PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Methods Mol Biol. Author manuscript; available in PMC 2017 August 10.
Published in final edited form as:
PMCID: PMC5552037
NIHMSID: NIHMS761923

Pulmonary antigen presenting cells; isolation, purification and culture

Summary

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.

Keywords: antigen presenting cells, dendritic cells, macrophages, monocytes, lung, lymph nodes, gradient centrifugation, flow cytometry, autofluorescence, sorting, culture

1. Introduction

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.

2. Materials

2-1. Tissue digestion

  1. Digestion buffer: PBS (Mg- Ca-) with 0.5% BSA (pH 7.2 - 7.4), filter-sterilized and stored at 4° C.
  2. Preparation buffer: PBS (Mg- Ca-) with 0.5% BSA and 2 mM EDTA (pH 7.2 - 7.4), filter-sterilized and stored at 4° C.
  3. 5 mg/ml Liberase TM (Roche) in PBS, stored at -20° C.
  4. 25 mg/ml Collagenase XI (approx. 12500 U/ml) in PBS, stored at -20° C
  5. 100 mg/ml Hyaluronidase type I-S (approx. 6000 U/ml) in PBS, stored at -20° C
  6. 20 mg/ml DNase I in water, stored at -20° C (Note 1)
  7. 120 mM EDTA in PBS (pH7.2), stored at 4° C.
  8. Nycodenz (Accurate Chemical)
  9. Incubator, 37° C
  10. Cell strainer 70 um

2-2. Staining of leukocytes

  1. Preparation buffer: PBS (Mg- and Ca-free) with 0.5% BSA and 2 mM EDTA (pH 7.2-7.4)
  2. FACS buffer: 0.5% BSA, 0.1% NaN3, and 2mM EDTA in PBS
  3. normal mouse serum
  4. normal rat serum
  5. antibody dilution buffer (5% normal mouse serum, 5% normal rat serum, and 5 ug/ml anti-CD16/32 in FACS buffer)
  6. Antibodies
    Fc block: anti-mouse CD16/CD32 (2.4G2)
    Pan DC markers: CD11c (N418 or HL3), MHC class II I-Ab (AF6-120.1) or I-Ad (AMS-32.1). (Note 2)
    DC subset markers: CD11b (M1/70), CD14 (Sa2-8), CD103 (M290 or 2E7), CD317 (JF05-1C2.4.1, 120G8, or eBio927), Ly-6C (AL-21), Siglec-H (eBio440c)
    Macrophage markers: CD11b (M1/70), CD11c (N418 or HL3), F4/80 (BM8), Siglec-F (E50-2440)
    Monocyte markers: CD115 (AFS98), Ly-6C (AL-21), CD11b (M1/70)
    Activation/maturation markers: CD40 (1C10), CD80 (16-10A1), CD86 (GL1), CD197/CCR7 (4B12),
    Lymphocyte markers: CD3e (145-2C11), CD19 (6D5 or eBio1D3), CD49b (DX5).
  7. Round (U) bottom 96 well plate
  8. Plate rotor
  9. 15 ml conical tubes
  10. FACS tubes
  11. Flow cytometer (e.g. FACS LSR-II (Becton Dickenson))

2-3. Cell sorting and culture

  1. Cell sorter (e.g. FACS Vantage or FACS-ARIA-II (Becton Dickenson))
  2. RPMI 1640
  3. Fetal bovine serum, certified (low endotoxin)
  4. β-mercaptoethanol
  5. Penicillin/Streptomycin
  6. Round (U) bottom 96 well plate
  7. Flat bottom 96 well plate
  8. CO2 incubator, 5 % CO2, 37° C

