Name: in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system
Uploaded: 2015-11-23T17:00:00.000+00:00
Duration: 8 min 18 s
Description: Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

We are grateful to Drs. Markus Manz and Irving Weissman for providing reagents. We also acknowledge the service provided by the Flow Cytometric Analysis and Cell Sorting Core Facility of the First and Second Core Laboratory at National Taiwan University College of Medicine and NTU hospital, respectively. This work was supported by the Ministry of Science and Technology, Taiwan (MOST 102-2320-B-002-030-MY3) and the National Health Research Institutes, Taiwan (NHRI-EX102-10256SI and NHRI-EX103-10256SI).

C57BL/6 wild-type mice were purchased from the National Laboratory Animal Center (NLAC), Taiwan. All mice were bred and kept under specific pathogen-free conditions. Protocols and animal use procedures were reviewed and approved by the Institutional Animal Care and Use Committee of National Taiwan University College of Medicine (Permit Number: 20120075). In addition, researchers made every effort to reduce the potential for pain, suffering, or distress in the animals while performing experiments. All procedures described were carried out at RT while wearing gloves.
1. Preparation of AC-6 Feeder cells
Note: The AC-6.21 (AC-6 in short) stromal cell line18 (provided by I. Weissman, Stanford University) should be maintained in RPMI supplemented with 15% heat-inactivated fetal bovine serum (FBS). To serve as feeder cells, AC-6 cells are &#947;-irradiated with 3,000 rad (30Gy) one day before the co-culture to prevent their proliferation. Note that AC-6 cells tend to lose their differentiation-supporting ability if left overcrowded during maintenance, or if their passage number is over 20.
<ol>
	<li>Wash AC-6 cells once with 1 ml Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) and remove DPBS by aspiration. Treat AC-6 cells with 0.8 ml trypsin solution (0.05 % trypsin and 0.5 mM EDTA in DPBS) for 3-5 min at 37oC, 5% CO2 and then stop the reaction by diluting it with 10 volumes of culture medium to make single cell suspension.</li>
	<li>Seed AC-6 cells at 5.9x104 cells/well into 12-well plates and incubate at 37oC, 5% CO2 O/N. 
	Note: The density of AC-6 cells is very important as a lower density will favor cDC generation. Seeding at a density of 5.9x104 cells/well will allow AC-6 to reach confluency by the next day, which is optimal for derivation of pDCs from CLPs.</li>
	<li>Irradiate AC-6 cells at 3,000 rad (30 Gy) using &#947;-irradiator. 
	Note: The dose used to irradiate AC-6 is optimized to prevent the cells from proliferating and yet keep them viable long enough to provide the cytokines and cell-cell contact required for differentiation of DCs from CLPs.</li>
	<li>Aspirate medium and replace with 1 ml complete RPMI (RPMI with 10 % heat-inactivated FBS, 50 &#181;g/ml gentamycin and 50 &#181;M &#946;-mercaptoethanol).</li>
</ol>
2. Isolate BM Cells from Mice
<ol>
	<li>Sacrifice mice by CO2 asphyxiation and cervical dislocation. Place the mice on a dissection tray and sterilize with 70% ethanol. Make an incision at the mid-abdomen and remove the skin from the distal part of the mouse including the skin covering the lower extremities.</li>
	<li>Dissect mice in a semi-sterile hood. To release the femur and tibiae, clip the femur above and tibiae below the knee joint. Detach the muscles from the bones using scissors and place the bones in a 6 cm petri dish containing 5 ml complete RPMI.</li>
	<li>Move to a sterile culture hood. Fill a 3 ml syringe connected to a 27G needle with complete RPMI. Cut off both ends of the bones using scissors, and insert the 27G needle to flush the marrow out of the bones by gently injecting complete RPMI once from each end.</li>
	<li>Prepare a single cell suspension by gentle pipetting the cells up and down 3-5 times in the culture dish using the syringe without the needle.</li>
	<li>Centrifuge the cells for 5 min at 500 x g and decant the supernatant.</li>
	<li>Lyse red blood cells with 1 ml ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) incubating for 1 min and stopping the reaction by diluting with 10 volumes of complete RPMI. Allow cells to stand for 5 min in order to pellet down dead cell clumps and tissue debris by gravity. 
