Name: determination of vaccine immunogenicity using bovine monocyte-derived dendritic cells
Uploaded: 2023-05-19T12:30:00
Description: Watch this Scientific Journal Video about Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells at JoVE.com

We thank Dr. Eveline Wodak and Dr. Angelika Loistch (AGES) for their support in determining the health status of the animals and for providing BTV, Dr. Bernhard Reinelt for providing bovine blood, and Dr. Bharani Settypalli and Dr. William Dundon of the IAEA for useful advice on the real-time PCR experiments and language editing, respectively.

Blood collection is performed by a certified veterinarian service in accordance with the ethical guidelines of the Austrian Agency for Health and Food Safety(AGES) and in compliance with the accepted animal welfare standards32. The study received ethical approval from the Austrian Ministry of Agriculture. The experimental design for MoDCs generation and its subsequent application is illustrated in Figure 1.
1. Production of naive MoDC...

This study demonstrates a standardized in vitro method for generating and phenotyping bovine MoDCs and their subsequent use in measuring the vaccine immunogenicity of a commercial vaccine (e.g., RV). Bovine MoDCs can be used as a tool for screening potential vaccine antigens against cattle diseases and predicting their potential clinical impact based on immune responses before proceeding toward in vivo animal trials. The MoDCs generated were identified based on their morphological, phenotypic, and funct...