3. Methods

3-1. Tissue digestion

  1. Collect lungs from mice and place in tissue culture dish (60 mm) or 6 well-plate containing 1 ml of digestion buffer (Reagent #1). Up to 4 lungs per dish can be included.
  2. Mince tissue using scissors, razor blade and/or forceps (Fig. 1). Scissors are recommended.
    Figure 1
    Minced lung tissue
  3. Add 1 ml of digestion buffer. (Note 3)
    Add:  40 ul of Liberase
      20ul of DNase I
      20 ul of collagenase XI
      20 ul of hyaluronidase
  4. Swirl the dish gently, then incubate dish at 37° C for 60 minutes.
  5. During the incubation, prepare Nycodenz solution. Weigh Nycodenz according to your target cell types (Fig. 2).
    Figure 2
    Gradient centrifugation for enrichment of dendritic cells and macrophages from the lung
    1.45 g: Dendritic cells (excluding pDCs) and macrophages
    1.6 g: Dendritic cells (including pDCs), macrophages, large monocytes, and large B cells
    1.8 g: Dendritic cells (including pDCs), macrophages, monocytes, and large T and B cells
    Add Nycodenz to 9.5 ml PBS in 15 ml tube. Place the tube on a shaker or a rotator.
  6. To stop tissue digestion, add 0.4 ml of cold 120 mM EDTA to dish.
  7. Add 5 ml of preparation buffer to 15 ml empty conical centrifugation tube (or 25 ml in 50 ml tube if you have multiple dishes). Keep the tubes on ice.
  8. Meanwhile, add 5 ml cold preparation buffer (Reagent #2) to dish. Transfer minced tissue onto a cell strainer in dish and using rubber tipped plunger of a 3 ml syringe, push tissues through the cell strainer onto the dish.
  9. Pipette the liquid in the dish back through the strainer several times to ensure a single cell suspension. Then pipette the cells several times to detach cells from dish, transfer cells (in 7 ml now) to 15 ml tube containing 5 ml of preparation buffer on ice. (or 50 ml tube with 25 ml preparation buffer).
  • 11
    Centrifuge at 450-g for 5 min at 4° C. This is equivalent to 1,600 rpm in a table top Sorvall centrifuge.
  • 12
    Resuspend cells in 10 ml preparation buffer. Carefully layer 2-3ml of gradient solution (e.g. 14.5-18% Nycodenz solution in PBS) under the cell suspension, and spin at 450-g for 20 min at room temperature with the brake OFF.
  • 13
    The enriched dendritic cells form a fuzzy white layer at the interface of the gradient solution and buffer. Remove the media until 1.5 ml of liquid is left above the interface. Collect the cell layer carefully. (Avoid the pellet in sample).
  • 14
    Wash cells with 5 ml preparation buffer. Spin cell suspension at 450-g for 5 min at 4°C with brake ON.
  • 15
    Resuspend cell pellets in 500~1000 ul of preparation buffer. Count cells.
  • 16
    For cell analysis, go to section 3-2. Staining of Dendritic cells.
  • For cell sorting, go to section 3-3. Sorting of Dendritic cells.

3-2. Staining of leukocytes

  1. Place 1×105~2×106 cells in each well of round bottom 96-well plate.
    Afterwards, use multichannel pipette.
    Spin the plate at 800 g for 3 min, and then discard supernatant.
  2. Add 50 ul of Ab dilution buffer, and then incubate the cells on ice for 5-10 min.
  3. Prepare Ab cocktail with Ab dilution buffer (2x of final concentration. Optimal final concentration is usually 0.5-2 ug/ml). Add 50 ul of 2x Ab solution to cells then mix well.
    The Ab composition of the cocktail depends on the goal of the experiment, but an example is shown below.
    • I-Ab – eFluor 450
    • CD11b – eFluor 605NC
    • CD103 – Phycoerythrin
    • CD11c – PerCP-Cy5.5
    • CD115 - APC
    • Ly-6C - APC-Cy7
    • (FITC-labeled Ab is not used because this channel will be used for detection of autofluorescence signals.) (Note 4)
    • Protect cells from light and incubate on ice for 30 min.
  4. Wash cells with FACS buffer twice. 1st time, add 100 ul FACS buffer, and 2nd time, resuspend pellet with 200 ul FACS buffer. Pipette cells every time to resuspend cells.
  5. Suspend cells in 200ul FACS buffer, and transfer cells to FACS tube.