	Note: Do not stand more than 5 min as this will result in cell loss due to settling of viable cells.</li>
	<li>Slowly decant cell-containing supernatant to a new tube leaving debris behind in the original tube.</li>
	<li>Centrifuge for 5 min at 500 x g to pellet the cells, then remove and discard the supernatant.</li>
	<li>Resuspend total BM cells (typically 4-6 x 107 BM cells are obtained from one mouse) in 100 &#181;l FACS buffer (1x PBS+ 2% FBS+ 1mM EDTA).</li>
</ol>
3. FACS Analysis and Sorting of CLPs
<ol>
	<li>Add anti-CD16/32 (hybridoma supernant clone 2.4G2, 50 &#181;l/reaction or 1-2 &#181;g/reaction) into cells in FACS buffer (step 2.9) for 1-2 min in order to block Fc receptors. 
	Note: Anti-CD16/32 is also called Fc block, which is added to prevent non-specific binding of antibodies to cells expressing Fc receptors including granulocytes, monocytes, and B lymphocytes.</li>
	<li>Simultaneously stain cells with the following fluorescent dye-labeled antibodies (for 4x107 cells) for 15 min on ice, avoiding ambient light. Antibodies: PE-conjugated lineage markers (0.2 &#181;g each) including anti-CD3 (17A2), anti-CD8 (53-6.7), anti-B220 (RA3-6B2), anti-CD19 (eBio1D3), anti-CD11b (M1/70), anti-Gr-1 (RB6-8C5), anti-Thy1.1 (HIS51), anti-NK1.1 (PK136), anti-TER119 (TER-119), and anti-MHC-II (NIMR-4), 0.2 &#181;g anti-c-Kit-PerCP-Cy5.5 (2B8), 1 &#181;g anti-Sca-1-FITC (D7), 0.4 &#181;g anti-M-CSFR-APC (AFS98), and 0.2 &#181;g anti-IL-7R&#945;-PE-Cy7 (A7R34).</li>
	<li>Wash cells with 3 ml FACS buffer, and centrifuge for 5 min at 500 x g.</li>
	<li>Resuspend the cells in 300 &#181;l FACS buffer and filter cells through a 40 &#181;M cell strainer.</li>
	<li>Wash the tube with an additional 100 &#181;l FACS buffer to recover any remaining cells and filter into a sterile FACS sorting tube using the same cell strainer.</li>
	<li>Perform flow cytometric analysis immediately after the staining using appropriate filters and voltages for signal detection. Use 488 nm laser to detect the FITC-, PerCP-Cy5.5-labeled antibodies, 561 nm laser to detect the PE- and PE-Cy7-labelded antibodies and 633 nm laser to detect the APC-labeled antibody.</li>
	<li>Sort out CLPs according to the following markers lin-c-kitintSca-1intM-CSFR-IL-7R&#945;+ (as stained in step 3.2) using a cell sorter. Collect the sorted cells in a 15 ml tube containing 8 ml of complete RPMI as a cushion.</li>
	<li>Record the absolute number of sorted cells as shown by the cell sorter at the end of the run. Typically, 5x104 CLPs can be obtained from the BM of one mouse.</li>
</ol>
4. Coculture CLPs and AC-6
<ol>
	<li>Centrifuge the sorted cells from step 3.7 for 10 min at 500 x g, remove the supernatant and resuspend the cell pellet with enough complete RPMI to obtain a cell density around 5x104 cells/ml. Seed 500 cells/well into the 12-well plate containing feeder cells prepared in step 1.4.</li>
	<li>Add 100 ng/ml FL19 (hu-Flt3L-Ig generated in-house using an expression system provided by M. Manz, University Hospital Z&#252;rich, Switzerland) and incubate at 37oC, 5% CO2 with periodic visual monitoring of DC development under a microscope. 
	Note: Commercially available recombinant human FL or mouse FL can be used as a substitute for hu-Flt3L-Ig to support DC development.</li>
	<li>Collect the cells in the supernatant at day 12 and wash the wells once with 0.5 ml complete RPMI medium combining the resulting supernatants. Add 0.5 ml fresh medium and scrap the adherent cells with a cell scraper.</li>
	<li>Combine the cell-containing medium from both parts and centrifuge for 5 min at 500 x g.</li>
	<li>Decant supernatant and resuspend cells in 50 &#181;l FACS buffer. Add 50 &#181;l anti-CD16/32 hybridoma supernatant and incubate for 1-2 min to block Fc receptors.</li>
	<li>Enumerate and stain all the cells with the following antibodies (0.05 &#181;g each) anti-CD11c-APC (N418), anti-CD11b-FITC, and anti-B220-PE.</li>
	<li>Wash and centrifuge the cells as described in step 3.3.</li>
	<li>Resuspend the cells in 100 &#181;l FACS buffer, gate on CD11c+ and analyze for cDCs (CD11c+CD11b+B220-) and pDCs (CD11c+CD11b-B220+)17.</li>
</ol>

