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Dendritic Cell Development and Subset Identification

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Dendritic Cell Development and Subset Identification Immunology Dendritic Cell Development and Subset Identification Legend on page 2. Dendritic Cell Development Dendritic cells (DCs) are a heterogeneous population of immune cells transcriptional regulators are necessary during the late phases of DC that are critical mediators of innate and adaptive immunity. DC differentiation.2,3 Late developmental factors act to promote or precursors develop in the bone marrow.1 While plasmacytoid DCs maintain the differentiation of specific DC subsets.2,3 The final stage in complete development in the bone marrow, most DCs complete DC development occurs upon activation. Activation of human and development in lymphoid and peripheral tissues.1 Knockout mouse mouse DCs can be detected by upregulation of MHC class II, B7-1/ models have identified several cytokines and transcription factors that CD80, B7-2/CD86, CD40/TNFRSF5, or CD83 expression.4 are critical for DC development in vivo.2,3 A limited number of cytokines Understanding DC developmental pathways has facilitated the in vitro and transcription factors, including Flt-3 Ligand, Ikaros, PU.1, and generation of DCs from bone marrow or blood-derived DC precursors GFI-1, are essential during early DC development, and their silencing using cytokine cocktails of Flt-3 Ligand, M-CSF, GM-CSF, and IL-4.5 results in global reductions in DC numbers.2,3 Additional cytokines and References 1. Liu, K. & M.C. Nussenzweig (2010) Immunol. Rev. 234:45. 2. Belz, G.T. & S.L. Nutt (2012) Nat. Rev. Immunol. 12:101. 3. Satpathy, A.T. et al. (2011) Semin. Immunol. 23:388. 4. Lipscomb, M.F. & B.J. Masten (2002) Physiol. Rev. 82:97. 5. Naik, S.H. et al. (2007) Nat. Immunol. 8:1217. Hematopoietic Hematopoietic Stem Cells Bone Marrow Stem Cells Common Myeloid Common Lymphoid Common Myeloid Progenitor Cells + Progenitor Cells Ly-6C Progenitor Cells Monocytes + CD14 Common Dendritic Monocytes Precursor Cells Ly-6C+ Dermal Langerhans Cells Inflammatory Monocyte- Monocytes derived Dendritic Cells Inflammatory Monocyte- Plasmacytoid derived Dendritic Cells Dendritic Cells Pre-conventional Langerhans Cells Dendritic Cells Dermal Dendritic Cells* Common Dendritic Precursor Cells Conventional Migratory CD4+, CD8− Conventional Dendritic Cells Lymphoid-resident Dendritic Cells Plasmacytoid Conventional Lymphoid- Dendritic Cells resident Dendritic Cells Dermal Dendritic Cells* CD4−, CD8+ Conventional Integrin αE/CD103+ Lymphoid-resident + + Conventional Migratory Integrin αM/CD11b Dendritic Cells CD1c/BDCA-1 Dendritic Cells Conventional/Myeloid Conventional Migratory Dendritic Cells Dendritic Cells Thrombomodulin/ − − + CD4 , CD8 Conventional CD141/BDCA-3 Lymphoid-resident Conventional/Myeloid Dendritic Cells Dendritic Cells Growth Factors for Research on Dendritic Cell Molecule Species Proteins Flt-3 Ligand Human On the Cover Mouse Staining of DC-SIGN in Human THP-1 derived Dendritic Cells. THP-1 cells were cultured in the presence of GM-CSF Human Recombinant Human IL-4 (Catalog # 204-IL), Recombinant Mouse Human GM-CSF (Catalog # 215-GM), and Recombinant Human TNF-α (Catalog # 210-TA) for 2 days. Differentiated IL-4 Human cells were fixed with PFA, and DC-SIGN was detected using Mouse Mouse Anti-Human DC-SIGN Monoclonal Antibody (Catalog # MAB161) at 25 µg/mL for 3 hours at room temperature. M-CSF Human Cells were then stained using the NorthernLights™ Mouse 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). learn more | RnDSystems.com/DCpathway Products for Research on Dendritic Cell Development Proteins Antibodies 2000 Molecule Species B/N E FC ICC/IHC IP WB B7-1/CD80 Human, Mouse 1500 B7-2/CD86 Human Mouse B7-H1 Human Mean RFU Mean 1000 Mouse BATF3 Human CD40/TNFRSF5 Human 500 Mouse 0 CD83 Human 10-2 10-1 100 101 Mouse Recombinant Mouse B7-H1/PD-L1 Fc Chimera (μg/mL) CSL/Rbp-J Human Recombinant Mouse B7 H1/PDL1 Fc Chimera (Catalog # Flt-3/Flk-2 Human, Mouse 1019-B7) inhibits anti-CD3-induced cell proliferation of stimulated mouse T cells. The ED50 for this effect is typically GFI-1 Human 0.15-0.75 μg/mL. GM-CSF Rα Human Mouse ID2 Human kDa R NR 190 Ikaros Human IRF2 Human Mouse 92.5 IRF4 Human 66 Mouse 55 IRF8 Human 43 M-CSF R Human 36 Mouse 29 Notch-2 Human 21 Mouse 18.4 12.4 PU.1/Spi-1 Human 6.3 Mouse RelB Human 1 μg/lane of Recombinant Mouse B7-H1/PD-L1 Fc Chimera was resolved with SDS PAGE under reducing (R) and non- Spi-B Human reducing (NR) conditions and visualized by silver staining, Mouse showing bands at 79 kDa and 150 kDa, respectively. Application Key: B/N Blocking/Neutralization E ELISA FC Flow Cytometry ICC Immunocytochemistry IHC Immunohistochemistry IP Immunoprecipitation WB Western blot A B 105 105 58.3 104 104 Detection of CD40 and CD83 on Human MoDCs by Flow 94.2 Cytometry. Human CD14+ cells were isolated from Ficoll separated peripheral blood mononuclear cells using the MagCellect Human CD14+ Cell Isolation Kit (Catalog # 103 CD83 103 MAGH105). Mature monocyte-derived dendritic cells were CD40/TNFRSF5 stimulated overnight with Recombinant Human TNF-α (Catalog # 210-TA) and further stained with A) PE- conjugated Mouse Anti-Human CD40 Monoclonal Antibody (Catalog # FAB6321P) or B) APC-conjugated Mouse Anti- Human CD83 Monoclonal Antibody (Catalog # FAB1774A) and Fluorescein-conjugated Mouse Anti-Human CD11c 100 100 (FAB1777F). Quadrants were set based on isotype controls. -103 0 103 104 105 -103 0 103 104 105 CD11c CD11c learn more | RnDSystems.com/DCDevelopment Dendritic Cell Subsets and Markers Several phenotypic and functional DC subsets have been skin, lung, and intestine.4 As part of the first line of defense against characterized including classical/conventional DCs (cDCs), exogenous pathogens, the skin houses a number of highly specialized Langerhans cells, inflammatory monocyte-derived DCs (MoDCs), and DC subsets.5,6 Langerhans cells are a unique subset of epidermal plasmacytoid DCs (pDCs).1 While there is functional homology DCs and appear to function similarly in mice and humans. Dermal between mouse and human DCs, differences between mouse and dendritic cell subsets also appear to function similarly in mice and human DC subset development and cell surface markers have humans, but their identifying markers differ significantly between the complicated their characterization.2 Most DCs have a characteristic two species.7,8 Two additional human and five additional mouse cDCs morphology defined by long dendrite extensions as well as high levels subsets have been identified that can be characterized by their of MHC class II expression. In contrast, pDCs have a rounded expression of specific cell surface markers, transcription factors, and morphology similar to plasma cells and low MHC class II expression. secreted factors. In contrast to cDCs, MoDCs develop from monocytes DCs initiate innate immunity via detection of pathogens by pattern at sites of inflammation and have been experimentally characterized recognition receptors and secretion of pro-inflammatory or in mice in vivo.9 Multiple MoDC subsets arise from Ly-6Chigh tolerogenic cytokines.3 They also initiate adaptive immune activity or monocytes.10 Endogenous human MoDCs that are pro-inflammatory tolerance by acting as professional antigen-presenting cells (APCs). in nature and likely correspond to mouse MoDCs have been identified All dendritic cells differentiate from a common myeloid progenitor and in peripheral blood.11 pDCs are inefficient APCs and instead function can be lymphoid-resident or reside in peripheral tissues and migrate in a primarily innate capacity through the detection of viral nucleic to lymphoid organs, where they activate T cells.4 High concentrations acids and the secretion of Type I IFNs, TNF-α, and IL-6.12 of DCs are present at areas of intense antigen exposure such as the References 7. Merad, M. et al. (2008) Nat. Rev. Immunol. 8:935. 1. Mildner, A. & S.Jung (2014) Immunity 40:642. 8. Zaba, L.C. et al. (2009) J. Invest. Dermatol. 129:302. 2. Crozat, K. et al. (2010) Immunol. Rev. 234:177. 9. Fekete, T. et al. (2012) Eur. J. Immunol. 42:458. 3. Lewis, K.L. & B. Reizis (2012) Cold Spring Harb. Perspect. Biol. 4:a007401. 10. Dominguez, P.M. & C. Ardavin (2010) Immunol. Rev. 234:90. 4. Merad, M. & M.G. Manz (2009) Blood 113:3418. 11. Satpathy, A.T. et al. (2012) Nat. Immunol. 13:1145. 5. Kaplan, D.H. (2010) Trends Immunol. 31:446. 12. Gilliet, M. et al. (2008) Nat. Rev. Immunol. 8:594. 6. Chu, C.C. et al. (2011) Semin. Immunol. 23:28. Dendritic Cell Subset Specific Markers Human CD1c/BDCA-1+ Conventional/Myeloid Dendritic Cells Cell Surface Markers Lin– (CD3–, CD14–, CD19–, CD20–, CD56–), MHC class II+, CD1c/BDCA-1+, Integrin αM/CD11b+, CD11c+, SIRPα/CD172a+ Secreted Molecules TNF-α, IL-6, IL-1β/IL-1F2, IL-10 Human Thrombomodulin/CD141/BDCA-3+ Conventional/Myeloid Dendritic Cells Cell Surface Markers Lin– (CD3–, CD14–, CD19-, CD20–, CD56–), MHC class II+, CD1a+, CLEC9a+, CD11c+, Thrombomodulin/CD141/BDCA-3+, XCR1+ Secreted Molecules IFN-β, IL-12 Mouse CD4-CD8α+ Conventional Lymphoid-resident Dendritic Cells Cell Surface Markers MHC class II+, CD1d1+, CD4–, CD8α+,CLEC9a+, DEC-205/CD205+, Integrin αM/CD11b+, CD11c+, Langerin/CD207+/–, XCR1+ Secreted Molecules IFN-g, IL-12 Mouse CD4+CD8α- Conventional Lymphoid-resident Dendritic Cells Cell Surface Markers MHC class II+, CD4+, CD8α–, SIRPα+/CD172a, Integrin αM/CD11b+, CD11c+ Secreted Molecules IFN-g, IL-2, IL-6 Mouse CD4-CD8α- Conventional Lymphoid-resident
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