Veterinary vaccination represents a crucial aspect of animal husbandry and health, as it contributes to improving food security and animal welfare by conferring protection against diseases that affect the livestock sector globally1. An effective in vitro method to assess the immunogenicity of possible vaccine candidates would help accelerating&#160;the process of vaccine development and production. It is, therefore, necessary to expand the field of immune assays with innovative methodologies based on in vitro studies, as this would help to unveil the complexity of the immune processes related to immunization and pathogen infection. Currently, in vivo animal immunization and challenge studies, which require periodic sampling (e.g., blood and spleen), are used to measure the immunogenicity of candidate vaccines and adjuvants. These assays are expensive, time-consuming, and have ethical implications, because in most cases, animal euthanasia is carried out by the end of the trials.
As an alternative to in vivo assays, peripheral blood mononuclear cells (PBMCs) have been used to evaluate vaccine-induced immune responses in vitro2. PBMCs are a heterogeneous population of cells composed of 70%-90% lymphocytes, 10%-20% monocytes, and a limited number of dendritic cells (DCs, 1%-2%)3. PBMCs harbor antigen-presenting cells (APCs) such as B cells, monocytes, and DCs, which constantly patrol the organism searching for signs of infection or tissue damage. Locally secreted chemokines facilitate the recruitment and activation of APCs to these sites by binding to cell surface receptors. In the case of monocytes, chemokines direct their fate to either differentiate into DCs or macrophages4. As soon as DCs encounter and capture a pathogen, they migrate to secondary lymphoid organs, where they can present the processed pathogen peptide antigens using major histocompatibility complex (MHC) class I or class II surface proteins to CD8+ T cells or CD4+ T cells, respectively, thus triggering an immune response5,6.
The key role played by DCs in orchestrating a protective immune response against various pathogens makes them an interesting research target for understanding intracellular immune mechanisms, especially when designing vaccines and adjuvants against infectious agents7. Since the fraction of DCs that can be obtained from PBMCs is rather small (1%-2%), monocytes have instead been used to generate DCs in vitro8. These monocyte-derived DCs (MoDCs) were initially developed as a possible treatment strategy in cancer immunotherapy9. More recently, MoDCs have been used for vaccine research10,11,12, and classical monocytes are the predominant subtype (89%) for MoDC production13. The production&#160;of MoDCs in vitro has previously been achieved through the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) given in combination with other cytokines such as interleukin-4 (IL-4), tumor necrosis factor &#945; (TNF-&#945;), or IL-1314,15,16.
The success of an in vitro MoDC assay relies on the capability of antigen-stimulated mature MoDCs to modulate the extent and type of the immune response specific to the type of antigen detected17. The type of pathogen recognized and presented by MoDCs determines the differentiation of CD4+ T helper (Th) cells into either Th1, Th2, or Th17 effector cells and is characterized by a pathogen-specific secretory cytokine profile. A Th1 response is elicited against intracellular pathogens and results in the secretion of interferon-gamma (IFN-&#947;) and tumor necrosis factor beta (TNF-&#946;), which modulates phagocytic-dependent protection. A Th2 response is triggered against parasitic organisms and is characterized by IL-4, IL-5, IL-10, and IL-13 secretion, which initiates phagocytic-independent humoral protection. Th17 offers neutrophil-dependent protection against extracellular bacterial and fungal infections mediated by the secretion of IL-17, IL-17F, IL-6, IL-22, and TNF-&#945;18,19,20,21. Based on previous studies, it has been noted that not all pathogens fall within the expected cytokine profile. For example, dermal MoDCs, in response to Leishmania parasitic infection, stimulate IFN-&#947; secretion from CD4+ T cells and CD8+ T cells, thus inducing a protective proinflammatory Th1 response22.
It has also been shown that, in chicken MoDCs primed with Salmonella lipopolysaccharide (LPS), can induce a variable response against Salmonella typhimurium by activating both Th1 and Th2 responses, whereas Salmonella gallinarum induces a Th2 response alone, which could explain the higher resistance of the latter&#160;toward MoDC clearance23. The activation of MoDCs against Brucella canis (B. canis) has also been reported in both canine and human MoDCs, meaning this could represent a zoonotic infection mechanism24. Human MoDCs primed with B. canis induce a strong Th1 response that confers resistance to severe infection, whereas canine MoDCs induce a dominant Th17 response with a reduced Th1 response, subsequently leading to the establishment of chronic infection25. Bovine MoDCs show an enhanced affinity for foot-and-mouth disease virus (FMDV) conjugated with immunoglobulin G (IgG) as compared to non-conjugated FMDV alone, as the MoDCs form a viral-antibody complex in response to the former10. Taken together, these studies show how MoDCs have been used to analyze the complexity of immune responses during pathogen infection. The adaptive immune responses can be evaluated by the quantification of specific markers associated with lymphocyte proliferation. Ki-67, an intracellular protein detected only in dividing cells, is regarded as a reliable marker for proliferation studies26, and similarly, CD25 expressed on the surface of T cells during the late phase of activation corresponds to lymphocyte proliferation27,28.
