Abstract
Dendritic cells (DC) are professional antigen-presenting cells (APCs) that modulate the outcome of the immune response toward immunity or tolerance. There are a large variety of DC subsets according to surface phenotype, function, and tissue distribution. Murine plasmacytoid DC (pDC) represent a distinctive DC population and are characterized by the expression of CD11c, B220, Gr-1, CD45RA, Ly49Q, BST2, and siglec-H on the cell surface. PDC act as immunogenic cell sentinels by secreting large amounts of type I interferon (IFN) in the lymph nodes in response to viral stimulation. PDC also act as tolerogenic cells when expressing the inducible tolerogenic enzyme indoleamine 2,3-dioxygenase (IDO), the inducible costimulator ligand (ICOS-L), and/or the programmed death 1 ligand (PD-L1), which mediate regulatory T-cell (Treg) development and suppression of self- and alloreactive cells. The PDC ability to induce Treg development is associated with capture and presentation of antigenic peptides associated with major histocompatibility complex (MHC) class I and II. Here, we provide the tools to study PDC development from bone marrow cultures, their antigen presentation properties, and their interactions with Treg under a tolerogenic setting of sterile inflammation.
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Acknowledgments
We acknowledge Dr. Tamar Hermesh for technical assistance. We also wish to acknowledge the efforts of the Mount Sinai Sorting Core Facility. This work was supported by the Programa Ramón y Cajal RYC-2006-1588, Ministerio de Educación y Ciencia SAF2007-63579, Programa José Castillejo JC2008-00065, and Programa de Investigación de Grupos Emergentes del ISCIII (to JCO), NIH R01 AI-41428, AI-72039 (to JSB), and Howard Hughes Medical Institute (to EG).
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Appendices
Appendix 1: 10 mL Solution of 2% Ketamine, 0.02% Acepromazine Solution
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2 mL Ketamine HCl (100 mg/mL) – Use 4 mL of 50 mg/mL ketamine HCl.
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0.2 mL Acepromazine (10 mg/mL).
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7.8 mL Sterile PBS.
Appendix 2: 500 mL of PDC Culture Medium
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435 mL RPMI 1640 supplemented with l-Glutamine.
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50 mL FCS (10%).
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5 mL 1 M HEPES (10 mM).
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10 mL (5,000 IU/μg) Penicillin/streptomycin (100 IU/mL penicillin and 100 μg/mL streptomycin).
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Filter and store at 4°C.
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Immediately before using, add β-mercaptoethanol (2-ME) to a concentration of 50 μM and cold filter a second time.
Appendix 3: FLT3-L Preparation
Use lyophilized FLT3-L (R&D Systems, Minneapolis, MN) to prepare 50 ng/mL aliquots:
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1.
Add 480 μL of PBS to 25 μg of lyophilized FLT3-L.
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2.
Add 20 μL of 2.5% fatty-acid-free BSA in PBS.
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3.
Wait 10 min for the powder to dissolve. Do not vortex to avoid bubbles. It takes about 10 min to dissolve.
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4.
Pipet up and down to mix the solution. Avoid adding bubbles.
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5.
Dispense into aliquots. 35 μL aliquot to be most efficient for 2-mouse experiments. Store at −20°C for up to 3 months.
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Gehrie, E., Van der Touw, W., Bromberg, J.S., Ochando, J.C. (2010). Plasmacytoid Dendritic Cells in Tolerance. In: Cuturi, M., Anegon, I. (eds) Suppression and Regulation of Immune Responses. Methods in Molecular Biology, vol 677. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-869-0_9
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DOI: https://doi.org/10.1007/978-1-60761-869-0_9
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