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 &#62; 90%, 4 mg/peptide) from a peptide synthesis service provider.</li>
	<li>Set up a square 6&#215;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 &#181;g/&#181;L for each peptide). Store at -80 &#176;C for analysis of HBV-specific CD4 T-cell responses.</li>
	<li>Dissolve the rest of the peptides separately (10 &#181;g/&#181;L) and store at -80 &#176;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 &#215; 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&#39;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 &#176;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 &#176;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 &#176;C).</li>
		<li>Transfer the thawed cell suspension to a 15 mL centrifuge tube. Add 1 mL of Benzonase RPMI 1640 (37 &#176;C) dropwise to the tube. Slowly add 6 mL of Benzonase RPMI 1640 (37 &#176;C) to the centrifuge tube, rinse cryovial with another 2 mL of Benzonase RPMI 1640 (37 &#176;C) to retrieve all cells. Continue with the rest of the samples as quickly as possible. 
		&#8203;NOTE: Slow dilution of cryopreserved samples is the key to maintain the viability of thawed cells.</li>
		<li>Centrifuge (400 &#215; 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 &#181;m cell strainer if needed (i.e., if any visible clump exists).</li>
		<li>Aliquot a 10 &#181;L suspension and dilute in Dulbecco&#39;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 &#176;C) to the tube, centrifuge (400 &#215; 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 &#956;g/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 &#956;g/mL for each single peptide) to each well. For wells of background control and positive control, add the same amount of solvent (DMSO, 1 &#956;L/mL). Add 10 U/mL IL-2 and 10 ng/mL IL-7. Incubate at 37 &#176;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 &#956;g/mL), IL-2 (100 U/mL), and IL-7 (20 ng/mL). 
	NOTE: To avoid disturbing the cells at the bottom, pipette about 90 &#956;L 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&#215;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 &#956;L (discarding excessive medium), and supplement culture with 100 &#956;L of fresh complete culture medium containing peptides (4 &#956;g/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&#215;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 &#215; g, 3 min). Use 200 &#181;L 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 &#181;L of complete culture medium supplemented with the same peptide pools (2 &#181;g/mL for each single peptide). For the well of background control, add complete culture medium supplemented with 1 &#181;L/mL of DMSO. For the well of positive control, add complete culture medium supplemented with 1 &#181;L/mL of DMSO, 150 ng/mL of phorbol 12-myristate 13-acetate (PMA), and 1 &#181;mol/L of ionomycin. 
		&#8203;NOTE: High dose of DMSO will block the cytokine secretion of T cells (most significant for TNF-&#945;). Dose of DMSO higher than 5 &#181;L/mL is not recommended. Generally, the dose of DMSO in our experiment does not exceed 1 &#181;L/mL.</li>
		<li>Incubate at 37 &#176;C and 5% CO2 for 6 h.</li>
		<li>After 1 h of incubation, add Monensin (1.37 &#181;g/mL) to the culture.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>After 6 h of incubation. Remove supernatant after centrifugation (550 &#215; g, 3 min), wash cells once with 200 &#181;L of DPBS (550 &#215; g, 3 min), stain surface markers (CD3, CD4, and CD8) and viability marker (using Fixable Viability Dye) in a 4 &#176;C refrigerator for 30 min.</li>
		<li>Wash once with 200 &#181;L of DPBS (550 &#215; g, 3 min). Fixate and permeabilize cells, and stain intracellular cytokines (TNF-&#945; and IFN-&#947;) in a 4 &#176;C refrigerator for 45 min.</li>
		<li>After the final wash in intracellular staining, resuspend cells in 150 &#181;L 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="Equation 1" src="/files/ftp_upload/62387/62387eq01.jpg" /> 
		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 &#215;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&#39; 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 &#215; 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&#215;104 cells/well in 80 &#181;L complete culture medium.</li>
		<li>Add a single peptide (10 &#181;g/mL), incubate at 37 &#176;C and 5% CO2 for 2 h. Set 2 background control: peptide pulsing with HLA-DR blocking (pretreatment with anti-HLA-DR (10 &#181;g/mL) for 1 h); DMSO (1 &#181;L/mL) pulsing. The final volume of complete culture medium in each well is 100 &#181;L.</li>
		<li>Add mitomycin C (100 &#181;g/mL), incubate at 37 &#176;C and 5% CO2 for 1 h.</li>
		<li>Wash 3 times with 200 &#181;L of RPMI 1640 (550 &#215; g, 3 min) in a plate to remove un-pulsed peptide and mitomycin C. For the first wash, supplement the incubation culture with 100 &#181;L of RPMI 1640.</li>
		<li>Resuspend cells in 120 &#181;L 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 &#215; g, 3 min) in a plate, an wash twice with 200 &#181;L of RPMI 1640 (550 &#215; g, 3 min) in a plate. 
		&#8203;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 &#181;L of complete culture medium.</li>
		<li>For the well of PBMCs chosen for epitope identification, mix the aliquot (70 &#181;L each) with peptide&#160;pulsed BLCLs (3 wells, including 2 background controls). 
		&#8203;NOTE: At day 12, the number of peptide pools expanded PBMCs will usually reach to above 5-7&#215;105 per well, so the ratio of PBMCs/BLCLs is about 6/1 to 4/1.</li>
		<li>Incubate at 37 &#176;C and 5% CO2 for 6 h.</li>
		<li>After 1 h of incubation, add Monensin (1.37 &#181;g/mL) to the culture. The final volume of complete culture medium in each well is 200 &#181;L.</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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Albumin Bovine V (BSA)</td>
			<td>Beyotime</td>
			<td>ST023</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated Anti-human TNF-&#945;</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&#160;&#956;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&#8217;s Phosphate Buffered Saline</td>
			<td></td>
			<td></td>
			<td>Prepare ddH2O (1000 ml) containing NaCl (8000 mg), KCl (200 mg), KH2PO4 (200 mg), and Na2HPO4.7H2O (2160&#160; 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-&#947;</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

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-&#945; secreting CD4 T cells and the frequency of IFN-&#947; secreting CD4 T cells in background control (DMSO) are 0.154% and 0.013% respectively. The frequency of TNF-&#945; secreting CD4 T cells and the frequency of IFN-&#947; secreting CD4 T cells specific for peptide pool Core11 are 0.206 and 0.017 respectively, so bot...

Watch this Scientific Journal Video about Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix at JoVE.com

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

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 ...

analysis-hbv-specific-cd4-t-cell-responses-identification-hla-dr

Research

JoVE Journal

Immunology and Infection

2.8K Views.  Southwest Hospital, Third Military Medical University (Army Medial University). 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.

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

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

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

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.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

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Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1^-/- Mice