3-3. Flow cytometric analysis

  1. Gate on single cells (P1 in FSC-A vs. FSC-H) and viable cells (P2 in FSC-A vs. SSC) (Fig. 3 ).
    Figure 3
    A gating strategy for lung APC analysis
  2. Set voltage of each channel (Note 5).
  3. Run compensation samples (unstained cells and cells stained with single dye).
  4. Adjust compensation manually (Note 5). We do not recommend using “Auto Comp”, which cannot adjust compensation for DCs or macrophages. Set gates for positive cells (not autofluorescent cells) and negative cells, then adjust the compensation value in each channel. Repeat same procedures for all channels.
  5. Gate on DCs (P4 and P6; e.g. CD11c+MHC-II+autofluorescence- cells for lung DCs, and CD3-CD19- cells for LN DCs and/or alveolar macrophages (P3; CD11chi autofluorescent)) (Fig. 2). (Note 4, Note 6)
  6. Gate on DC subsets (e.g. P9: CD11b+, P10: CD103+) (Fig. 4). (Note 6)
    Figure 4
    Analysis of lung DC subsets
  7. Collect 10,000 cells (or as many as possible) in P3.

3-4. Sorting of dendritic cells

  1. Place up to 1×108 cells in 15 ml conical tube. Fill the tube with preparation buffer (Reagent #2).
    Spin the tube at 500 g for 5 min, and then remove supernatant.
  2. Resuspend cells with 1ml Ab dilution buffer containing antibodies. Protect cells from light and incubate on ice for 30 min.
  3. Meanwhile, add 4 ml complete culture medium to each FACS collection tube.
  4. Wash cells with preparation buffer (Reagent #2) twice. 1st time, add 14 ml buffer, and 2nd time, resuspend pellet with 15 ml buffer. Resuspend cells well every time.
  5. Suspend cells in 1 ml preparation buffer (Reagent #2), and transfer cells to FACS filter cap tube.
  6. Place 1 ml preparation buffer (Reagent #2) on the top of tube 3 times to rinse filter.
  7. Remove 3 ml complete culture medium from each collection tube.
  8. Sort cells on FACS Vantage or FACS ARIA-II (Fig. 5). Note 7)
    Figure 5
    DC subset sorting by flow cytometry

3-4. Culture of dendritic cells with T cells

  1. Transfer sorted dendritic cells or macrophages from FACS tube to 15 ml conical tube containing 10 ml culture medium.
    Spin the plate at 500 g for 5 min.
  2. Wash cells twice with culture medium, then count them.
  3. Resuspend dendritic cells with culture medium at 5×105/ml. (Note 8)
    Plate 5×104 dendritic cells (100 ul) in each well of a round bottom, 96-well plate.
  4. Add 50 ul of culture medium containing antigens, cytokines or antibodies.
  5. Adjust concentration of purified T cells to 2×106 cells/ml.
    Add 50 ul of T cell suspension to each well (final number: 1×105 cell/well). (Note 9)
  6. Culture cells in CO2 incubator (5 % CO2, 37 °C). (Note 10)
  7. On day 3, split cells from one well into two wells. Add 100ul of fresh culture medium to each well.
  8. T cell proliferation can be assessed on day 3-5 by counting cell number, CFSE-dilution assay, or [3H]-thymidine incorporation assay.
  9. On day 5-6, collect cells with supernatant and centrifuge at 500 g for 5 min. Save supernatant for cytokine assay.
    If desired, T cells can be restimulated as follows. (Note 11)
  10. Wash cells with culture medium twice then count.
  11. Resuspend cells with culture medium and adjust cell concentration to 5×105/ml.
  12. Put 200 ul of T cell suspension (1×105) into flat-bottom 96 well culture plate coated with anti-CD3e (1 ug/ml) and anti-CD28 (1-5 ug/ml) mAbs.
  13. Culture cells in CO2 incubator (5 % CO2, 37 °C).
  14. 24 hours later, collect supernatants for cytokine assay.

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.

Footnotes

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.

  • Alveolar macrophages: CD11blo,CD11chi, F4/80int, Siglec-Fhi, autofluorescencehi
  • Interstitial macrophages: CD11bhi, CD11clo, F4/80+
  • Monocytes: CD11bhi, CD115hi, Ly-6Chi, MHC-IIlo, autofluorescence
  • Inflammatory DCs: CD11bhi, CD11cint, Ly-6Chi, MHC-II+
  • Monocyte-derived DCs: CD11bhi, CD11cint, CD14hi, Ly-6Clo, MHC-IIhi
  • PreDC-derived CD11bhi DCs: CD11bhi, CD11cint, CD14hi, Ly-6Clo, MHC-IIhi
  • CD103+ DCs: CD11blo, CD11chi, CD103+, CD117+, CD207+, MHC-IIhi
  • Plasmacytoid DCs: CD11blo, CD11cint/lo, CD45R/B220+, CD317+, Ly-6C+, MHC-IIlo, Siglec-H+
  • CD8+ DCs (LNs): CD8a+, CD11blo, CD11chi, MHC-IIhi
  • B cells: CD19+, CD45R/B220+, sIgM+

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.

References

1. Sertl K, Takemura T, Tschachler E, Ferrans VJ, Kaliner MA, Shevach EM. Dendritic cells with antigen-presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura. The Journal of experimental medicine. 1986;163:436–451. [PMC free article] [PubMed]
2. Lambrecht BN, Hammad H. Biology of lung dendritic cells at the origin of asthma. Immunity. 2009;31:412–424. [PubMed]
3. Sung SS, Fu SM, Rose CE, Jr, Gaskin F, Ju ST, Beaty SR. A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins. J Immunol. 2006;176:2161–2172. [PubMed]
4. Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ, Ley K. Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent. The Journal of experimental medicine. 2006;203:1273–1282. [PMC free article] [PubMed]
5. Nakano H, Free ME, Whitehead GS, Maruoka S, Wilson RH, Nakano K, Cook DN. Pulmonary CD103(+) dendritic cells prime Th2 responses to inhaled allergens. Mucosal immunology. 2012;5:53–65. [PMC free article] [PubMed]
6. Inaba K, Witmer-Pack MD, Inaba M, Muramatsu S, Steinman RM. The function of Ia+ dendritic cells and Ia- dendritic cell precursors in thymocyte mitogenesis to lectin and lectin plus interleukin 1. The Journal of experimental medicine. 1988;167:149–162. [PMC free article] [PubMed]
7. Stevens WW, Kim TS, Pujanauski LM, Hao X, Braciale TJ. Detection and quantitation of eosinophils in the murine respiratory tract by flow cytometry. Journal of immunological methods. 2007;327:63–74. [PMC free article] [PubMed]
8. Jakubzick C, Randolph GJ. Methods to study pulmonary dendritic cell migration. Methods in molecular biology. 2010;595:371–382. [PubMed]
9. Vermaelen K, Pauwels R. Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights. Cytometry A. 2004;61:170–177. [PubMed]
10. 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. Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. Nat Immunol. 2007;8:1217–1226. [PubMed]
11. Onai N, Obata-Onai A, Schmid MA, Ohteki T, Jarrossay D, Manz MG. Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow. Nat Immunol. 2007;8:1207–1216. [PubMed]
12. 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. Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas. Cell. 2010;143:416–429. [PMC free article] [PubMed]
13. Nakano H, Lin KL, Yanagita M, Charbonneau C, Cook DN, Kakiuchi T, Gunn MD. Blood-derived inflammatory dendritic cells in lymph nodes stimulate acute T helper type 1 immune responses. Nat Immunol. 2009;10:394–402. [PMC free article] [PubMed]