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Authors have nothing to disclose.

Here we describe an in vitro culture system for the robust generation of DCs, and pDCs in particular, from a small number of CLPs. The uniqueness of this culture system is due to the use of AC-6 cells, a stromal cell line, as feeders. This approach has been shown to provide not only the cytokines, such as IL-7, SCF, M-CSF and FL20, but also the cell-cell contact21 necessary to support DC development. AC-6 cells have been used previously to facilitate the study of in vitro DC develo...

Dendritic cells (DCs) are professional antigen-presenting cells that play an important role in controlling immune responses1. While DCs are heterogeneous, they can be broadly classified into two populations, conventional DCs (cDCs) and plasmacytoid DCs (pDCs)2,3. In addition to lymphoid organs, cDCs and pDCs are also found in tissues including lung, intestine, and skin2. The morphology of cDCs and pDCs differs, with cDCs exhibiting dendrite-like projections and the shape of pDCs being more plasma cell-like. In addition, the common mouse DC marker, CD11c, is more highly expressed on cDCs than on pDCs. Moreover, cDCs can be further divided into CD11b+CD24- cDCs and CD11b-CD24+ cDCs, both of which express higher levels of MHC class II and costimulatory molecules than do pDCs2. Mature pDCs, on the other hand, have been shown to selectively express Siglec-H and BST24. Functionally, cDCs are better antigen presenting cells than are pDCs; however, pDCs can produce a vast amount of type I interferon upon virus infection or inflammatory stimulation5.
Both cDCs and pDCs are short-lived, and therefore, must be constantly replenished from progenitors within the bone marrow (BM)6,7. Adoptive transfer of common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) into lethally-irradiated mice demonstrates that cDCs and pDCs can be generated from both lineages8-10. However, there is a subset of pDCs that express RAG1/2 and possess rearranged D-J segments at the IgH locus11,12. These cells also share molecular similarities with B lymphocytes, including expression of the B220 marker, nucleic acid-sensing receptors (TLR7/TLR9), and transcription factors (Spib and Bcl11a)13, features all believed to be signatures of the lymphoid lineage. Therefore, CLPs may be good choices for in vitro generation of pDCs because of lineage similarity.
While the frequency of both cDCs and pDCs in humans and mice is very low6, DCs, particularly cDCs, can be generated in vitro from BM or progenitors in the presence of cytokines, such as GM-CSF11,14 or Flt3 ligand (FL) using feeder-free systems 11,15,16. Unfortunately, however, it is not possible to produce large numbers of pDCs in vitro using FL11,15,16. Previously we demonstrated that pDCs can be efficiently generated in vitro from CLPs using the AC-6 feeder system17. The advantage of using the AC-6 stromal cell line in the culture system is that it provides cell-cell contact and secretion of cytokines that support the generation of large numbers of pDCs from CLPs. Although production with this system is quite robust, careful replication of the procedures described below is required in order to ensure efficient generation of pDCs.

