2B). The TLR profile of langerin+ DCs was similar to that of CD1a+ dermal DCs, although all myeloid cells expressed a similar array of receptors (Fig. 2C). Langerin+ CD1c+ DCs were also detectable in tissues normally devoid of LCs, including the lung, liver, and tonsil. There was variable expression of CD1a; higher levels were detected in the lung than liver or tonsil (Fig. 3A). We examined skin and lung with matched draining LNs to determine whether the phenotype of langerin+ DCs in draining LNs was concordant with the tissues (Fig. 3B). Matched populations of langerin+ DCs and LCs, separable by CD11c and Figure 4. In situ expression of langerin by dermal DCs. (A) Sorted CD1a+ + CD1a expression, were observed in skin and axillary LNs. In the dermal DCs, langerin DCs (LDC) and LCs from freshly digested whole lung and bronchial LNs, only langerin+ DCs but no LCs were skin were spun onto cytopsin slides and reprobed with antibodies for + HLA-DR and langerin. One of 2 independent experiments is shown. (B) found. The langerin DCs from both tissue and corresponding + In situ detection of langerin DCs in a whole-mount stain of dermal sheet LN had a similar phenotype (Fig. 3B). (apical surface). The field contains a migratory LC with high langerin + + Immunofluorescence staining of sorted LCs, langerin CD1a expression but no CD11c, a CD11c+ langerin+ DC (arrow) and a number 2 2 DCs, and langerin CD1a+ DCs revealed diffuse cytoplasmic staining of langerin CD11c+ myeloid DCs. Data taken from 1 of 5 experiments.

630 Journal of Leukocyte Biology Volume 97, April 2015 www.jleukbio.org Bigley et al. Langerin+ human dendritic cells

Figure 5. Homeostasis of langerin+ DCs compared with CD1a+ DCs and LCs. (A) Comparison of the level of donor engraftment at day 28 post-HSCT among dermal CD1a+ DCs (green gate and symbols), langerin+ DCs (blue gate and symbols), LCs (red gate and symbols), and peripheral blood monocytes and DCs (black). Cells were sorted as indicated on the dot plot and probed with X-Y FISH, as shown in the example. Four samples were analyzed. pDC, Plasmacytoid DC. (B) Analysis of LCs and dermal DCs in 2 patients with failure of bone marrow-derived DC hematopoiesis as a result of GATA-2 mutation. A healthy control is shown on the left for comparison. (Upper) LC in situ in the epidermis; (lower) a small number of LCs crossing the dermis (red gates), in the absence of any detectable langerin+ or CD1a+ dermal DCs (blue gates).

models of human DC homeostasis: HSCT and human DC crossing the dermis, further supports the conclusion that deficiency as a result of GATA2 mutation. langerin+ dermal DCs are unlikely to be derived from LCs In patients receiving HSCT, LCs remain predominantly (Fig. 5B). recipient in origin for the first 28 d post-transplant, whereas + donor-derived cells replace the blood and interstitial DC Induction of langerin expression on CD1c blood compartments [9, 25]. Langerin+ DCs and LCs were isolated myeloid DCs from skin biopsies at day 28 of sex-mismatched transplants Having observed langerin+ DCs in human tissues, peripheral and probed with X-Y FISH. This indicated that langerin+ DCs blood was examined for langerin expression. None was detect- were replaced rapidly by donor-derived cells (80–90%) in able on freshly isolated cells, but langerin was induced at a low parallel with CD1a+ dermal DCs, blood monocytes, and blood level in serum-containing medium and by TGF-b after 18 h of DCs. All of these populations engrafted with much more culture of unfractionated PBMC. Flow analysis after 18 h rapid kinetics than LCs, which were 10–20% donor at the same indicated that langerin was expressed selectively by CD1c+ time-point. These results indicate that langerin+ DCs are unlikely myeloid DCs (Fig. 6A). Addition of TNF-a or GM-CSF, cytokines to be derived from maturing epidermal LCs crossing the dermis, that are known to derive langerin+ DCs, did not further augment as the latter were mostly recipient at the time of analysis the expression of langerin, and langerin induction was not (Fig. 5A). observed on the small number of CD141+ DCs present in these Patients with DC deficiency as a result of GATA2 mutations experiments (not shown). Sorted CD1c+ myeloid DCs, but not lack interstitial DC populations but maintain epidermal LCs [26, other populations, also expressed langerin after 18 h with serum- 27]. Closer analysis of these patients revealed that langerin+ DCs containing medium or with TGF-b (Fig. 6B). qPCR for langerin are also missing in parallel with CD1a+ dermal DCs. In contrast, showed that freshly isolated CD1c+ DCs express a low level of the persistence of LCs, including the small afferent population langerin message that was up-regulated in culture with TGF-b.

