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...

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+cloning+human+autoantigen-specific+T+cells.">An efficient method for cloning human autoantigen-specific T cells.</a> Journal of Immunological Methods. 298, 83-92 (2005).</li><li>So, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proinsulin+C-peptide+is+an+autoantigen+in+people+with+type+1+diabetes.">Proinsulin C-peptide is an autoantigen in people with type 1 diabetes.</a> Proceedings of the National Academy of Sciences of the United States of America. 115, (2018).</li><li>Hui-Yuen, J., Mcallister, S., Koganti, S., Hill, E., Bhaduri-Mcintosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+Epstein-Barr+Virus+Growth-transformed+Lymphoblastoid+Cell+Lines.">Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines.</a> Journal of Visualized Experiments. , 2-7 (2011).</li><li>Alexander-Miller, M. 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The authors declare no conflicts of interest.

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 ...

This protocol provides a platform for studying CD4T cell responses that are often weak and difficult to detect. This technique allows for the detection, enumeration, and isolation of CD4T cells without prior knowledge regarding antigen presentation. The main advantage is the analysis of CD4T cells that are quite often rare populations associated with autoimmune conditions such as type one diabetes.Upon obtaining the peripheral blood sample from a healthy donor, dilute the blood in PBS in at least one to two ratio before layering 35 milliliters of blood over 15 milliliters of density gradient medium in a 50 milliliter tube. Separate the cells by density gradient centrifugation and remove approximately 20 milliliters of the resulting top plasma layer. Then collect the white blood cell layer taking care to avoid the red blood cell pellet and wash the cells three times in fresh PBS.After counting, dilute the cells to a one times 10 to the sixth peripheral blood mononuclear cell or PBMC per milliliter of PBS concentration. Add 300 microliters of cells for each control into individual 10 milliliter tubes. After topping each tube with PBS, sediment the cells by centrifugation and resuspend the cells at a one times 10 to the sixth cells per milliliter of RP5 medium concentration.Then plate 100 microliters of cells per control to each of three wells of a 96 well plate containing 100 microliters of RP5 medium per well. Place the plate in the cell culture incubator for seven days. Next, transfer the remaining PBMCs into a new 50 milliliter tube and add one microliter of CFSE working stock solution per one milliliter of cell suspension to the side of the tube.Quickly invert the tube several times to mix and place the cells in a cell culture incubator for five minutes. At the end of the incubation, arrest the reaction with five milliliters of RP5 medium and collect the cells by centrifugation. Resuspend the pellet at one times 10 to the sixth PBMCs per milliliter of fresh RP5 medium and add one milliliter of cells to one 10 milliliter tube per antigen to be tested.Then add 100 microliters of cells per antigen to each of three wells of a 96 well plate well containing 100 microliters of RP5 medium supplemented with the diluted antigen of interest per well. Place the plate in the cell culture incubator for seven days. To stain the CD4+T cell populations for flow cytometric analysis, at the end of the culture transfer the entire volume of cells from each well into individual fluorescence activated cell sorting or FACS tubes.Wash each sample with one milliliter of PBS supplemented with 0.1%fetal calf serum or FCS. Resuspend the pellets in an appropriate anti-human CD4 antibody in 100 microliters of PBS plus FCS per tube. Incubate the cells at four degrees Celsius for 20 minutes protected from light.At the end of the incubation, wash the samples in one milliliter of fresh PBS plus FCS per tube and resuspend the pellets in 100 microliters of fresh PBS plus FCS. Immediately before the analysis, add one microliter of propidium iodide to each tube to allow exclusion of dead cells. Gate the lymphocytes according to their forward and side scatter areas.To exclude the apoptotic cells, gate the forward scatter area versus propidium iodide negative cells and use the unstained cells to set a voltage baseline for the non-fluorescent cells. Set the voltages for the CD4 antibody fluorophore and the CFSE signal so that the fluorescent signal is below 1000 for each. Use the single color control CFSE and CD4 samples to confirm a positive fluorescent signal for each color using the voltages set with the unstained cells.As CFSE and propidium iodide have some spectral overlap, adjust the compensation to subtract the propidium iodide fluorescence from the CFSE fluorescence until the CFSE only sample does not yield a signal in the propidium iodide channel. When all of the samples have been analyzed, calculate the cell division index by dividing the number of divided cells by 5000 undivided cells from the antigen stimulated group by the mean number of divided cells per 5000 undivided cells from the cells cultured without antigen. Almost all donors demonstrate a strong T-cell response to Tetanus toxoid after in vitro stimulation because the donors have been vaccinated making Tetanus toxoid a useful positive control antigen.The proliferation of CD4+T cells from unstimulated PBMCs is minimal however. After seven days of stimulation with human pro-insulin C-peptide, pro-insulin C-peptide specific CD4+T cells can be detected in the peripheral blood of an individual with type one diabetes. PBMC stimulated with influenza A virus matrix protein also demonstrate proliferation.Taken together, these data demonstrate that the assay can be performed using full length proteins as well as short synthetic peptides. The FACS component of this method can be performed using a cell sorting flow cytometer. Using a cell sorter, the antigen-specific T-cells can be isolated at the single cell level for downstream analysis.This method has allowed for the discovery of CD4T cell responses in individuals with early onset type one diabetes. Analyzing these rare T-cell populations utilizing other methods presents various technical challenges.

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Immunology and Infection

8.3K Views.  St. Vincent's Institute of Medical Research. This protocol provides a platform for studying CD4T cell responses that are often weak and difficult to detect. This technique allows for the detection, enumeration, and isolation of CD4T cells without prior knowledge regarding antigen presentation. The main advantage is the analysis of CD4T cells that are quite often rare populations associated with autoimmune conditions such as type one diabetes.Upon obtaining the peripheral blood sample from a healthy donor, dilute the blood in PBS ...