Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix

Summary

Based on a hepatitis B virus (HBV)-derived peptide matrix, HBV-specific CD4 T-cell responses could be evaluated in parallel with identification of HBV-specific CD4 T-cell epitopes.

Abstract

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct functional subsets based on the cytokines they secreted: for example, IFN-γ for CD4 T helper 1 cells, IL-4 and IL-13 for CD4 T helper 2 cells, IL-21 for CD4 T follicular helper cells, and IL-17 for CD4 T helper 17 cells. Analysis of hepatitis B virus (HBV)-specific CD4 T cells based on cytokine secretion after HBV-derived peptides stimulation could provide information not only about the magnitude of HBV-specific CD4 T-cell response but also about the functional subsets of HBV-specific CD4 T cells. Novel approaches, such as transcriptomics and metabolomics analysis, could provide more detailed functional information about HBV-specific CD4 T cells. These approaches usually require isolation of viable HBV-specific CD4 T cells based on peptide-major histocompatibility complex-II multimers, while currently the information about HBV-specific CD4 T-cell epitopes is limited. Based on an HBV-derived peptide matrix, a method has been developed to evaluate HBV-specific CD4 T-cell responses and identify HBV-specific CD4 T-cell epitopes simultaneously using peripheral blood mononuclear cells samples from chronic HBV infection patients.

Introduction

Currently, there are 3 main approaches to analyze antigen-specific T cells. The first approach is based on the interaction between the T-cell receptor and the peptide (epitope). Antigen-specific T cells could be directly stained with peptide-major histocompatibility complex (MHC) multimers. The advantage of this method is that it could obtain viable antigen-specific T cells, suitable for downstream transcriptomics/metabolomics analysis. A limitation of this method is that it could not provide information about the whole T-cell response to a specific antigen, as it requires validated epitope peptides while the number of identified epitopes for a specific antigen is lim....

Protocol

Written informed consent was obtained from each patient included in the study. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the medical ethics committee of Southwest Hospital.

1. Design of the HBV-derived peptide matrix

1. Download amino acid sequences of the HBV core antigen from NCBI databases (GenBank: AFY98989.1).
2. Purchase HBV core antigen derived peptides (a panel of 35 15-mer peptides overlapping by 10 residues, purity > 90%, 4 mg/peptide) from a peptide synthesis service provider.
3. Set up a square 6×6 peptide matrix with each....

Results

The frequency of cytokine secreting CD4 T cells are calculated as the sum of both single producers and double producers. As demonstrated in Figure 1, the frequency of TNF-α secreting CD4 T cells and the frequency of IFN-γ secreting CD4 T cells in background control (DMSO) are 0.154% and 0.013% respectively. The frequency of TNF-α secreting CD4 T cells and the frequency of IFN-γ secreting CD4 T cells specific for peptide pool Core11 are 0.206 and 0.017 respectively, so both TNF-α secreting CD.......

Discussion

The most critical steps in this protocol are listed as follows: 1) enough PBMCs of high viability to start PBMCs expansion; 2) appropriate environment for PBMCs expansion; and 3) complete removal of residual peptide pools in PBMCs culture before epitope identification.

All the analysis in this protocol depends on the robust proliferation of CD4 T cells. In general, the number of PBMCs after 10-day expansion will be 2-3 times of the initial number. The cell number and the viability of PBMCs are.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Albumin Bovine V (BSA)	Beyotime	ST023
APC-conjugated Anti-human TNF-α	eBioscience	17-7349-82	Keep protected from light
Benzonase Nuclease	Sigma-Aldrich	E1014	Limit cell clumping
B lymphoblastoid cell lines (BLCLs)	FRED HUTCHINSON CANCER RESEARCH CENTER	IHW09126	HLA-DRB1*0803 homozygote
B lymphoblastoid cell lines (BLCLs)	FRED HUTCHINSON CANCER RESEARCH CENTER	IHW09121	HLA-DRB1*1202 homozygote
Cell Culture Flask (T75)	Corning	430641
Cell Culture Plate (96-well, flat bottom)	Corning	3599	Flat bottom
Cell Culture Plate (96-well, round bottom)	Corning	3799	Round bottom
Cell Strainer	Corning	CLS431751	Pore size 70 μm, white, sterile
Centrifuge Tube (15 mL)	KIRGEN	KG2611	Sterile
Centrifuge Tube (50 mL)	Corning	430829	Sterile
Centrifuge, Refrigerated	Eppendorf	5804R
Centrifuge, Refrigerated	Thermo	ST16R
Centrifuge, Refrigerated	Thermo	Legend Micro 21R
Cytofix/Cytoperm Kit (Transcription Factor Buffer Set)	BD Biosciences	562574	Prepare solution before use
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	D2650	Keep at room temperature to prevent crystallization
Dulbecco’s Phosphate Buffered Saline			Prepare ddH2O (1000 ml) containing NaCl (8000 mg), KCl (200 mg), KH2PO4 (200 mg), and Na2HPO4.7H2O (2160  mg). Adjust PH to 7.4. Sterilize through autoclave.
Ficoll-Paque Premium	GE Healthcare	17-5442-03
Filter Tips (0.5-10)	Kirgen	KG5131	Sterile
Filter Tips (100-1000)	Kirgen	KG5333	Sterile
Filter Tips (1-200)	Kirgen	KG5233	Sterile
FITC-conjugated Anti-human CD4	BioLegend	300506	Keep protected from light
Fixable Viability Dye eFluor780	eBioscience	65-0865-14	Keep protected from light
GolgiStop Protein Transport Inhibitor (Containing Monensin)	BD Biosciences	554724	Protein Transport Inhibitor
Haemocytometer	Brand	718620
HBV Core Antigen Derived Peptides	ChinaPeptides
HEPES	Gibco	15630080	100 ml
Human Serum AB	Gemini Bio-Products	100-51	100 ml
Ionomycin	Sigma-Aldrich	I0634
KCl	Sangon Biotech	A100395-0500
KH2PO4	Sangon Biotech	A100781-0500
LSRFortessa Flow Cytometer	BD
L-glutamine	Gibco	25030081	100 ml
Microcentrifuge Tube (1.5 mL)	Corning	MCT-150-C	Autoclaved sterilization before using
Microplate Shakers	Scientific Industries	MicroPlate Genie
Mitomycin C	Roche	10107409001
Na2HPO4.7H2O	Sangon Biotech	A100348-0500
NaCl	Sangon Biotech	A100241-0500
PCR Tubes (0.2 mL)	Kirgen	KG2331
PE/Cy7-conjugated Anti-human CD8	BioLegend	300914	Keep protected from light
PE-conjugated Anti-human IFN-γ	eBioscience	12-7319-42	Keep protected from light
Penicillin Streptomycin	Gibco	15140122	100 ml
PerCP-Cy5.5-conjugated Anti-human CD3	eBioscience	45-0037-42	Keep protected from light
Phorbol 12-myristate 13-acetate (PMA)	Sigma-Aldrich	P1585
Recombinant Human IL-2	PeproTech	200-02
Recombinant Human IL-7	PeproTech	200-07
RPMI Medium 1640	Gibco	C11875500BT	500 ml
Sodium pyruvate,100mM	Gibco	15360070
Trypan Blue Stain (0.4%)	Gibco	15250-061
Ultra-LEAF Purified Anti-human HLA-DR	BioLegend	307648
Wizard Genomic DNA Purification Kit	Promega	A1125