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...

The authors have no conflicts of interest to disclose.

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

1.6K visualizações.  International Atomic Energy Agency. Este protocolo representa um ensaio funcional in vitro utilizando células dendríticas derivadas de monócitos bovinos para medir a imunogenicidade vacinal antes de estudos in vivo. Portanto, preenchendo uma lacuna existente na vacinologia animal. É a geração e aplicação de células dendríticas.Como as células apresentadoras de antígenos mais potentes, as células dendríticas tornaram-se um alvo chave de pesquisa para a compreensão dos mecanismos imunológicos intracelulares...