<table><tbody><tr><td>Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5</td><td>Biolegend</td><td>108408</td><td>linage marker</td></tr><tr><td>Anti-mouse NK1.1 (PE), clone PK136</td><td>Biolegend</td><td>108708</td><td>linage marker</td></tr><tr><td>Anti-mouse&amp;nbsp; CD11b (PE), cloneM1/70</td><td>Biolegend</td><td>101208</td><td>linage marker</td></tr><tr><td>Anti-mouse CD19 (PE), clone eBio1D3</td><td>Biolegend</td><td>115508</td><td>linage marker</td></tr><tr><td>Anti-mouse B220 (PE), clone RA3-6B2</td><td>Biolegend</td><td>103208</td><td>linage marker/FACS</td></tr><tr><td>Anti-mouse CD3 (PE), clone 17A2</td><td>Biolegend</td><td>100308</td><td>linage marker</td></tr><tr><td>Anti-mouse CD8a (PE), clone 53-6.7</td><td>Biolegend</td><td>100707</td><td>linage marker</td></tr><tr><td>Anti-mouse MHC-II (PE), clone NIMR-4</td><td>Biolegend</td><td>107608</td><td>linage marker</td></tr><tr><td>Anti-mouse Ter119 (PE), clone TER-119</td><td>Biolegend</td><td>116208</td><td>linage marker</td></tr><tr><td>Anti-mouse Thy1.1 (PE), clone HIS51</td><td>eBioscience</td><td>12-0900-83</td><td>linage marker</td></tr><tr><td>Anti-mouse M-CSFR (APC), clone AFS98</td><td>Biolegend</td><td>135510</td><td>FACS</td></tr><tr><td>Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8</td><td>Biolegend</td><td>105824</td><td>FACS</td></tr><tr><td>Anti-mouse Sca-1 (FITC), clone D7</td><td>Biolegend</td><td>108106</td><td>FACS</td></tr><tr><td>Anti-mouse IL-7Ra (PE/Cy7), clone A7R34</td><td>Biolegend</td><td>135014</td><td>FACS</td></tr><tr><td>Anti-mouse CD11c (PerCP/Cy5.5), clone N418</td><td>Biolegend</td><td>117328</td><td>FACS</td></tr><tr><td>Anti-mouse CD11b (FITC), clone M1/70</td><td>Biolegend</td><td>101206</td><td>FACS</td></tr><tr><td>FACSAria III</td><td>BD Biosciences</td><td></td><td>Cell sorter</td></tr><tr><td>FACS sorting tube&amp;nbsp;</td><td>BD Biosciences</td><td>352054</td><td></td></tr><tr><td>FlowJo</td><td>FlowJo LLC</td><td></td><td>Flow analysis sofrware</td></tr></tbody></table>

in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

A total of 4-6x107 BM cells are typically isolated from femurs and tibiae of one 6-8 wk-old, wild-type C57BL/6 mouse. To sort out CLPs, total BM cells are stained with PE-conjugated antibodies against lineage markers (CD3, CD8, B220, CD19, CD11b, Gr-1, Thy1.1, NK1.1, TER119, and MHC-II), anti-c-Kit-PerCP/Cy5.5, anti-Sca-1-FITC, anti-M-CSFR-APC and anti-IL-7R&#945;-PE/Cy7, analyzed and sorted with a cell sorter. The sorting strategy for CLP is shown in Figure 1. Typically, 5x104 CLPs...

Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

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Research

JoVE Journal

Immunology and Infection

8.4K Views.  National Taiwan University College of Medicine. In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand. 

Video: In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Biology

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Medicine

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Source: Chang, S., et al. <a href="https://app.jove.com/v/53211">In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with &#947;-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

Source: Chang, S., et al. In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System. J. ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Summary

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Chapters in this video

Murine Model of CD40-activation of B cells

Culture of myeloid dendritic cells from bone marrow precursors

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors