Abstract
Antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages comprise a relatively small fraction of leukocytes residing in lymphoid and non-lymphoid tissues. Accordingly, functional analyses of these cells have been hampered by low cell yields. Also, alveolar macrophages share several physical properties with DCs, and this has complicated efforts to prepare pure populations of lung APCs. To overcome these difficulties, we have developed improved flow cytometry-based methods to analyze and purify APCs from the lung and its draining lymph nodes (LNs). In this chapter, we describe these methods in detail, as well as methods for culturing APCs and characterizing their interactions with T cells.
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References
Sertl K, Takemura T, Tschachler E, Ferrans VJ, Kaliner MA, Shevach EM (1986) Dendritic cells with antigen-presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura. J Exp Med 163:436–451
Lambrecht BN, Hammad H (2009) Biology of lung dendritic cells at the origin of asthma. Immunity 31:412–424
Sung SS, Fu SM, Rose CE Jr, Gaskin F, Ju ST, Beaty SR (2006) A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing langerin and tight junction proteins. J Immunol 176:2161–2172
Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ, Ley K (2006) Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent. J Exp Med 203:1273–1282
Nakano H, Free ME, Whitehead GS, Maruoka S, Wilson RH, Nakano K, Cook DN (2012) Pulmonary CD103(+) dendritic cells prime Th2 responses to inhaled allergens. Mucosal Immunol 5:53–65
Inaba K, Witmer-Pack MD, Inaba M, Muramatsu S, Steinman RM (1988) The function of Ia + dendritic cells and Ia- dendritic cell precursors in thymocyte mitogenesis to lectin and lectin plus interleukin 1. J Exp Med 167:149–162
Stevens WW, Kim TS, Pujanauski LM, Hao X, Braciale TJ (2007) Detection and quantitation of eosinophils in the murine respiratory tract by flow cytometry. J Immunol Methods 327:63–74
Jakubzick C, Randolph GJ (2010) Methods to study pulmonary dendritic cell migration. Methods Mol Biol 595:371–382
Vermaelen K, Pauwels R (2004) Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights. Cytometry A 61:170–177
Naik SH, Sathe P, Park HY, Metcalf D, Proietto AI, Dakic A, Carotta S, O’Keeffe M, Bahlo M, Papenfuss A, Kwak JY, Wu L, Shortman K (2007) Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. Nat Immunol 8:1217–1226
Onai N, Obata-Onai A, Schmid MA, Ohteki T, Jarrossay D, Manz MG (2007) Identification of clonogenic common Flt3 + M-CSFR + plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow. Nat Immunol 8:1207–1216
Cheong C, Matos I, Choi JH, Dandamudi DB, Shrestha E, Longhi MP, Jeffrey KL, Anthony RM, Kluger C, Nchinda G, Koh H, Rodriguez A, Idoyaga J, Pack M, Velinzon K, Park CG, Steinman RM (2010) Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas. Cell 143:416–429
Nakano H, Lin KL, Yanagita M, Charbonneau C, Cook DN, Kakiuchi T, Gunn MD (2009) Blood-derived inflammatory dendritic cells in lymph nodes stimulate acute T helper type 1 immune responses. Nat Immunol 10:394–402
Nakano H, Burgents JE, Nakano K, Whitehead GS, Cheong C, Bortner CD, Cook DN (2013). Migratory properties of pulmonary dendritic cells are determined by their developmental lineage. Mucosal Immunol. 2012 Nov 21. doi: 10.1038/mi.2012.106. [Epub ahead of print]
Acknowledgments
We thank Rhonda Wilson, Keiko Nakano, Seddon Thomas, Maria Sifre, and Carl Bortner for help with flow cytometric analysis. This work was supported by the Intramural Research Program of the National Institutes of Health and the National Institute of Environmental Health Sciences.
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Nakano, H., Cook, D.N. (2013). Pulmonary Antigen Presenting Cells: Isolation, Purification, and Culture. In: Allen, I. (eds) Mouse Models of Allergic Disease. Methods in Molecular Biology, vol 1032. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-496-8_2
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DOI: https://doi.org/10.1007/978-1-62703-496-8_2
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