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).

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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 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 Integrin αE/CD103+ Conventional Migratory Dendritic Cells Cell Surface Markers MHC class II+, CD1d1+, CD8α+/–, CLEC9a+/–, DEC-205/CD205+/–, EpCAM/TROP1+/–, Integrin αE/CD103+, CD11c+, Langerin/CD207+/–, XCR1+/– Secreted Molecules IL-12, IL-23 Mouse Integrin αM/CD11b+ Conventional Migratory Dendritic Cells Cell Surface Markers MHC class II+, DEC-205/CD205+, Integrin αM/CD11b+, CD11c+ Secreted Molecules IL-6, IL-10, IL-23 Human Dermal Dendritic Cells CD14+ Cells Cell Surface Markers MHC class II+, CD1c/BDCA1+, CD14+, CD45+, DC-SIGN/CD209+, Integrin αM/CD11b+, CD11c+, Thrombomodulin/CD141/BDCA-3+/– CD14- Cells Cell Surface Markers MHC class II+, CD1a+, CD1c/BDCA-1+, CD14–, CD45+, Integrin αM/CD11b+, CD11c+ Mouse Dermal Dendritic Cells Langerin+ Cells Cell Surface Markers MHC class II+, EpCAM/Trop1–, CD11c+, Integrin αE/CD103+, Integrin αM/CD11blow, Langerin/CD207+, SIRPα/CD172a+/– Langerin- Cells Cell Surface Markers MHC class II+, DEC-205/CD205+, F4/80/EMR1+, CD11c+, Integrin αM/CD11bhigh, Langerin/CD207- Dendritic Cell Subset Specific Markerscontinued Human Langerhans Cells Cell Surface Markers MHC class II+, CD1a+, CD14–, CD45+, CD83+, E-Cadherin+, EpCAM/TROP1+,Integrin αM/CD11b+/–, CD11c+, Langerin/CD207+ Mouse Langerhans Cells Cell Surface Markers MHC class II+, CD45+, DEC-205/CD205+, E-Cadherin+, EpCAM/TROP1+, F4/80/EMR1+, Integrin αM/CD11b+, CD11c+, Langerin/CD207+ Human Inflammatory/Monocyte-derived Dendritic Cells Cell Surface Markers MHC class II+, CD14–, Fcγ RIII/CD16+, CD11c+ Secreted Molecules IL-23, TNF-α Mouse Inflammatory/Monocyte-derived Dendritic Cells Cell Surface Markers MHC class II+, DC-SIGN/CD209+, Integrin αM/CD11b+, CD11c+, Ly-6C+, MAC3+ Secreted Molecules IL-12, NO, TNF-α Human Plasmacytoid Dendritic Cells Cell Surface/Membrane-associated B220/CD45R+, DLEC/CLEC4C/BDCA-2+, IL-3 Rα/CD123+, CD11clow, Neuropilin-1/BDCA-4+, TLR7+, TLR9+; E2-2+, IRF7+, IRF8+, Spi-B+ Markers; Transcription Factors Secreted Molecules IFN-α, IFN-β, IL-6 , TNF-α Mouse Plasmacytoid Dendritic Cells Cell Surface/Membrane-associated B220/CD45R+, Gr-1/Ly-6G+, CD11c+, Ly-6C+, PDCA-1/Bst2+, Siglec-H+, TLR7+, TLR9+, E2-2+, IRF7+, IRF8+, Spi-B+ Markers; Transcription Factors Secreted Molecules IFN-α, IFN-β, IL-6, IL-12, TNF-α

4 4 10 10 104 A B C

3 10 103 103

2 10 102 102 γ RIII/CD16 CD1c/BDCA-1 Fc

1 Thrombomodulin/CD141/BDCA-3 10 101 101

0 10 100 100 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 100 101 102 103 104 CD11c CD11c CD11c Detection of Myeloid Dendritic Cells by Multi-Color Flow Cytometry. Human peripheral or C) PerCP-conjugated Mouse Anti-Human Fcγ RIII/CD16 and Fluorescein-conjugated blood mononuclear cells were stained simultaneously with the indicated antibodies CD11c. Quadrants were set based on isotype controls. Note: In addition to the population included in the Human Myeloid Dendritic Cell Multi-Color Flow Cytometry Kit (Catalog # of CD11c, Fcg RIII/CD16+ myeloid dendritic cells that are detected in part C, other FMC016). Dot plots show the relative populations of cells stained for A) APC-conjugated populations of Fcg RIII/CD16+ cells present in peripheral blood including granulocytes and Goat Anti-Human CD1c/BDCA-1 and Fluorescein-conjugated CD11c, or B) PE-conjugated natural killer cells may also be detected. Mouse Anti-Human Thrombomodulin/CD141/BDCA-3 and Fluorescein-conjugated CD11c,

104 kDa

kDa 103 116 116 80 80 53 53 37 CLEC9a 102 37

HLA-DR 29 29 Detection of CLEC9a by Flow Cytometry and Western Blot. Whole human blood was stained with an APC-conjugated hCLEC 20 -9A 20 Mouse Anti-Human CLEC9a Monoclonal Antibody (Catalog # FAB6049A) and a PE-conjugated Mouse Anti-Human HLA-DR 101 Monoclonal Antibody (Catalog # FAB4869P). The data shown is gated on monocytes based on forward and side scatter

parameters. Quadrants were set based on staining with an APC-conjugated Mouse IgG1 Isotype Control (Catalog # IC002A). 7 7 CLEC9a was also detected by Western blot in lysates from human CD14+ peripheral blood mononuclear cells (inset). The membrane was probed with a Sheep Anti-Human CLEC9a Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6049) 100 followed by an HRP-conjugated Donkey Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). 100 101 102 103 C 104 M

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Dendritic Cell Surface Markers Fluorochrome Conjugated Antibodies for Proteins Antibodies for Other Applications 40 Flow Cytometry* CFS/ Alexa Fluor® ICC/ Molecule Species APC PE PerCP B/N E IP WB FITC 488 / 700 IHC B220/CD45R Mouse        30 CD1a Human        CD1c/BDCA-1 Human    CD1d1 Mouse   20 CD3 Human  

Mouse         Number Cell Relative CD4 Human           10 Mouse          CD8α Human        Mouse        0 0 1 2 3 4 CD14 Human           10 10 10 10 10 Mouse       Integrin αM/CD11b CD19 Human      Detection of Integrin αM/CD11b in Mouse Peripheral Mouse  Blood Leukocytes by Flow Cytometry. Mouse peripheral CD45 Human        blood leukocytes were stained with a PE-conjugated Rat Anti-Mouse Integrin αM/CD11b Monoclonal Antibody Mouse         (Catalog # FAB1124P; filled histogram) or PE-conjugated CLEC9a Human         Rat IgG2B Isotype Control (Catalog # IC013P; open Mouse        histogram). DC-SIGN/CD209 Human          DEC-205/CD205 Human    Mouse    E-Cadherin Human         Mouse        EpCAM/Trop1 Human           69.4 F4/80/EMR1 Mouse     105 Fcγ RIII/CD16 Human      Mouse                 GR-1/Ly-6G Mouse 104 HLA-DR Human      IL-3 Rα/CD123 Human         

Mouse     /CD123 α 3 Integrin αE/ Mouse     10

CD103 IL-3R Integrin αM/ Human       CD11b Mouse        CD11c Human         Mouse        Langerin/CD207 Human        100 MHC class II Mouse   -103 0 103 104 105 (I-A/I-E) CD11c MS4A1/CD20 Human    Detection of IL-3 Rα/CD123 on Human MoDCs by Flow NCAM-1/CD56 Human       Cytometry. Human CD14+ cells were isolated from Ficoll Mouse      separated peripheral blood mononuclear cells using the + Neuropilin-1/ Human          MagCellect Human CD14 Cell Isolation Kit (Catalog # MAGH105). Mature monocyte-derived dendritic cells were BDCA-4 Mouse           stimulated overnight with Recombinant Human TNF-α    Siglec-H Mouse (Catalog # 210-TA) and further stained with APC-conjugated SIRPa/CD172a Human        Mouse Anti-Human IL-3 Rα/CD123 Monoclonal Antibody Mouse   (Catalog # FAB301A) and Fluorescein-conjugated Mouse Thrombomodu- Human         Anti-Human CD11c (Catalog # FAB1777F). Quadrants were lin/CD141/ Mouse         set based on isotype controls. BDCA-3 XCR1 Human     

Application Key: Fluorochrome Key: B/N Blocking/Neutralization, E ELISA APC Allophycocyanin ICC/IHC Immunocytochemistry/Immunohistochemistry CFS/FITC Carboxyfluorescein/Fluorescein isothiocyanate IP Immunoprecipitation PE Phycoerythrin WB Western blot PerCP Peridinin-chlorophyll-protein complex *Biotinylated and unlabeled forms are also available for most antibodies. Products for Research on Dendritic Cell Subsets

Dendritic Cell Secreted Factors

ELISpot/ Multiplex FluoroSpot Kits Array/ Assay 2.5 Molecule Species Proteins ELISAs Antibodies & Development Kits & Modules Reagents 2.0 IFN-α Human    Mouse    1.5 IFN-β Human   

Mouse    Mean OD IFN-γ Human      1.0 Mouse      β IL-1 /IL-1F2 Human      0.5 Mouse     IL-6 Human      0 Mouse      10-3 10-2 10-1 100 101 IL-10 Human      Recombinant Human IFN-γ (ng/mL) Mouse      Recombinant Human IFN-γ (Catalog # 285-IF)      IL-12 Human demonstrates anti-viral activity in HeLa human cervical Mouse      epithelial carcinoma cells infected with     IL-12/IL-23 p40 Human encephalomyocarditis (EMC) virus. The ED50 for this effect Mouse     is typically 0.15-0.75 ng/mL. IL-23 Human      Mouse     TNF-α Human      kDa R Mouse      190

