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

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).