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 limited for now. Compared to hepatitis B virus (HBV)-specific CD8 T-cell epitopes, fewer HBV-specific CD4 T-cell epitopes have been identified^1,2, which made this method less applicable for analysis of HBV-specific CD4 T cells currently.

The second approach is based on the upregulation of a series of activation-induced markers after antigen peptide stimulation³. The commonly used markers include CD69, CD25, OX40, CD40L, PD-L1, 4-1BB⁴. This method has now been used to analyze antigen-specific T-cell responses in vaccinated individuals^5,6, Human Immunodeficiency Virus infection patients⁷, and Severe Acute Respiratory Syndrome Coronavirus 2 infection patients^8,9. Unlike the peptide-MHC multimers based assay, this method is not restricted by validated epitopes and could obtain viable cells for downstream analysis. A limitation of this method is that it could not provide information about the cytokine profile of antigen-specific T cells. Also, the expression of these activation-induced markers by some activated antigen-non-specific cells might contribute to the background signals in analysis, which could be a problem especially when the target antigen-specific T cells are rare. Currently, there is limited application of this method on HBV-specific CD4 T cells⁴. Whether this method could be utilized to analyze HBV-specific CD4 T cells in a reliable way needs further investigation.

The third approach is based on the cytokine secretion after antigen peptide stimulation. Like activation-induced marker-based analysis, this method is not restricted by validated epitopes. This method could directly reveal the cytokine profile of antigen-specific T cells. The sensitivity of this method is lower than the activation-induced marker-based method as it relies on the cytokine secretion of antigen-specific T cells and the number of cytokines tested is usually limited. Currently, this method is widely used in analysis of HBV-specific T cells. As cytokine secreting HBV-specific T cells could hardly be detected by direct ex vivo peptide stimulation^10,11, the cytokine profile of HBV-specific T cells is usually analyzed after 10-day in vitro peptide stimulated expansion^{12,13,14,15,16}. Arrangement of peptide pools in a matrix form has been utilized to facilitate identification of antigen-specific epitopes^17,18. With the combination of peptide matrix and cytokine secretion analysis, a method has been developed to evaluate HBV-specific CD4 T-cell responses and identify HBV-specific CD4 T-cell epitopes simultaneously¹⁶. In this protocol, the details of this method are described. HBV core antigen is chosen as an example of demonstration in this protocol.

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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 position in the matrix containing only 1 peptide. There are 12 peptide pools: 6 row peptide pools and 6 column peptide pools, 5-6 peptides in each pool¹⁶. The row peptide pools and the column peptide pools in the matrix represent 2 separate formations of HBV core antigen.
4. For 3/4 of the purchased peptides, mix peptides in the same row/column of the matrix into 12 separate peptide pools by dissolving them together in dimethyl sulfoxide (DMSO) (2 µg/µL for each peptide). Store at -80 °C for analysis of HBV-specific CD4 T-cell responses.
5. Dissolve the rest of the peptides separately (10 µg/µL) and store at -80 °C for epitope identification.

2. Isolation of peripheral blood mononuclear cells (PBMCs)

1. Sample 5 mL of venous blood from chronic HBV infection patients.
   NOTE: The blood volume should be roughly estimated according to the number of peptide pools plus 1 background control and 1 positive control. Analysis of 1 peptide pool needs 3 x 10⁵ PBMCs. On average, 1 x 10⁶ PBMCs could be obtained from 1 mL of blood.
2. Isolate PBMCs from blood using Ficoll density gradient centrifugation (800 × g, 20 min) and cryopreserve isolated PBMCs in liquid nitrogen for later use.
3. Use a Pasteur pipette to collect granulocytes between the clear Ficoll layer and the red blood cell layer. Extract genomic DNA from granulocytes using a genomic DNA purification kit according to the manufacturer's protocol.
4. Send the DNA sample to genotyping service providers to determine the HLA-DRB1 genotype.

3. In Vitro Expansion of PBMCs Using a HBV Peptide Matrix

1. Thaw PBMCs.
   1. Warm RPMI 1640 supplemented with 1:10,000 benzonase (25 U/mL) to 37 °C in a water bath.
      NOTE: Benzonase helps to limit cell clumping during thawing. Each sample will require 20 mL of RPMI 1640 with benzonase. Calculate the amount needed to thaw all samples, and prepare a separate aliquot of media (37 °C) with 1:10,000 Benzonase (25 U/mL). Thaw no more than 5 samples at a time.
   2. Remove samples from liquid nitrogen and quickly thaw frozen vials in a water bath (37 °C).
   3. Transfer the thawed cell suspension to a 15 mL centrifuge tube. Add 1 mL of Benzonase RPMI 1640 (37 °C) dropwise to the tube. Slowly add 6 mL of Benzonase RPMI 1640 (37 °C) to the centrifuge tube, rinse cryovial with another 2 mL of Benzonase RPMI 1640 (37 °C) to retrieve all cells. Continue with the rest of the samples as quickly as possible.
      ​NOTE: Slow dilution of cryopreserved samples is the key to maintain the viability of thawed cells.
   4. Centrifuge (400 × g, 10 min), remove the supernatant, and loosen the pellet by tapping the tube.
   5. Gently resuspend the pellet in 1 mL of warm Benzonase RPMI 1640. Mix gently, and filter cells through a 70 µm cell strainer if needed (i.e., if any visible clump exists).
   6. Aliquot a 10 µL suspension and dilute in Dulbecco's phosphate-buffered saline (DPBS), add Trypan blue (0.04%), load onto a hemocytometer, wait for 1 min, and count the number of viable cells (clear cells).
   7. Add 9 mL of Benzonase RPMI 1640 (37 °C) to the tube, centrifuge (400 × g, 10 min), remove the supernatant, and loosen the pellet by tapping the tube.
2. Resuspend PBMCs in RPMI 1640 supplemented with 25 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% human AB serum (complete culture medium). Adjust cell density to 1.5 x 10⁶ cells/mL. Plate PBMCs in 96-well plates (flat bottom) at a density of 3 x 10⁵ cells/well.
3. Add HBV derived peptide pools (2 μg/mL for each single peptide) to each well. For wells of background control and positive control, add the same amount of solvent (DMSO, 1 μL/mL). Add 10 U/mL IL-2 and 10 ng/mL IL-7. Incubate at 37 °C and 5% CO₂.
4. At day 3, supplement culture medium with 50 U/mL of IL-2 and 10 ng/mL of IL-7.
   NOTE: During day 1 to day 3, no obvious T-cell proliferation will be observed. The total cell number will usually decrease by 1/3 to 1/2, due to the death of non-T cells such as B cells, NK cells, NKT cells, and monocytes.
5. At day 7, replace half of the culture medium with fresh medium containing peptides (4 μg/mL), IL-2 (100 U/mL), and IL-7 (20 ng/mL).
   NOTE: To avoid disturbing the cells at the bottom, pipette about 90 μL of culture medium carefully from the top of the medium. During day 3 to day 7, robust T-cell proliferation will be observed, and proliferating T-cells usually aggregate to form clusters.
6. At day 10, gently pipette cell culture in each well 7-9 times to disaggregate cell clusters, count the number of viable cells, and transfer 2×10⁵ cells in each well to a 96 well plate (round bottom) for HBV-specific CD4 T-cell response analysis.
7. Continue culturing the rest of cells for epitope identification at day 12, adjust the volume of culture medium to 100 μL (discarding excessive medium), and supplement culture with 100 μL of fresh complete culture medium containing peptides (4 μg/mL), IL-2 (100 U/mL), and IL-7 (20 ng/mL).
   NOTE: During day 7 to day 10, T cells continue proliferating vigorously. Replace the culture medium as in step 3.5 if the medium turns yellow. In general, the cell number will exceed 6×10⁵ at day 10. Each well usually shows similar cell number, count cell number in 3 wells and use the average value as an estimate of cell number for all the wells.

4. Analysis of HBV-specific CD4 T-Cell responses by intracellular flow cytometry

1. Stimulating PBMCs with peptide pools
   1. For the cells transferred to the 96 well plate (round bottom), wash 3 times in a plate (550 × g, 3 min). Use 200 µL of medium for each wash (RPMI 1640 for the first 2 washes, complete culture medium for the last wash). Discard the supernatants.
      NOTE: Removal of residual cytokines in the culture by repeated washing could effectively decrease the background in intracellular flow cytometry analysis.
   2. For each well of cells stimulated with a specific peptide pool, add 200 µL of complete culture medium supplemented with the same peptide pools (2 µg/mL for each single peptide). For the well of background control, add complete culture medium supplemented with 1 µL/mL of DMSO. For the well of positive control, add complete culture medium supplemented with 1 µL/mL of DMSO, 150 ng/mL of phorbol 12-myristate 13-acetate (PMA), and 1 µmol/L of ionomycin.
      ​NOTE: High dose of DMSO will block the cytokine secretion of T cells (most significant for TNF-α). Dose of DMSO higher than 5 µL/mL is not recommended. Generally, the dose of DMSO in our experiment does not exceed 1 µL/mL.
   3. Incubate at 37 °C and 5% CO₂ for 6 h.
   4. After 1 h of incubation, add Monensin (1.37 µg/mL) to the culture.
2. Flow cytometry
   1. After 6 h of incubation. Remove supernatant after centrifugation (550 × g, 3 min), wash cells once with 200 µL of DPBS (550 × g, 3 min), stain surface markers (CD3, CD4, and CD8) and viability marker (using Fixable Viability Dye) in a 4 °C refrigerator for 30 min.
   2. Wash once with 200 µL of DPBS (550 × g, 3 min). Fixate and permeabilize cells, and stain intracellular cytokines (TNF-α and IFN-γ) in a 4 °C refrigerator for 45 min.
   3. After the final wash in intracellular staining, resuspend cells in 150 µL of flow cytometry buffer (DPBS + 0.5% BSA).
   4. Acquire flow cytometry data on a flow cytometer.
3. Analysis of flow cytometry results
   1. Definition of positive well: consider a well as positive if it has a frequency of cytokine secreting T cells at least two times of the background control well (Figure 1).
   2. According to the following formula, calculate the response rate for each cytokine analyzed (Figure 2):
      
      NOTE: The row peptide pool and the column peptide pools in the matrix represent 2 distinct formations of HBV core antigen, so the final response rate should be divided by 2.

5. Identification of HBV-specific HLA-DR Restricted CD4 T-cell Epitopes

1. Thaw and maintain allogenic B lymphoblastoid cell lines (BLCLs) in T-75 flask (5-20 ×10⁶ cells, 20 mL of complete culture medium).
   NOTE: To guarantee the good state of BLCLs, this step should be initiated 2 weeks before thawing of patients' PBMCs. BLCLs must be homozygous in HLA-DRB1 allele. According to the genotyping result, patients should share the same HLA-DRB1 allele as BLCLs.
2. Screening of candidate peptides for identification (Figure 3)
   1. According the T-cell response rate results at day 10, screen out 2 peptide pools with the highest response rate (1 row peptide pool and 1 column peptide pool).
   2. Set the peptide in those 2 pools as a candidate peptide if the other peptide pool containing this peptide also shows a positive result in T-cell response analysis. Use the PBMCs expanded with the other peptide pool for epitope identification later.
3. Pulsing BLCLs with peptide
   1. At day 12, count the number of viable BLCLs, transfer cells to 15 mL centrifuge tubes, centrifuge (350 × g, 10 min) and remove the supernatant. Resuspend the cell pellet in complete culture medium, and aliquot BLCLs to a 96-well plate (round bottom) at 4×10⁴ cells/well in 80 µL complete culture medium.
   2. Add a single peptide (10 µg/mL), incubate at 37 °C and 5% CO₂ for 2 h. Set 2 background control: peptide pulsing with HLA-DR blocking (pretreatment with anti-HLA-DR (10 µg/mL) for 1 h); DMSO (1 µL/mL) pulsing. The final volume of complete culture medium in each well is 100 µL.
   3. Add mitomycin C (100 µg/mL), incubate at 37 °C and 5% CO₂ for 1 h.
   4. Wash 3 times with 200 µL of RPMI 1640 (550 × g, 3 min) in a plate to remove un-pulsed peptide and mitomycin C. For the first wash, supplement the incubation culture with 100 µL of RPMI 1640.
   5. Resuspend cells in 120 µL of complete culture medium.
4. Stimulating PBMCs with peptide pulsed BLCLs.
   1. At day 12, transfer PBMCs to a 96-well plate (round bottom).
   2. Remove the supernatant after centrifugation (550 × g, 3 min) in a plate, an wash twice with 200 µL of RPMI 1640 (550 × g, 3 min) in a plate.
      ​NOTE: Removal of residual cytokines and peptides in the culture by repeated washing is the key step to decrease the background in intracellular flow cytometry analysis. Especially for residual peptides, it will bind to BLCLs and significantly increase the background.
   3. Resuspend PBMCs at each well with 210 µL of complete culture medium.
   4. For the well of PBMCs chosen for epitope identification, mix the aliquot (70 µL each) with peptide pulsed BLCLs (3 wells, including 2 background controls).
      ​NOTE: At day 12, the number of peptide pools expanded PBMCs will usually reach to above 5-7×10⁵ per well, so the ratio of PBMCs/BLCLs is about 6/1 to 4/1.
   5. Incubate at 37 °C and 5% CO₂ for 6 h.
   6. After 1 h of incubation, add Monensin (1.37 µg/mL) to the culture. The final volume of complete culture medium in each well is 200 µL.
5. Flow cytometry
   1. Repeat the same operations as in step 4.2.
6. Analysis of flow cytometry results
   1. Verify a peptide as an HLA-DR restricted CD4 T-cells epitope if PBMCs incubated with this peptide pulsed BLCLs show a frequency of cytokine secreting CD4 T cells at least two times of the PBMCs incubated with background controls (peptide pulsing with HLA-DR pre-blocking; DMSO pulsing) (Figure 4).

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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 bot...

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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...

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Materials

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