www.jleukbio.org Volume 97, April 2015 Journal of Leukocyte Biology 631 Figure 6. Induction of langerin in CD1c+ blood myeloid DCs. (A) Langerin induction on whole-cultured PBMC after 18 h (T18) of culture in vitro, with or without TGF-b, compared with isotype control. DCs and monocytes were identiﬁed by the gating strategy shown and the expression of langerin displayed below. Representative example of 4 experiments. (B) Langerin induction on sorted monocytes and blood DCs after 18 h of culture in serum containing medium and TGF-b.*P , 0.05; **P , 0.01. Mean of 4 experiments. (C) Expression of langerin mRNA by CD14+ monocytes (blue bars) and CD1c+ blood DCs (red bars) at 0 h and 18 h with TGF-b. One of 3 experiments is shown. (D) Inhibition of langerin induction in serum containing medium and TGF-b by an ALK-3-Fc chimeric-blocking agent. One of 3 experiments is shown.

This is in contrast to CD14+ monocytes, which remain negative have parallel homeostasis with blood monocytes and DCs and are (Fig. 6C). It was possible to reduce the level of langerin not derived from LCs. Finally, freshly isolated blood CD1c+ DCs induction by TGF-b with the use of an ALK-3-Fc chimeric-blocking contain langerin mRNA and up-regulate langerin surface-antigen reagent (Fig. 6D). expression in response to serum or TGF-b within 18 h. Together, these results indicate that CD1c+ myeloid DCs can be induced to express langerin and that in vivo, a fraction of these cells is + DISCUSSION langerin , possibly through the action of local environmental factors, such as TGF-b. These results demonstrate the existence of widely distributed Langerin has not been detected on tissue DCs of the skin and langerin+ DCs that have variable expression of CD1a+ and are lung by previous studies [7, 10], although langerin+ DCs were closely related to CD1c+ DCs. In the recently proposed reported in human colon [11], and there is reference to trace classification of mammalian DCs, this subset is related to CD11b+ levels of langerin expression in human blood [28, 29]. TGF-b is DCs of mice and is known as DC2 [2]. The expression of langerin able to up-regulate expression by CD1c+ blood DCs, suggesting is low but distinct and is found in sites that do not normally that langerin expression in tissues may be induced by epithelial contain LCs, including lymphoid and nonlymphoid tissues. or autocrine TGF-b [30–32]. The use of an ALK-3-blocking Langerin is not expressed by the CD141high XCR1+ cross- reagent implicates similar TGF-b signaling pathways to those presenting DC [3], and conversely, langerin+ CD1c+ DCs do not involved in the induction of langerin on progenitor cell express the characteristic markers of cross-presenting DCs. The populations [24]. One caveat of our experiments is that in vitro absence of langerin from human DC1 is an important species preparation of skin by enzymatic digest may have enhanced the difference between mice and humans, contrasting with the level of langerin expression. However, our results also show that consistent expression of langerin by mouse CD8+/CD103+ DC1 langerin+ DCs are detectable in situ by immunofluorescence and [18–20]. It should be noted, however, that approximately one- also found in lung, liver, and LN, tissues that do not require long third of langerin+ DCs in murine nonlymphoid tissues does not preparative manipulation. express the DC1 marker CD103, suggesting expression on Expression of langerin by CD34+ progenitors and CD14 additional DC populations that may include some DC2 [20]. monocytes is well described after prolonged in vitro culture In vivo experiments with HSCT recipients and patients with [8, 21–24, 31–33]. These techniques are usually designed to make DC deficiency show that langerin+ DCs and CD1a+ dermal DCs LCs, although they may also generate langerin+ DCs similar to