This study demonstrates a standardized method for the in vitro generation of cattle MoDCs followed by their application in an in vitro immune assay used for testing the immunogenicity of vaccines. A commercially available rabies vaccine (RV) was used to validate the efficacy of this assay. T lymphocyte activation and proliferation were measured by flow cytometry, real-time quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent&#160;assay (ELISA) through the analysis of well-established cell activation markers such as Ki-67 and CD25 and the secretion of IFN-&#947;28,29,30,31. No animal or human experimental trials are performed during the MoDC assay.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Gibco, Thermo Fisher</td>
			<td>A1049201</td>
			<td>Ammonium-Chloride-Potassium buffer for lysis of residual RBCs in harvested PBMC Fraction</td>
		</tr>
		<tr>
			<td>BD Vacutainer Heparin Tubes</td>
			<td>Becton, Dickinson (BD) and Company</td>
			<td>366480</td>
			<td>10 mL, additive sodium heparin 158 USP units, glass tube, 16 x 100 mm size</td>
		</tr>
		<tr>
			<td>Bovine Dendritic Cell Growth Kit</td>
			<td>Bio-Rad, UK</td>
			<td>PBP015KZZ</td>
			<td>Cytokine cocktail composed of recombinant bovine IL-4 and GM-CSF</td>
		</tr>
		<tr>
			<td>Bovine IFN-&#947; ELISA Kit</td>
			<td>Bio-Rad</td>
			<td>MCA5638KZZ</td>
			<td>Kit use for measuring IFN-&#947; expression in culture supernatant</td>
		</tr>
		<tr>
			<td>CD14 Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA2678F</td>
			<td>Mouse anti-bovine CD14 monoclonal antibody, clone CC-G33, isotype IgG1</td>
		</tr>
		<tr>
			<td>CD25 Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA2430PE</td>
			<td>Mouse anti bovine CD25 monoclonal antibody, clone IL-A11, isotype IgG1</td>
		</tr>
		<tr>
			<td>CD4 Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA1653A647</td>
			<td>Mouse anti bovine CD4 monoclonal antibody, clone CC8, isotype IgG2a</td>
		</tr>
		<tr>
			<td>CD40 Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA2431F</td>
			<td>Mouse anti-bovine CD40 monoclonal antibody, clone IL-A156, isotype IgG1</td>
		</tr>
		<tr>
			<td>CD8 Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA837F</td>
			<td>Mouse anti bovine CD8 monoclonal antibody, clone CC63, isotype IgG2a</td>
		</tr>
		<tr>
			<td>CD86 Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA2437PE</td>
			<td>Mouse anti-bovine CD86 monoclonal antibody, clone IL-A190, isotype IgG1</td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>-</td>
			<td>Thermal cycler PCR machine</td>
		</tr>
		<tr>
			<td>Corning Centrifuge Tube</td>
			<td>Falcon Corning&#160;</td>
			<td>352096 &#38; 352070</td>
			<td>15 mL and 50 mL, high-clarity poypropylene conical bottom, graduated, sterial, seal screw cap, falcon tube</td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm Plus</td>
			<td>BD Bio Sciences</td>
			<td>555028</td>
			<td>Fixation/permeabilization kit with BD golgiPlug, use for flow cytometer cell staining</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>1009832500</td>
			<td>Absolute for analysis EMSURE&#160;ACS,ISO, Reag. Ph Eur</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco, Thermo Fisher</td>
			<td>10500064</td>
			<td>Qualified, heat inactivated</td>
		</tr>
		<tr>
			<td>Ficoll Plaque PLUS</td>
			<td>GE Health care Life Sciences, USA</td>
			<td>341691</td>
			<td>Lymphocyte-isolation medium</td>
		</tr>
		<tr>
			<td>FlowClean Cleaning Agent</td>
			<td>Beckman Coulter, Life Sciences</td>
			<td>A64669</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo, Becton, Dickinson (BD) and Company, LLC, USA</td>
			<td>-</td>
			<td>Flow cytometer Histogram software</td>
		</tr>
		<tr>
			<td>FlowTubes/ FACS&#160; (Fluorescence-activated single-cell sorting) Tube</td>
			<td>Falcon Corning&#160;</td>
			<td>352235</td>
			<td>5 mL, sterial, round bottom polystyrene test tube with cell strainer snap cap, use in flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Fluoresceinisothiocynat-Dextran</td>
			<td>Sigma Aldrich, Germany</td>
			<td>60842-46-8</td>
			<td>FITC-dextran MW</td>
		</tr>
		<tr>
			<td>Gallios Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>-</td>
			<td>Flow cytometer machine</td>
		</tr>
		<tr>
			<td>Hard-Shell 96-Well PCR Plates</td>
			<td>Bio-Rad</td>
			<td>HSP9601</td>
			<td>96 well, low profile, thin wall, skirted, white/clear</td>
		</tr>
		<tr>
			<td>Human CD14 MicroBeads</td>
			<td>Miltenyi Bioteck, Germany</td>
			<td>130-050-201</td>
			<td>2 mL microbeads conjugated to monoclonal anti-human CD14 antibody isotype IgG2a, used for selection of bovine monocytes from PBMCs</td>
		</tr>
		<tr>
			<td>Kaluza</td>
			<td>Beckman Coulter, Germany</td>