Multi-Color Flow Cytometry Kits for Dendritic Cell Research Kits Kit Antibodies 92.5 Human Myeloid Dendritic Cells CD1c/BDCA-1-APC, Thrombomodulin/CD141/BDCA-3-PE, CD11c- 66 Fluorescein, Fcg RIII/CD16-PerCP 55 Human Plasmacytoid Dendritic Cells CD45-APC, DLEC/CLEC4C/BDCA-2-PE, IL-3 R/CD123-PerCP, Neuropilin-1/ BDCA-4-Fluorescein 43 36 29

21 104 104 18.4 12.4 6.3 103 103 1 μg/lane of Recombinant Human IFN-γ was resolved with SDS PAGE under reducing (R) conditions and visualized by 102 102 silver staining, showing a single band at 17 kDa Neuropilin-1/BDCA-4 DLEC/CLEC4C/BDCA-2 101 101

100 100 100 101 102 103 104 100 101 102 103 104 CD45 IL-3 Rα/CD123 Detection of Plasmacytoid Dendritic Cells by Multi-Color Flow Cytometry. Plasmacytoid dendritic cells, enriched from human peripheral blood mononuclear cells, were stained with the indicated antibodies provided in the Human Plasmacytoid Dendritic Cell Multi-color Flow Cytometry Kit (Catalog # FMC017). Dot plots show the relative populations of cells co- stained with APC-conjugated Mouse Anti-Human CD45 and PE-conjugated Goat Anti-Human DLEC/CLEC4C/BDCA-2 or with PerCP-conjugated Mouse Anti-Human IL-3 Ra/CD123 and Fluorescein-conjugated Mouse Anti-Human Neuropilin-1/ BDCA-4. Quadrants were set based on isotype controls.

learn more | RnDSystems.com/DendriticCells New kit for Dendritic Cell Differentiation and Characterization The CellXVivo™ Human Monocyte-derived Dendritic Cell Differentiation Immature and TNF-a treated mature dendritic cells can be Kit (Catalog # CDK004) is specially formulated and optimized for the characterized using antibodies directed against specific cell surface culture and differentiation of human dendritic cells. This kit contains markers (B). Mature dendritic cells generated under these conditions high quality R&D Systems cytokines for generating immature and are biologically active (C). mature dendritic cells from an enriched population of CD14+ cells (A).

A. Isolation of Human Dendritic Cell B. Phenotypic Analysis of Cultured Monocyte- (DC) Precursors derived Dendritic Cells (MoDCs) Immature 80 60 40 70 50 30 20 60 40 10 50

Relative Cell Number Cell Relative 0 100 101 102 103 104 30 CD14 40 30 20 Relative Cell Number Cell Relative

Relative Cell Number Cell Relative 20 10 10

0 0 100 101 102 103 104 100 101 102 103 104 CD14 B7-2/CD86

+ + Isolation of CD14 Monocytes using the MagCellect™ Human CD14 Cell Isolation Kit. Mature + Human CD14 cells were isolated from Ficoll separated peripheral blood mononuclear cells 80 using the MagCellect Human CD14+ Cell Isolation Kit (Catalog # MAGH105). All viable cells were stained before (inset) and after (green) isolation using a PE-conjugated Mouse Anti- 70 Human CD14 Monoclonal Antibody (Catalog # FAB3832P). 60

50 C. Assessment of the Biological 40 a 30

Activity of Mature TNF- -treated MoDCs Number Cell Relative 20

8 10

7 0 100 101 102 103 104 6 B7-2/CD86 Phenotypic Analysis of Cultured Immature and Mature Monocyte-derived Dendritic Cells. 5 Immature MoDC (top histograms) were cultured for seven days in Serum-free Dendritic Cell 4 Base Media supplemented with Recombinant Human GM-CSF (Catalog # 215-GM) and Recombinant Human IL-4 (Catalog # 204-IL). Mature MoDC (bottom histograms) were grown 3 under the same conditions for seven days and then induced with Recombinant Human TNF-α (Catalog # 210-TA) for 3 additional days. Both sets of cells were stained with the 2 Mouse Anti-Human B7-2/CD86 Monoclonal Antibody (MAB141) (open histograms) or an

H-TdR Incorporation (cpm x 1000) (cpm Incorporation H-TdR appropriate isotype control antibody (filled histograms). Stained cells were analyzed using a 3 1 Becton Dickinson FACSCalibur™.

0 1e-4 0.001 0.01 0.1 1 10 100 1000 Dendritic Cell Number (x 1000)

Mature Dendritic Cells Induce Proliferation of Allogeneic T-cells. Serial dilutions of day 10 TNF-α treated mature dendritic cells were incubated with allogeneic (blue) or autologous (orange) human T cells for 3 days. 3H-Thymidine (3H-TdR) was added to the culture for the To view a complete description of the final 18 hours. Cells were harvested and the incorporation of 3H-TdR was measured using a generation of monocyte-derived dendritic cells scintillation counter. Results are presented as the mean cpm of triplicates. from CD14+ monocytes, please visit our website at RnDSystems.com/Products/CDK004

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