Name: analysis of hbv-specific cd4 t-cell responses and identification of hla-dr-restricted cd4 t-cell epitopes based on a peptide matrix
Uploaded: 2021-10-20T14:00:00
Description: 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

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 ove...

<ol>
	<li>Desmond, C. P., Bartholomeusz, A., Gaudieri, S., Revill, P. A., Lewin, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+T-cell+epitopes+in+hepatitis+B+virus:+identification,+genotypic+variation+and+relevance+to+antiviral+therapeutics.">A systematic review of T-cell epitopes in hepatitis B virus: identification, genotypic variation and relevance to antiviral therapeutics.</a> Antiviral Therapy. 13, 161-175 (2008).</li><li>Mizukoshi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+immune+responses+to+the+hepatitis+B+virus+polymerase.">Cellular immune responses to the hepatitis B virus polymerase.</a> Journal of Immunology. 173, 5863-5871 (2004).</li><li>W&#246;lfl, M., Kuball, J., Eyrich, M., Schlegel, P. G., Greenberg, P. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successive+annual+influenza+vaccination+induces+a+recurrent+oligoclonotypic+memory+response+in+circulating+T+follicular+helper+cells.">Successive annual influenza vaccination induces a recurrent oligoclonotypic memory response in circulating T follicular helper cells.</a> Science Immunology. 2, (2017).</li><li>Bowyer, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation-induced+Markers+Detect+Vaccine-Specific+CD4++T+Cell+Responses+Not+Measured+by+Assays+Conventionally+Used+in+Clinical+Trials.">Activation-induced Markers Detect Vaccine-Specific CD4+ T Cell Responses Not Measured by Assays Conventionally Used in Clinical Trials.</a> Vaccines. 6, (2018).</li><li>Morou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+differentiation+is+central+to+HIV-specific+CD4(+)+T+cell+dysfunction+in+progressive+disease.">Altered differentiation is central to HIV-specific CD4(+) T cell dysfunction in progressive disease.</a> Nature Immunology. 20, 1059-1070 (2019).</li><li>Grifoni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targets+of+T+Cell+Responses+to+SARS-CoV-2+Coronavirus+in+Humans+with+COVID-19+Disease+and+Unexposed+Individuals.">Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals.</a> Cell. 181, 1489-1501 (2020).</li><li>Meckiff, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imbalance+of+Regulatory+and+Cytotoxic+SARS-CoV-2-Reactive+CD4++T+Cells+in+COVID-19.">Imbalance of Regulatory and Cytotoxic SARS-CoV-2-Reactive CD4+ T Cells in COVID-19.</a> Cell. , (2020).</li><li>Boni, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hepatitis+B+virus+(HBV)-specific+T-cell+dysfunction+in+chronic+HBV+infection.">Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection.</a> Journal of Virology. 81, 4215-4225 (2007).</li><li>Chang, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+hepatitis+B+virus+(HBV)-specific+CD4++T-cell+responses+in+human+immunodeficiency+virus+type+1-HBV-coinfected+individuals+receiving+HBV-active+antiretroviral+therapy.">Reduced hepatitis B virus (HBV)-specific CD4+ T-cell responses in human immunodeficiency virus type 1-HBV-coinfected individuals receiving HBV-active antiretroviral therapy.</a> Journal of Virology. 79, 3038-3051 (2005).</li><li>Boni, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restored+Function+of+HBV-Specific+T+Cells+After+Long-term+Effective+Therapy+With+Nucleos(t)ide+Analogues.">Restored Function of HBV-Specific T Cells After Long-term Effective Therapy With Nucleos(t)ide Analogues.</a> Gastroenterology. 143, 963-973 (2012).</li><li>Kennedy, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserved+T-cell+function+in+children+and+young+adults+with+immune-tolerant+chronic+hepatitis+B.">Preserved T-cell function in children and young adults with immune-tolerant chronic hepatitis B.</a> Gastroenterology. 143, 637-645 (2012).</li><li>de Niet, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+T+cell+function+in+chronic+hepatitis+B+patients+upon+treatment+with+interferon+based+combination+therapy.">Restoration of T cell function in chronic hepatitis B patients upon treatment with interferon based combination therapy.</a> Journal of Hepatology. 64, 539-546 (2016).</li><li>Rinker, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus-specific+T+cell+responses+after+stopping+nucleos(t)ide+analogue+therapy+in+HBeAg-negative+chronic+hepatitis+B.">Hepatitis B virus-specific T cell responses after stopping nucleos(t)ide analogue therapy in HBeAg-negative chronic hepatitis B.</a> Journal of Hepatology. 69, 584-593 (2018).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-&#945;/IFN-&#947;+profile+of+HBV-specific+CD4+T+cells+is+associated+with+liver+damage+and+viral+clearance+in+chronic+HBV+infection.">TNF-&#945;/IFN-&#947; profile of HBV-specific CD4 T cells is associated with liver damage and viral clearance in chronic HBV infection.</a> Journal of Hepatology. 72, 45-56 (2020).</li><li>Hoffmeister, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+T+cell+epitopes+by+flow+cytometry.">Mapping T cell epitopes by flow cytometry.</a> Methods. 29, 270-281 (2003).</li><li>Anthony, D. D., Lehmann, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+epitope+mapping+using+the+ELISPOT+approach.">T-cell epitope mapping using the ELISPOT approach.</a> Methods. 29, 260-269 (2003).</li></ol>

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 ...

