Name: quantification of proliferating human antigen-specific cd4+ t cells using carboxyfluorescein succinimidyl ester
Uploaded: 2019-06-04T13:00:00
Description: Watch this Scientific Journal Video about Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester at JoVE.com

This work was supported by: The Juvenile Diabetes Research Foundation [JDRF 5-CDA-2014-210-A-N] (S. M.). The National Health and Medical Research Council (NHMRC GNT123586) (S. M.), Diabetes Australia Research Trust Millennium Award (Y17M1-MANS) (S. M.), Operational Infrastructure Support Program of the Victorian Government (S. M., A. D., E. T., M. S.), and NHMRC Postgraduate Scholarship APP1094337 and JDRF PhD Top-up Scholarship (M. S.).

All subjects gave informed consent prior to the collection of peripheral blood. Ethical approval for experiments using PBMC was given by St. Vincent&#8217;s Hosptial (HREC-A 135/08, and HREC-A 161/15).
1. Reagent Preparation
<ol>
	<li>Human T cell media
	<ol>
		<li>Prepare RP-5 media for culturing PBMC, which consists of RPMI 1640, 1x non-essential amino acids, L-alanyl-L-glutamine dipeptide (2 mM), penicillin (100 U/mL)/streptomycin (0.1 mg/mL), and 5% pooled human serum (P...

<ol>
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CFSE-based proliferation is a simple and robust method for detecting and enumerating antigen-specific human CD4+ T cells. It has been previously demonstrated that using the optimal concentration of CFSE is essential for optimal results4. Too much CFSE abrogates proliferation, whereas too little does not allow for divided and undivided cells to be distinguished. In contrast, relatively high concentrations (5.0 &#181;M) of CFSE are used to label purified murine T cells3<...

The ability to detect and study antigen-specific T cells is important in studies of cell-mediated immunity. However, doing so is particularly challenging for autoantigen-specific CD4+ T-cell responses, which are very weak and difficult to detect. A common method used for the detection of antigen-specific lymphocyte proliferation is [3H]-thymidine, which is a radiolabeled nucleotide incorporated into the DNA of proliferating cells. Although the [3H]-thymidine assay can detect DNA synthesis, this method is an indirect measure of cell division, because DNA synthesis can initiate independently of mitosis (i.e., during gene duplication and apoptosis1). This issue is compounded by the fact that antigen-specific proliferation of cells can result in considerable apoptosis2, leading to potential overestimation of antigen-specific proliferation. Furthermore, the [3H]-thymidine method does not provide phenotypic information for proliferating lymphocytes, such as CD4+ or CD8+ lineage proliferation in PBMCs stimulated with antigenic peptides.
In 2003, we published the first dye dilution assay using CFSE, called the CFSE-based proliferation assay3,4. CFSE is a fluorescent dye that binds stably to intracellular proteins by forming a covalent bond to intracellular lysine residues. Since CFSE-labeled proteins are divided equally among daughter cells3, cells that have divided can be distinguished from undivided cells by flow cytometry, which also allows for the quantitative phenotyping of lymphocyte populations. Indeed, the number of divisions a cell has undergone from the time of CFSE-staining can be measured to some degree5. More recently, many similar dyes such as CellTrace Violet proliferation dye (VPD) and CytoTrack dye have been developed, which work in a similar way6. This protocol focuses on CFSE, but the principles apply equally to other related dyes.
Peptide-MHC tetramer staining is a widely used method for detecting and cloning antigen-specific CD8+ T cells. This is a well-established method7,8,9,10; however, tetramer-based cloning requires existing knowledge of the epitope/MHC restriction and each epitope requires its own tetramer11, which limits the scope of discovery and cloning of novel epitope-specific T cells. The CFSE-based proliferation can be used with peptides, proteins, or cell lysates. The protocol described herein is both simple and robust, and the responding CD4+ T cells can be sorted for use in downstream functional and biochemical characterization assays12,13.

<table>
	<tbody>
		<tr>
			<td>Anti-human CD4-AlexaFluor647</td>
			<td>Biolegend</td>
			<td>317422</td>
			<td>RRID:AB_2716180</td>
		</tr>
		<tr>
			<td>Carboxyfluorescein succinimidyl ester (CFSE)</td>
			<td>ThermoFisher</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>71-7167-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax (1x)</td>
			<td>Gibco</td>
			<td>35050</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A H1N1 (PR8) Matrix protein 1</td>
			<td>Sino Biological</td>
			<td>40010-V07E</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential amino acids (1x)</td>
			<td>Gibco</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin (1x)</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Pooled human serum</td>
			<td>Australian Red Cross</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Proinsulin C-peptide PI33-63</td>
			<td>Purar Chemicals</td>
			<td>N/A</td>
			<td>Custom made synthetic peptide</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetanus Toxoid protein</td>
			<td>Statens Serum Intitut</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of proliferating human antigen-specific cd4+ t cells using carboxyfluorescein succinimidyl ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

In vitro stimulation of human PBMCs with tetanus toxoid protein: PBMCs were stained with CFSE and stimulated for 7 days in the presence of tetanus toxoid. Almost all donors showed a strong T cell response to tetanus toxoid because they had been vaccinated, which makes tetanus toxoid a useful positive control antigen. Figure 2 demonstrates in triplicate, that the CFSE proliferation of CD4+ T cells from unstimulated PBMCs was minimal (~12 CFSEdi...

Watch this Scientific Journal Video about Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester at JoVE.com

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

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