			<td>-</td>
			<td>Flow cytometer multicolor data analysis software</td>
		</tr>
		<tr>
			<td>MACS Column</td>
			<td>Miltenyi Bioteck, Germany</td>
			<td>130-042-401</td>
			<td>Magnetic activated cell sorting or immune magentic cell separation colum for separation of various CD14 cell population based on cell surface antigens</td>
		</tr>
		<tr>
			<td>MHC Class II DQ DR Polymorphic Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA2228F</td>
			<td>Mouse anti-sheep MHC Class II DQ DR Polymorphic:FITC, clone 49.1, isotype IgG2a, cross reactive with bovine</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tube</td>
			<td>Sigma Aldrich</td>
			<td>HS4325</td>
			<td>1.5 mL, conical bottom, graduated, sterial tube</td>
		</tr>
		<tr>
			<td>Microsoft Power Point</td>
			<td>Microsoft</td>
			<td>-</td>
			<td>The graphical illustrations of experimental design</td>
		</tr>
		<tr>
			<td>Mouse IgG1 Negative Control:FITC for CD14, CD40 Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA928F</td>
			<td>Isotype control CD14 and CD40 monoclonal antibody&#160;</td>
		</tr>
		<tr>
			<td>Mouse IgG1 Negative Control:PE for CD86 Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA928PE</td>
			<td>Isotype control CD86 monoclonal antibody&#160;</td>
		</tr>
		<tr>
			<td>Mouse IgG1 Negative Control:RPE for CD25 Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA928PE</td>
			<td>Isotype control CD25 monoclonal antibody&#160;</td>
		</tr>
		<tr>
			<td>Mouse IgG2a Negative Control:FITC for MHC Class II Antibody</td>
			<td>Bio-Rad</td>
			<td>MCA929F</td>
			<td>Isotype control for MHC class II monoclonal antibody&#160;</td>
		</tr>
		<tr>
			<td>Nobivac Rabies</td>
			<td>MSD Animal Health, UK</td>
			<td>-</td>
			<td>1 &#181;L/mL of cell cultured inactivated vaccine containing &#62; 2 I.U./mL Rabies virus strain</td>
		</tr>
		<tr>
			<td>Optical seals</td>
			<td>Bi0-Rad</td>
			<td>TCS0803</td>
			<td>0.2 mL flat PCR tube 8-cap strips, optical, ultraclear, compatible for qPCR machine</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco, Thermo Fisher</td>
			<td>15140122</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Gibco, Thermo Fisher</td>
			<td>10010023</td>
			<td>pH 7.4, 1x concentration</td>
		</tr>
		<tr>
			<td>Prism - GraphPad 5 Software&#160;</td>
			<td>Dotmatics</td>
			<td>-</td>
			<td>Statistical software</td>
		</tr>
		<tr>
			<td>Purified Anti-human Ki-67 antibody</td>
			<td>Biolegend, USA</td>
			<td>350501</td>
			<td>Monoclonal antibody, cross reactive with cow, clone ki-67</td>
		</tr>
		<tr>
			<td>Purified Mouse IgG1 k Isotype Ctrl Antibody</td>
			<td>Biolegend</td>
			<td>400101</td>
			<td>Isotype control for Ki-67 monoclonal antibody</td>
		</tr>
		<tr>
			<td>READIDROP Propidium Iodide</td>
			<td>BD Bio Sciences</td>
			<td>1351101</td>
			<td>Live/dead cell marker used for flow cytometry, amine reactive dye</td>
		</tr>
		<tr>
			<td>Recombinant Human IL-2 Protein</td>
			<td>R&#38;D System, USA</td>
			<td>202-IL-010/CF</td>
			<td>Interleukin-2, 20 ng/ml</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74106</td>
			<td>Kit use for extraction of total RNA; RLT buffer = lysis buffer; RW1 buffer = stringent guanidine-containing washing buffer; RDD buffer = DNase buffer; RPE buffer = mild wash buffer; RNaseOUT = RNase inhibitor.</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Sigma Aldrich</td>
			<td>R8758</td>
			<td>Cell culture media with L-glutamine and sodium bicarbonate</td>
		</tr>
		<tr>
			<td>SMART-servier medical art&#160;</td>
			<td>Les Laboratories Servier</td>
			<td>-</td>
			<td>Licensed under a creative commons attribution 3.0 unported license</td>
		</tr>
		<tr>
			<td>SsoAdvanced Universal SYBR Green Supermix</td>
			<td>Bio-Rad</td>
			<td>172-5270</td>
			<td>2x qPCR mix conatins dNTPs, Ss07d fusion polymerase, MgCl2, SYBR Green I supermix = supermix, ROX normalization dyes.</td>
		</tr>
		<tr>
			<td>SuperScript III First-Strand Synthesis System</td>
			<td>Invitrogen, Thermo Fisher</td>
			<td>18080051</td>
			<td>Kit for cDNA synthsis</td>
		</tr>
		<tr>
			<td>Tissue Culture Test plate 24</td>
			<td>TPP, Switzerland</td>
			<td>92024</td>
			<td>24 well plate, sterilized by radiation , growth enhanced treated, volume 3.18 mL</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Gibco, Thermo Fisher</td>
			<td>15250061</td>
			<td>0.4%, 100 mL, dye to assess cell viability</td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water</td>
			<td>Invitrogen, Thermo Fisher</td>
			<td>10977023</td>
			<td>0.1 &#181;m membrane filtered distilled water</td>
		</tr>
		<tr>
			<td>VACUETTE Heparin Blood Collection Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>15206067</td>
			<td>VACUETTE Heparin Blood Collection Tubes have a green top and contain spray-dried lithium, sodium or ammonium heparin on the inner walls and are usedin clinical chemistry, immunology and serology. The anticoagulant heparin activates antithrombin, which blocks the clotting cascade and thus produces a whole blood/plasma sample.</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma Aldrich</td>
			<td>W3500-1L</td>
			<td>Sterile-filtered, bioReagent suitable for cell culture</td>
		</tr>
	</tbody>
</table>

determination of vaccine immunogenicity using bovine monocyte-derived dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

This methodology describes the in vitro generation of cattle MoDCs for the evaluation of candidate vaccine antigens prior to performing in vivo studies. Figure 1 illustrates the experimental scheme of bovine MoDC generation and the application of the MoDCs for the in vitro assay. Using the magnetic-based cell sorting technique, it was possible to collect approximately 26 million CD14+ myocytes from the harvested PBMCs, which were previously isolated from...

Watch this Scientific Journal Video about Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells at JoVE.com

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

This protocol represents a functional in vitro assay using bovine monocyte-derived dendritic cells to measure vaccine immunogenicity prior to in vivo studies. Therefore, filling an existing gap in livestock vaccinology. It is the generation and application of dendritic cells.As the most potent antigen-presenting cells, dendritic cells have become a key research target for understanding intracellular immune mechanisms including vaccine efficacy. This assay can be implemented as a tool for screening vaccine candidates, as a quality control state for vaccine production, and for antigen and adjuvant selection. Demonstrating the procedure will be Richard Kangethe, scientist in the animal production and health laboratory.Begin by inverting and mixing the blood obtained from the calf's jugular vein puncture with heparinized vacutainers. Then transfer 20 milliliters of heparinized blood to a sterile 50 milliliter tube and dilute it with 10 milliliters of phosphate buffered saline or PBS. Pipette 15 milliliters of lymphocyte isolation medium to a sterile 50 milliliter tube and tilt the tube to a 45 degree position.Carefully layer 30 milliliters of blood PBS medium on top of the lymphocyte isolation using a 25 milliliter pipette and slowly return the tube to a vertical position before centrifuging it. Then collect the thin white layer of peripheral blood mononuclear cells or PBMC layer using a Pasteur pipette and transfer it into a new 50 milliliter tube. Wash the harvested PBMCs two times using 40 milliliters of PBS per wash and mix it thoroughly.After centrifuging, resuspend the pellet in 15 milliliters of ammonium chloride potassium buffer or ACK buffer. After 10 to 15 minutes of incubation at room temperature, add up to 40 milliliters of PBS and centrifuge the tube with maximum acceleration and deceleration. Resuspend the pellet in 10 milliliters of complete culture medium.Then add five microliters of CD14 MicroBead buffer per 10 million cells and mix thoroughly using a pipette. For flow cytometry analysis, add 10 microliters of CD14 fluorescein isothiocyanate or FITC staining antibody to the PBMCs and incubate it for 15 minutes at four to eight degrees Celsius before proceeding with the flow cytometry. To the unstained PBMCs, add one milliliter of fax buffer and centrifuge it for seven minutes at 500g and four degrees Celsius.Then resuspend the pellet in 500 microliters of fax buffer per 100 million cells. Next, insert the immunomagnetic cell separation column into the separator and place a 50 milliliter collection tube under the column outlet for collecting the flow-through. After washing the column with one milliliter degassed fax buffer, replace the used 15 milliliter tube with a new one.Pipette a hundred million cells in 500 microliters of buffer at a time allowing the cell suspension to pass through the column. Then rinse the column three times with three milliliters of degassed fax buffer each time. After collecting the flow-through, label the first eluted naive lymphocyte fraction as the CD14 negative cell fraction.Remove the column from the separator and place a new sterile 15 milliliter tube under the column for the effluent collection. Add five milliliters of fax buffer into the column and immediately push it through using the plunger. Collect and label the second flow-through or the naive monocyte fraction as the CD14 positive cell fraction.Add complete culture medium to the harvested CD14 positive monocytes to attain 1 million cells per milliliter. Next, add one milliliter cell suspension to each well of a sterile 24-well plate before supplementing each well with 40 microliters of the cytokine cocktail provided in the kit. On day two, transfer half of each well's content to individual 1.5 milliliter tubes using a pipette.After centrifuging the 1.5 milliliter tubes at 500g for seven minutes, discard the supernatant and resuspend the pellet in 500 microliters of fresh complete culture medium. Transfer 500 microliters of this resuspended cell suspension back to the corresponding wells so that the final volume in the well becomes