a vaccine-pulsed dendritic cell-based in vitro assay to induce lymphocyte proliferation

A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation

To generate antigen-pulsed MoDCs, add 1 microliter per milliliter of rabies virus vaccine or RV suspension to 1 milliliter of na&#239;ve 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 1 milliliter of ice ice-cold PBS per well, and mixing it thoroughly. Transfer the suspension to a 15-milliliter tube.
Wash the wells with 2 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 500 g for 7 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 1 milliliter of the na&#239;ve lymphocyte cell suspension and 1 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 1 milliliter of the co-culture to a sterile 1.5-milliliter tube, and centrifuge the tube. Then, resuspend the cell pellet with 1 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 na&#239;ve lymphocytes and monocytes, transfer 1 milliliter of the cell suspension to a sterile 1.5-milliliter tube using a pipette, and centrifuge for 10 minutes at 500 g. Then, resuspend the pellet in 1 milliliter of PBS and transfer it to a 15-milliliter tube. Also, collect the residual cells and the corresponding tubes using 1 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 7 minutes at 850 g, 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 real-time 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.

a-vaccine-pulsed-dendritic-cell-based-vitro-assay-to-induce

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

EoE Sub Articles

eoe sub articles

<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>Bovine Dendritic Cell Growth Kit</td>
			<td>Bio-Rad, UK</td>
			<td>PBP015KZZ</td>
			<td>Cytokine cocktail composed of recombinant bovine IL-4 and GMCSF</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>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>Phosphate Buffer Saline (PBS)</td>
			<td>Gibco, Thermo Fisher</td>
			<td>10010023</td>
			<td>pH 7.4, 1x concentration</td>
		</tr>
		<tr>
			<td>FlowTubes/ FACS (Fluorescenceactivated single-cell sorting) Tube</td>
			<td>Falcon Corning</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>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>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>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>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>
	</tbody>
</table>

Source: Liaqat, F., et al., <a href="https://app.jove.com/v/64874">Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells.</a> J. Vis. Exp. (195), (2023).
This video demonstrates a monocyte-derived dendritic cell, MoDC-based assay to measure the immunogenicity of commercially available vaccines. MoDC assays investigate dendritic cell responses to antigens, pathogens, or treatments and can provide insights into immune responses, vaccine development, and immunotherapy research.

1. Production of Na&#239;ve MoDCs
NOTE: Whole blood samples were obtained from a single pathogen-free calf by jugular vein puncture with heparinized ...

Begin by adding the test vaccine antigens to the dendritic cell culture.
The dendritic cell recognizes the antigen, internalizes it via receptor-mediated endocytosis, fragments it into smaller peptides, and transfers the resulting antigen-MHC complex to the cell surface for presentation.
Co-culture antigen-presenting dendritic cells with na&#239;ve T lymphocytes to enable interaction between the T cell receptor and the antigen-MHC complex, along with their co-receptors.
This leads to immunological synapse formation,&#160; activating T lymphocytes via intracellular signaling.
Activated T lymphocytes undergo clonal expansion, generating effector T lymphocytes.
Add interleukin-2, or IL-2, cytokine molecules to the culture and incubate. The binding of IL-2 triggers intracellular signaling, enabling the proliferation and survival of lymphocytes.
Take a small volume of the culture and centrifuge. Discard the supernatant. Resuspend the cell pellet with antigen-presenting dendritic cells and transfer it to the same well.
Restimulation with antigen increases lymphocyte proliferation.
Perform downstream analysis to quantify specific markers associated with lymphocyte proliferation.

29 visualizações. Um ensaio in vitro baseado em células dendríticas pulsadas por vacina para induzir a proliferação de linfócitos

Vídeo: Um ensaio in vitro baseado em células dendríticas pulsadas por vacina para induzir a proliferação de linfócitos - Experimento

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

In vivo Triagem de imunogenicidade de vesículas extracelulares derivadas do tumor por citometria de fluxo de células T esplênicas

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

JoVE Journal

Pesquisa do câncer

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

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

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

Geração de imatura, madura e tolerogênicas células dendríticas com diferentes fenótipos Metabolic

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

Imunologia e Infecção

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.

Novo protocolo para a geração de células dendríticas imunogênicas fisiológicas

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

A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation

Transcrição

A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation