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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.
Pulmonary APCs take up inhaled antigens, process them, and present antigen-derived peptides to T and B lymphocytes to initiate adaptive immune responses (1). In keeping with their ability to acquire antigens from the airspace or parenchymal tissue, DCs and macrophages are located within the airway epithelium, lung parenchyma and alveolar spaces (2, 3). To maintain their positions within the lung, many DCs and macrophages adhere tightly to tissue stromal cells. Protocols that yield large numbers of lung APCs must therefore disrupt molecular interactions that hold APCs and stromal cells together. Although collagenase D has been widely used for this purpose, the yield of DCs obtained from procedures that employ this enzyme has been suboptimal. To improve cell yields, we have modified a tissue digestion method that was originally designed for cardiovascular tissue digestion (4), and found that this new protocol dramatically improves the yield of APCs from the lung (5).
Lung APCs are highly diverse in terms of both size and density. For example, alveolar macrophages are large and light, while monocytes are relatively small and dense, with lymphocytes and non-leukocytes having even higher densities. Therefore, gradient centrifugation provides a convenient and effective method to enrich for APCs (6). We have developed simple methods that enrich for different APCs, depending on which type is needed for the individual experiment at hand. After this enrichment step, APCs are often analyzed by flow cytometry to determine their frequency and their display of cell surface molecules. Unlike most other macrophages in the body, alveolar macrophages display the pan-DC marker, CD11c, as well as MHC class II (7, 8). Consequently, if other markers are not used, alveolar macrophages can be easily mistaken for pulmonary DCs. According, many investigators now use the autofluorescent properties of macrophages and their display of high levels of Siglec-F to distinguish them from DCs (7, 9). In addition, pulmonary DCs are heterogeneous (2) and include plasmacytoid, inflammatory, and conventional DCs. The latter category includes the two major lung DC subsets, which express high levels of CD11b and CD103, respectively. CD11bhi DCs can be further segregated into pre-DC-derived and monocyte-derived DCs (moDCs) (10, 11). In this chapter, we describe how to distinguish each DC subset from the others by flow cytometry. This technology is not only useful for characterizing APCs, but also for purifying individual APC populations. Purified APCs can be subsequently studied ex vivo to identify their biologic functions. Here, we describe methods to culture lung APCs and culture them with naïve T cells to study APC-mediated T helper cell differentiation.
|Add:||40 ul of Liberase|
|20ul of DNase I|
|20 ul of collagenase XI|
|20 ul of hyaluronidase|
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.
1Use distilled water to dissolve DNase I. Do not use PBS.
2Because binding of anti-I-A/I-E mAb (M5/114) alters the phenotype and function of APCs, we recommend using anti-I-Ab (AF6-120.1), anti-I-Ad (AMS-32.1) or anti-I-E (14-4-S) mAb to detect MHC class II.
31.1 ml premixed enzymes in digestion buffer can be added.
4Because alveolar macrophages are autofluorescent, they display positive signals in channels in which cells were not stained. Autofluorescence signal is detected in channels with violet and blue lasers (e.g. Pacific blue, AmCyan, FITC and PE channels).
5We recommend eliciting advice from an expert in flow cytometry to set voltage and compensation. Because different cell populations have different signal backgrounds (including autofluorescence), Auto-comp cannot adjust the compensation appropriately
6Surface makers of pulmonary APC populations are shown below.
7After sorting lung DCs by flow cytometry, approximately 1-2 ×104 CD11bhi DCs and 2-4 ×104 CD103+ DCs are usually obtained per mouse, although cell yields vary among different experiments depending on the treatments the mice received. Multiply mouse number based on DC number needed for experiment.
8Complete RPMI 1640 with 10 % FBS (low endotoxin) is recommended.
9A 1:2 ratio of DCs to T cells induces robust T cell proliferation and differentiation, although T cell responses can be detected with wide range of ratio (1:1 - 1:100) of DCs to T cells.
10T cell response is affected by pH of medium. Check concentration of CO2 in incubator and pH of culture medium prior to culture.
11Restimulation allows assessment of T cell responses following their differentiation without transfer of cytokines produced by naïve or differentiating T cells during the primary culture. In addition, because an equal number of T cells are typically restimulated, this method allows measurements of T cell responses on a per cell basis.