one milliliter. Enrich each well with 20 microliters of cytokine cocktail.To generate antigen-pulsed MoDCs, add one microliter per milliliter of rabies virus vaccine or RV suspension to one milliliter of naive MoDC culture in the 24-well plate and incubate the plate for 48 hours at 5%carbon dioxide and 37 degrees Celsius. On day seven, keep the 24-well plate containing the antigen-pulsed MoDCs on ice for 10 minutes before adding one milliliter of ice cold PBS per well and mixing it thoroughly. Transfer the suspension to a 15 milliliter tube.Wash the wells with two milliliters of ice cold PBS. Collect the residual cells within each well and transfer the washed contents into their respective tubes. Centrifuge the cell suspension at 500g for seven minutes.After discarding the supernatant, resuspend the antigen-pulsed MoDCs cell pellet in a complete culture medium to adjust the final concentration of 100, 000 cells per milliliter. For MoDC-lymphocyte co-culture, seed the wells of a sterile 24-well plate with one milliliter of the naive lymphocyte cell suspension and one milliliter of antigen-pulsed or non-antigen-pulsed MoDC suspension on the seventh day of the antigen pulse. After two days of incubation, supplement each well with 20 nanograms per milliliter recombinant interleukin-2 and continue the incubation for another 120 hours.On day 14, transfer one milliliter of the co-culture to a sterile 1.5 milliliter tube and centrifuge the tube. Then resuspend the cell pellet with one milliliter of antigen-pulsed or non-pulsed MoDCs. Mix well and transfer the cell suspensions to their corresponding wells.After cell surface staining of the PBMCs and naive lymphocytes and monocytes, transfer one milliliter of the cell suspension to a sterile 1.5 milliliter tube using a pipette and centrifuge for 10 minutes at 500g. Then resuspend the pellet in one milliliter of PBS and transfer it to a 15 milliliter tube. Also collect the residual cells and the corresponding tubes using one milliliter of PBS.Then add 10 milliliters of PBS to the 15 milliliter tube containing the cells and mix by pipetting. After centrifuging the tube for seven minutes at 850g, remove the supernatant and carefully resuspend the cell pellet with residual suspension left after decanting the supernatant. Transfer the cell suspension to a V-bottom 96-well plate and seal the wells to avoid spillage.Once the staining is complete, perform the flow cytometer analysis. From the realtime preview, adjust the threshold of fluorescence including voltage and gain and cell size to eventually draw gates around the desired cell population while excluding any cellular debris. CD14 cell staining and flow cytometry showed that the naive monocyte fraction comprised 98%CD14 positive monocyte cells and was functionally capable of antigen uptake.The naive MoDCs phenotypically characterized by assessing the expression of MHC Class 2 and co-stimulatory CD86 and CD40 cell surface markers validated the dendritic cells or DC-like phenotype. During the MoDC lymphocyte co-culture on day nine, a morphological change showing dendrite extension was observed in the RV pulse to MoDCs. Compared to the non-pulsed MoDC lymphocyte culture, a significant increase in lymphocyte proliferation was demonstrated by the upregulation of the Ki67 and CD25 activation markers on the CD4 positive and CD8 positive T-cells on day 16 in the pulsed MoDC lymphocyte co-culture.The CD8 positive T-cells from the mature RV pulsed MoDC co-cultures exhibited an eightfold upregulation of Ki67 compared to the non-specific group. The CD4 positive cells in the same co-culture showed a sevenfold increase in Ki67 compared to controls, demonstrating the ability of RV primed MoDCs to present the RV antigen to naive lymphocytes and activating them in an in vitro condition. The qPCR quantification of co-cultures showed more than 30%increase in interferon gamma expression and more than 5%increase in Ki67 expression in all the co-cultures using GAPDH as a calibrator.A significantly higher concentration of secreted interferon gamma was observed in the RV pulsed MoDC lymphocyte co-culture compared to the non-specific treatment group. Perform the layering step very slowly and carefully, ensuring that blood is laid on top of the lymphocyte isolation medium. Be extra careful to collect all PBMCs without collecting too much material from the other layers.Other methods such as flow cytometry, ELISA and quantitative PCR, among others, can be applied after this procedure to evaluate the activation of immune markers.

determination-vaccine-immunogenicity-using-bovine-monocyte-derived

Research

JoVE Journal

Immunology and Infection

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

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 ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...