Complement Receptor C5aR1/CD88 and Dipeptidyl Peptidase-4/CD26 Define Distinct Hematopoietic Lineages of Dendritic Cells This information is current as Hideki Nakano, Timothy P. Moran, Keiko Nakano, Kevin E. of September 28, 2021. Gerrish, Carl D. Bortner and Donald N. Cook J Immunol 2015; 194:3808-3819; Prepublished online 13 March 2015; doi: 10.4049/jimmunol.1402195 http://www.jimmunol.org/content/194/8/3808 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/03/13/jimmunol.140219 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 53 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/194/8/3808.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021 • 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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Complement Receptor C5aR1/CD88 and Dipeptidyl Peptidase-4/CD26 Define Distinct Hematopoietic Lineages of Dendritic Cells Hideki Nakano,* Timothy P. Moran,*,†,1 Keiko Nakano,* Kevin E. Gerrish,‡ Carl D. Bortner,x and Donald N. Cook* Differential display of the integrins CD103 and CD11b are widely used to distinguish two major dendritic cell (DC) subsets in nonlymphoid tissues. CD103+ DCs arise from FLT3-dependent DC precursors (preDCs), whereas CD11bhi DCs can arise either from preDCs or FLT3-independent monocytes. Functional characterization of these two lineages of CD11bhi DCs has been hindered by the lack of a widely applicable method to distinguish between them. We performed gene expression analysis of hi lo fractionated lung DCs from C57BL/6 mice and found that monocyte-derived DCs (moDCs), including CD11b Ly-6C tissue- Downloaded from resident and CD11bhiLy-6Chi inflammatory moDCs, express the complement 5a receptor 1/CD88, whereas preDC-derived con- ventional DCs (cDCs), including CD103+ and CD11bhi cDCs, express dipeptidyl peptidase-4/CD26. Flow cytometric analysis of multiple organs, including the kidney, liver, lung, lymph nodes, small intestine, and spleen, confirmed that reciprocal display of CD88 and CD26 can reliably distinguish FLT3-independent moDCs from FLT3-dependent cDCs in C57BL/6 mice. Similar results were obtained when DCs from BALB/c mice were analyzed. Using this novel approach to study DCs in mediastinal lymph nodes, we observed that most blood-derived lymph node–resident DCs, as well as tissue-derived migratory DCs, are cDCs. Furthermore, http://www.jimmunol.org/ cDCs, but not moDCs, stimulated naive T cell proliferation. We anticipate that the use of Abs against CD88 and CD26 to distinguish moDCs and cDCs in multiple organs and mouse strains will facilitate studies aimed at assigning specific functions to distinct DC lineages in immune responses. The Journal of Immunology, 2015, 194: 3808–3819. endritic cells (DCs) induce adaptive immunity by ac- to CD8a+ DCs, which are primarily found in lymphoid tissues. quiring Ags and presenting peptides derived from them to Both of these DC types are derived exclusively from FLT3- naive T cells (1). In nonlymphoid tissues such as the dependent DC precursors (preDCs) and are therefore termed D + lung, two major CD11c DC subsets can be identified based on conventional DCs (cDCs) (3–7). In contrast, CD11bhi DCs in the by guest on September 28, 2021 their display of the integrins aE (CD103) and aM (CD11b) (2, 3). lung are a heterogeneous population of cells comprising both + Lung CD103 DCs are a homogeneous population and are similar cDCs and monocyte-derived DCs (moDCs). The latter include short-lived Ly-6Chi inflammatory DCs as well as longer lived Ly- lo *Immunity, Inflammation and Disease Laboratory, Division of Intramural Research, 6C resident moDCs that are seen both in the steady-state and National Institute of Environmental Health Sciences, National Institutes of Health, during inflammation (3, 4, 8–11). † Research Triangle Park, NC 27709; Division of Allergy and Immunology, De- Considerable progress has been recently made toward under- partment of Pediatrics, Duke University Medical Center, Durham, NC 27705; ‡Molecular Genetics Core Laboratory, Division of Intramural Research, National standing the molecular basis of DC development. In particular, Institute of Environmental Health Sciences, National Institutes of Health, Research x several transcription factors have been found to have important Triangle Park, NC 27709; and Signal Transduction Laboratory, Division