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 apoptosis

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 ....

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

List of materials used in this article

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