632 Journal of Leukocyte Biology Volume 97, April 2015 www.jleukbio.org Bigley et al. Langerin+ human dendritic cells the phenotype that we describe in vivo. Several investigators have Dimmick,I.,Jarrett,R.F.,Renia,L.,Tam,J.,Song,C.,Connolly,J., Chan, J. K., Gehring, A., Bertoletti, A., Collin, M., Ginhoux, F. (2012) demonstrated Birbeck granules in in vitro-derived LCs. We were Human tissues contain CD141hi cross-presenting dendritic cells with + unable to find these organelles in primary langerin DCs. This functional homology to mouse CD103+ nonlymphoid dendritic cells. – may have been hampered by effacement of ultrastructural Immunity 37, 60 73. + 4. Lenz, A., Heine, M., Schuler, G., Romani, N. (1993) Human and murine features following cell sorting, although it is notable that langerin dermis contain dendritic cells. Isolation by means of a novel method and DCs in mice also express less langerin than LCs and to our phenotypical and functional characterization. J. Clin. 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(2007) CD14+ antigen-presenting cells in human dermis are that LCH cells are quite different to LCs [32, 34–37]. The lack of less mature than their CD1a+ counterparts. Int. Immunol. 19, 1271–1279. EpCAM, lower CD1a, and expression of CD11b, CD13, and CD33 8. Klechevsky, E., Morita, R., Liu, M., Cao, Y., Coquery, S., Thompson- + Snipes, L., Briere, F., Chaussabel, D., Zurawski, G., Palucka, A. K., Reiter, by LCH cells is also a feature more in keeping with langerin Y., Banchereau, J., Ueno, H. (2008) Functional specializations of human CD1c+ DCs than LCs. A langerin+ DC origin for LCH is perhaps epidermal Langerhans cells and CD14+ dermal dendritic cells. Immunity 29, 497–510. more consistent with the widespread distribution of multisystem 9. Haniffa, M., Ginhoux, F., Wang, X. N., Bigley, V., Abel, M., Dimmick, I., LCH and absence of Birbeck granules in the liver and Bullock, S., Grisotto, M., Booth, T., Taub, P., Hilkens, C., Merad, M., gastrointestinal tract [38]. 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(2013) Intestinal lamina propria dendritic cells maintain T cell confined to human LCs in vivo but is expressed at low level by homeostasis but do not affect commensalism. J. Exp. Med. 210, 2011–2024. a fraction of nonlymphoid and lymphoid CD1c+ DCs and is 12. Valladeau,J.,Ravel,O.,Dezutter-Dambuyant,C.,Moore,K.,Kleijmeer,M., + Liu,Y.,Duvert-Frances,V.,Vincent,C.,Schmitt,D.,Davoust,J.,Caux,C., induced rapidly on blood CD1c DCs in vitro. Lebecque, S., Saeland, S. (2000) Langerin, a novel C-type lectin specificto Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules. Immunity 12, 71–81. 13. Hunger, R. E., Sieling, P. A., Ochoa, M. T., Sugaya, M., Burdick, A. E., AUTHORSHIP Rea, T. H., Brennan, P. J., Belisle, J. T., Blauvelt, A., Porcelli, S. A., Modlin, R. L. (2004) Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide antigens to T cells. J. Clin. Invest. 113, V.B. designed and performed experiments, analyzed data, and 701–708. wrote the paper. N.M., P.M., and R.D. performed experiments 14. Kissenpfennig, A., A¨ıt-Yahia, S., Clair-Moninot, V., Stossel,¨ H., Badell,E.,Bordat,Y.,Pooley,J.L.,Lang,T.,Prina,E.,Coste,I., and analyzed data. S.P. and S.C. performed experiments. M.H. Gresser,O.,Renno,T.,Winter,N.,Milon,G.,Shortman,K.,Romani, designed and performed experiments and analyzed data. M.C. N.,Lebecque,S.,Malissen,B.,Saeland,S.,Douillard,P.(2005) Disruption of the langerin/CD207 gene abolishes Birbeck granules designed experiments, analyzed data, and wrote the paper. without a marked loss of Langerhans cell function. Mol. Cell. Biol. 25,88–99. 15. Poulin, L. F., Henri, S., de Bovis, B., Devilard, E., Kissenpfennig, A., Malissen, B. (2007) The dermis contains langerin+ dendritic cells that ACKNOWLEDGMENTS develop and function independently of epidermal Langerhans cells. J. Exp. Med. 204, 3119–3131. This work was supported by the Histiocytosis Research Trust, the 16. Bursch, L. S., Wang, L., Igyarto, B., Kissenpfennig, A., Malissen, B., fi Histiocytosis Association, the Medical Research Council, the Kaplan, D. H., Hogquist, K. A. (2007) Identi cation of a novel population of Langerin+ dendritic cells. J. Exp. Med. 204, 3147–3156. British Society for Haematology, Bright Red, The George Walker 17. Ginhoux, F., Collin, M. P., Bogunovic, M., Abel, M., Leboeuf, M., Helft, J., Foundation, and The Wellcome Trust (Grant WT088555MA). Ochando, J., Kissenpfennig, A., Malissen, B., Grisotto, M., Snoeck, H., Randolph, G., Merad, M. (2007) Blood-derived dermal langerin+ dendritic cells survey the skin in the steady state. J. Exp. Med. 204, 3133–3146. 18. Ginhoux, F., Liu, K., Helft, J., Bogunovic, M., Greter, M., Hashimoto, D., DISCLOSURES Price, J., Yin, N., Bromberg, J., Lira, S. A., Stanley, E. R., Nussenzweig, M., The authors declare no conflict of interest. Merad, M. 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