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Immunology and Infection

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

Published: October 20th, 2021

DOI:

10.3791/62387

1Department of Infectious Diseases, Southwest Hospital, Third Military Medical University (Army Medial University), 2Chongqing Key Laboratory for Research of Infectious Diseases

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.

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.

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....

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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 ove.......

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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 bot.......

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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.......

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This work was supported by National Natural Science Foundation of China (81930061), Chongqing Natural Science Foundation (cstc2019jcyj-bshX0039, cstc2019jcyj-zdxmX0004), and Chinese Key
Project Specialized for Infectious Diseases (2018ZX10723203).

....

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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

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