Name: Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix (Video) | JoVE Journal
Uploaded: 2021-10-20T14:00:00.000+00:00
Duration: 10 min 37 s
Description: Learn to evaluate HBV-specific CD4 T cell responses and identify HLA-DR-restricted epitopes using PBMCs from chronic hepatitis B patients. This method combines peptide stimulation with flow cytometry analysis.

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5 minutes

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).

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
<ol>
	<li>Download amino acid sequences of the HBV core antigen from NCBI databases (GenBank: AFY98989.1).</li>
	<li>Purchase HBV core antigen derived peptides (a panel of 35 15-mer peptides overlapping by 10 residues, purity &gt; 90%, 4 mg/peptide) from a peptide synthesis service provider.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Dissolve the rest of the peptides separately (10 µg/µL) and store at -80 °C for epitope identification.</li>
</ol>
2. Isolation of peripheral blood mononuclear cells (PBMCs)
<ol>
	<li>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.</li>
	<li>Isolate PBMCs from blood using Ficoll density gradient centrifugation (800 × g, 20 min) and cryopreserve isolated PBMCs in liquid nitrogen for later use.</li>
	<li>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.</li>
	<li>Send the DNA sample to genotyping service providers to determine the HLA-DRB1 genotype.</li>
</ol>
3. In Vitro Expansion of PBMCs Using a HBV Peptide Matrix
<ol>
	<li>Thaw PBMCs.
	<ol>
		<li>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.</li>
		<li>Remove samples from liquid nitrogen and quickly thaw frozen vials in a water bath (37 °C).</li>
		<li>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.</li>
		<li>Centrifuge (400 × g, 10 min), remove the supernatant, and loosen the pellet by tapping the tube.</li>
		<li>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).</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Analysis of HBV-specific CD4 T-Cell responses by intracellular flow cytometry
<ol>
	<li>Stimulating PBMCs with peptide pools
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Incubate at 37 °C and 5% CO2 for 6 h.</li>
		<li>After 1 h of incubation, add Monensin (1.37 µg/mL) to the culture.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>After the final wash in intracellular staining, resuspend cells in 150 µL of flow cytometry buffer (DPBS + 0.5% BSA).</li>
		<li>Acquire flow cytometry data on a flow cytometer.</li>
	</ol>
	</li>
	<li>Analysis of flow cytometry results
	<ol>
		<li>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).</li>
		<li>According to the following formula, calculate the response rate for each cytokine analyzed (Figure 2): 
		<img alt="Response rate equation: Σ(frequency of cytokine secreting cells in positive well - background)/2." src="/files/ftp_upload/62387/62387eq01.jpg" data-altupdatedat="1788282455000" data-alt="Equation 1"> 
		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.</li>
	</ol>
	</li>
</ol>
5. Identification of HBV-specific HLA-DR Restricted CD4 T-cell Epitopes
<ol>
	<li>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.</li>
	<li>Screening of candidate peptides for identification (Figure 3)
	<ol>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Pulsing BLCLs with peptide
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Add mitomycin C (100 µg/mL), incubate at 37 °C and 5% CO2 for 1 h.</li>
		<li>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.</li>
		<li>Resuspend cells in 120 µL of complete culture medium.</li>
	</ol>
	</li>
	<li>Stimulating PBMCs with peptide pulsed BLCLs.
	<ol>
		<li>At day 12, transfer PBMCs to a 96-well plate (round bottom).</li>
		<li>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.</li>
		<li>Resuspend PBMCs at each well with 210 µL of complete culture medium.</li>
		<li>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.</li>
		<li>Incubate at 37 °C and 5% CO2 for 6 h.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Repeat the same operations as in step 4.2.</li>
	</ol>
	</li>
	<li>Analysis of flow cytometry results
	<ol>
		<li>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).</li>
	</ol>
	</li>
</ol>

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<li>Grifoni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targets+of+T+Cell+Responses+to+SARS-CoV-2+Coronavirus+in+Humans+with+COVID-19+Disease+and+Unexposed+Individuals%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals.</a> Cell. 181, 1489-1501 (2020).</li>
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</ol>

The authors have nothing to disclose.

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...

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 identified1,2, 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 individuals5,6, Human Immunodeficiency Virus infection patients7, and Severe Acute Respiratory Syndrome Coronavirus 2 infection patients8,9. 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 stimulation10,11, the cytokine profile of HBV-specific T cells is usually analyzed after 10-day in vitro peptide stimulated expansion12,13,14,15,16. Arrangement of peptide pools in a matrix form has been utilized to facilitate identification of antigen-specific epitopes17,18. 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.

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

analysis of hbv-specific cd4 t-cell responses and identification of hla-dr-restricted cd4 t-cell epitopes based on a peptide matrix

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

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-&#947; 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.

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...

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 ...

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Research

JoVE Journal

Immunology and Infection

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.<br...

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry.  This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

The video demonstrates an assay using human induced pluripotent stem cell (iPSC)-derived CD8+ T cells to inhibit hepatitis B virus (HBV) replication in mice. These T cells, engineered with HBV-specific receptors, are transferred into transgenic mice. Replication-competent HBV genome plasmids are then injected directly into the liver, enabling viral DNA uptake. Once viral antigens are expressed and presented by hybrid MHC-I molecules, the engineered T cells recognize and bind to these complexes.

Suppressing Hepatitis B Virus Replication Using Viral Antigen-Specific T Cells

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design.  Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Learn to evaluate HBV-specific CD4 T cell responses and identify HLA-DR-restricted epitopes using PBMCs from chronic hepatitis B patients. This method combines peptide stimulation with flow cytometry analysis.

Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix (Video) | JoVE Journal

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

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