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.

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><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-&amp;gamma; ELISA Kit</td><td>Bio-Rad</td><td>MCA5638KZZ</td><td>Kit use for measuring IFN-&amp;gamma; 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&amp;nbsp;</td><td>352096 &amp;amp; 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&amp;nbsp;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&amp;nbsp; (Fluorescence-activated single-cell sorting) Tube</td><td>Falcon Corning&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Nobivac Rabies</td><td>MSD Animal Health, UK</td><td>-</td><td>1 &amp;micro;L/mL of cell cultured inactivated vaccine containing &gt; 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&amp;nbsp;</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&amp;amp;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&amp;nbsp;</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 &amp;micro;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 ...

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

Research

JoVE Journal

Immunology and Infection

1.8K Views.  International Atomic Energy Agency. Dit protocol vertegenwoordigt een functionele in vitro test met behulp van van rundermonocyten afgeleide dendritische cellen om de immunogeniciteit van het vaccin te meten voorafgaand aan in vivo studies. Daarom wordt een bestaande leemte in de veevaccinatie opgevuld. Het is het genereren en toepassen van dendritische cellen.Als de krachtigste antigeen-presenterende cellen zijn dendritische cellen een belangrijk onderzoeksdoel geworden voor het begrijpen van intracellulaire immuunmecha...

Video: Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Cancer Research

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

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

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 ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

De-Sialylated Monocyte-Derived-DCs from Isolated PBMCs Using Sialidase Treatment

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

Author Spotlight: Dendritic Cells Maturation Using Sialidases-Based Enzymatic Treatment of the Cell Surface 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from na&#239;ve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.
Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from na&#239;ve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

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