of Intra- + mural Research, National Institute of Environmental Health Sciences, National Insti- roles in the progression of progenitor cells to mature DCs. CD103 tutes of Health, Research Triangle Park, NC 27709 cDC development requires several such factors, including inhibi- 1Current address: Department of Pediatrics, University of North Carolina School of tor of DNA binding 2, IFN regulatory factor 8, BATF3, and NFIL3 Medicine, Chapel Hill, NC. (4). A different set of transcription factors, including IFN regu- Received for publication August 27, 2014. Accepted for publication February 13, latory factor 4, contributes to CD11bhi cDC development, al- 2015. though mice lacking these proteins retain substantial numbers of This work was supported by the Intramural Research Program of the National In- hi stitutes of Health, National Institute of Environmental Health Sciences Grant ZIA CD11b DCs in some tissues. In contrast to transcription factors ES102025-09. that affect either CD103+ cDCs or CD11bhi cDCs, ZBTB46 is The microarray data presented in this article have been submitted to the Gene Ex- a transcription factor found in both types of cDCs (12). Mice pression Omnibus (http://www.ncbi.nlm.nih.gov/geo/info/linking.html) under acces- expressing a Zbtb46 promoter–driven GFP or diphtheria toxin sion number GSE64896. receptor allow ready identification and purification of all cDCs, at Address correspondence and reprint requests to Dr. Hideki Nakano, Immunity, In- flammation and Disease Laboratory, National Institute of Environmental Health least during steady-state conditions (12, 13). The lectin DNGR-1/ Sciences, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709. E-mail CLEC9A is also produced in common DC precursors, allowing address: [email protected] fate mapping of their progeny, which include CD8a+ and CD103+ The online version of this article contains supplemental material. cDCs, as well as some CD11bhi cDCs (14). Although these novel Abbreviations used in this article: BM, bone marrow; BMDC, BM-derived DC; reporter mouse strains have improved our ability to identify C5aR1, complement 5a receptor 1; cDC, conventional DC; DC, dendritic cell; hi DPP4, dipeptidyl peptidase-4; FLT3L, FLT3 ligand; HDE, house dust extract; LN, cDCs, it is often necessary to discriminate between CD11b lymph node; MHC-II, MHC class II; mLN, mediastinal lymph node; moDC, cDCs and CD11bhi moDCsincommonlyusedstrainsthatdo monocyte-derived DC; preDC, DC precursor; qPCR, quantitative PCR. not carry reporter genes. For such experiments, Abs must be used Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 to discriminate among the various DC subsets, and considerable www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402195 The Journal of Immunology 3809 progress has also been made in this area. Abs against CD14 and conjugated streptavidin. Stained cells were analyzed on a five-laser LSR II or CD64 are widely used to identify macrophages, and recent studies sorted on a five-laser FACSAria II flow cytometer (BD Biosciences), and the data were analyzed using a FACSDiva (BD Biosciences) and FlowJo have shown that these molecules are also displayed by some hi + hi hi software (Tree Star, Ashland, OR). For gated CD11b DCs, we analyzed CD11c MHC class II (MHC-II) CD11b DCs, presumably from 5 3 103 to 2 3 104 cells. Only single cells were analyzed, and dead CD11bhi moDCs (15). Accordingly, display of CD14 and CD64 cells were excluded based on their forward and side scatter. has been used to identify moDCs, together with the marker CD24, Purification of monocytes and preDCs and their differentiation which is displayed on some CD11bhi cDCs (16, 17). This is in vitro and in vivo a useful strategy for discriminating between cDCs and moDC in the lung and small intestine, but it is of limited utility for dis- Bone marrow (BM) was collected and RBCs were lysed with 0.15 M ammonium chloride/1 mM potassium bicarbonate. Inflammatory mono- tinguishing DC lineages in other organs (15, 17). For example, in 2 2 2 2 2 cytes (CD3 CD11b+CD11c CD19 CD49b CD115+F4/80+I-A Ly-6A/ skin-draining lymph nodes (LNs), cDCs identified by their high E2Ly-6ChiLy-6G2TER11927-aminoactinomycin D2) or preDCs (B2202 expression of Zbtb46 have relatively high levels of CD14 (12), CD32CD11b2CD11c+CD192CD49b2CD135+CD172aintI-A2Ly-6A/E2 which is associated