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

Summary

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.

Abstract

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.

Introduction

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 assay^3,4. 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 method^7,8,9,10; 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 assays^12,13.

Protocol

All subjects gave informed consent prior to the collection of peripheral blood. Ethical approval for experiments using PBMC was given by St. Vincent’s Hosptial (HREC-A 135/08, and HREC-A 161/15).

1. Reagent Preparation

1. Human T cell media
   1. 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 (PHS).
2. CFSE stock solutions
   1. Prepare a master stock by dissolving 25 mg of CFSE in ~9 mL of DMSO to achieve a final stock solution with a concentration of 5 mM.
   2. Prepare a working stock by diluting the master stock in PBS to achieve a working concentration of 10 µM.

2. Preparation of Human PBMCs from Whole Blood

1. There are generally between 0.5–1.5 x 10⁶ PBMCs/mL of human peripheral blood. Therefore, the amount of blood required depends upon the desired number of PBMCs. Dilute human peripheral blood with PBS at least 1:2. Separate the PBMCs by adding 15 mL of density gradient medium to a 50 mL tube, then overlay 35 mL of diluted whole blood.
2. Centrifuge at 850 x g for 15 min without deceleration at room temperature (RT). This will result in three clear layers: the bottom layer containing the red blood cell pellet, middle layer of density gradient medium with white blood cells lining its top interface, and top plasma layer¹⁴.
3. Remove approximately 20 mL of the top plasma layer. Collect the white blood cell layer, being careful to avoid the red blood cell pellet. Wash 3x with PBS and count viable cells using trypan blue exclusion on a hemocytometer. Dilute to 1 x 10⁶ PBMCs/mL in PBS.
4. Non-CFSE-stained cells
   1. These cells are used as compensation controls for flow cytometry, comprising of unstained and CD4⁺ single-stained cells. Add 300 µL of each control sample PBMC suspension to a 10 mL tube, top with PBS, and centrifuge at 550 x g for 5 min at RT.
   2. Resuspend 1 x 10⁶ cells/mL in RP-5 media. Incubate these unlabeled cells for 7 days in a 37 °C/5% CO₂ incubator with the CFSE-labeled cells (step 2.6.1).
5. CFSE-stained cells
   1. Transfer the cells from step 2.3 into a 50 mL tube. Add 1.0 µL of CFSE working stock solution (10 µM) per 1 mL of cell suspension to the side of the tube. Mix quickly by inverting the tube several times. The final concentration of CFSE is 10 nM.
   2. Incubate for 5 min in a 37 °C/5% CO₂ incubator. To stop the staining, add 5 mL of RP-5 media, pellet the cells by centrifuging for 5 min at 550 x g. Resuspend the PBMCs at 1 x 10⁶/mL in RP-5 media.
   3. Add 1.0 mL of cell suspension to a 10 mL tube. Use one tube for each antigen to be tested.
6. Antigenic stimulation of human PBMCs and cell culture
   1. Culture human CFSE-labelled PBMCs with antigens in RP-5 media for 7 days in a 37 °C/5% CO₂ incubator. Culture 1 x 10⁵ cells/well (100 µL) in a 96 well plate with 100 µL/well of RP-5 media containing diluted antigen.
      NOTE: Antigens used, including working concentrations, are summarized in Table 1.

3. Anti-CD4 Staining for FACS Analysis

1. Pipette 200 µL of the cultured cells into FACS tubes, wash cells 1x in 1.0 mL of PBS containing 0.1% FCS, and centrifuge for 5 min at 550 x g.
2. Stain with anti-human CD4 Alexa Fluor 647 (0.25 μg/mL) in 100 µL of PBS/0.1% FCS. Keep aside a sample of CFSE-labelled cells, unstained with any other fluorophores, to use for setting the FACS CFSE compensation. Incubate the cells at 4 °C protected from light for 20 min.
3. Wash the cells by adding 1 mL of PBS/0.1% FCS, centrifuge at 550 x g for 5 min at RT, and resuspend in 100 µL of PBS/0.1% FCS. Immediately before FACS analysis, add 1 µL of propidium iodide (PI, 0.1 mg/mL) to all tubes to allow the dead cells to be excluded by flow cytometry.

4. Flow Cytometric Configuration and Gating Strategy

NOTE: Figure 1 shows the FACS configuration including compensation controls and gating strategy.

1. Gate the forward scatter (FSC) vs. side scatter (SSC) (Figure 1A) population to include all lymphocytes.
2. Gate the FSC vs. PI (Figure 1B) population on PI negative cells to exclude apoptotic cells.
3. Use unstained cells to set a voltage baseline for non-fluorescent cells. Set the voltages for CD4-A647 and CFSE so that the fluorescence signal is below 1,000 for each (Figure 1C). Use the single color controls CFSE (Figure 1D) and CD4-A647 (Figure 1E) to confirm positive fluorescent signals (~10,000) for each color, using the voltages set with unstained cells.
4. CFSE and PI have some spectral overlap; adjust the compensation to subtract PI fluoresence from CFSE fluoresence until the CFSE-only sample does not yield a signal in the PI channel.
   NOTE: These gates were applied to all samples analyzed herein.

5. Calculation of Cell Division Index to Enumerate Antigen-specific CD4⁺ T Cell Proliferation

NOTE: Cell division index (CDI) refers to the number of divided cells (CD4⁺/CFSE^dim) per 5,000 undivided cells (CD4⁺/ CFSE^bright). When the number of undivided CD4⁺ cells is not exactly 5,000, the number of divided cells is corrected to express the number of divided cells per 5,000 undivided cells. For example, using the tetanus toxoid-specific proliferation (Figure 2D), there were 4,930 undivided cells (CFSE^bright) and 3,268 divided cells (CFSE^dim); therefore, the corrected number of divided cells is (5,000/4,930) x 3,268 = 3,304.3.

1. Calculate the CDI (Table 1) by dividing the number of divided cells/5,000 undivided cells from the antigen-stimulated group by the mean number of divided cells (per 5,000 undivided cells) from the cells cultured without antigen (Table 2).

Results

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 CFSE^di...

Discussion

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 µM) of CFSE are used to label purified murine T cells^3<...

Materials

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