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><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

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

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Biochemistry

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

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.

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

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 ...

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

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

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.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

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

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

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.

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

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

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

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 is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

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

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

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

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.

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

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

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

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.

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

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

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;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.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

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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