In a Chronic HBV infection, CD4 T cells play important roles in both viral clearance and the returners. This method enables us to evaluate HBV specific CD4 T cell responses and identify HBV specific CD4 T cell epitopes, simultaneously. Demonstrating the procedure will be Jianmei Xiao, a research assistant.And Xing Wan, a technician from Bilao. To begin, isolate peripheral blood mononuclear cells, or PBMCs, from blood, using Ficoll density gradient centrifugation at 800 times G for 20 minutes. Then, collect the granulocytes between the and red blood cell layer, using a pasteur pipette.Transfer the thawed cell suspension to a 15 milliliter centrifuge tube, Add one milliliter of the pre warmed Benzonase RPMI 1640, drop wise, then slowly add another six milliliters. Rinse the cryo vial with two milliliters of Benzonase RPMI 1640 to retrieve the remaining cells. Then centrifuge the tube at 400 times G for 10 minutes.Remove the supernatant and loosen the pellet by tapping the tube. Gently re-suspend the pellet in one milliliter of warm Bensenase NACE RPMI 1640. Mix the cells gently, and filter them through a 70 micrometer cell strainer, if any cell clumps are visible.Count viable cells using trypan blue and a hemocytometer. Re-suspend the PBMCs and complete culture medium, containing 10 units per milliliter IL2, and 10 nanograms per milliliter IL7. Adjust the cell density to 1.5x10 to the 6 cells per milliliter.Then plate the cells in 96-Well flat bottom plates at a density of 3x10 to the fifth cells per well. Add hepatitis B virus or HBV derived peptide pools to each Well. For the background and positive control Wells, add the same amount of solvent.Incubate the plates at 37 degrees Celsius and 5%carbon dioxide. On day three, supplement the culture medium with 50 units per milliliter of IL2, and 10 nanograms per milliliter of IL7. On day seven, replace half of the culture medium with fresh medium containing:four micrograms per milliliter peptides, 100 units per milliliter IL2, and 20 nanograms per milliliter IL7.On day 10, gently pipette the cells in each Well seven to nine times to dis-aggregate cell clusters. Count the number Of viable cells and transfer 2x10 to the fifth cells into each Well of a 96-Well round bottom plate for HPV specific CD4 T cell response analysis. For the residual cells in the 96-Well flat bottom plate, adjust the volume of the culture medium to 100 microliters by discarding excessive medium.Then, supplement the culture with 100 microliters of fresh, complete culture medium, containing:four micrograms per milliliter peptides, 100 units per milliliter IL2, ans 20 nanograms per milliliter IL7. Continue culturing the cells at 37 degrees Celsius and 5%carbon dioxide for epitope identification on day 12. Wash the cells in the 96-Well round bottom plate by adding 200 microliters of RPMI 1640, centrifuging the plate at 550 times G for three minutes, and discarding the supernatant.Repeat the wash twice using complete culture medium for the last wash. For each well of cells stimulated with a specific peptide pool, add 200 microliters of complete culture medium supplemented with the same peptide pool. For the Background Control Well, add complete culture medium, supplemented with one microliter per milliliter of DMSO.For the Positive Control Well, add complete culture medium supplemented with one microliter per milliliter DMSO, 150 nanograms per milliliter PMA, and one micromole per liter of ionomycin. Incubate the plate at 37 degrees Celsius in 5%carbon dioxide for six hours. After one hour of incubation, add 1.37 micrograms per milliliter monensin to the culture.After the six hour incubation is complete, centrifuge the plate at 550 times G for three minutes. Remove the supernatant and wash the cells once with 200 microliters of DPPs, as previously demonstrated. Stain for surface markers CD3, CD4 and CD8, and a viability marker.After suspending the cells with vibrator, then refrigerate the plate at four degrees Celsius for 30 minutes. After washing the plate once with 200 microliters of DPBS, fix and permeabilize the cells, stain for intracellular cytokines, TNF Alpha and Interferon-gamma, and refrigerate the plate at four degrees Celsius for 45 minutes. Wash the cells once again, and re-suspend them in 150 microliters of flow cytometry buffer.Then, acquire the flow cytometry data using a flow cytometer. Maintain allergenic B-lymphoblastoid cell lines, or BLCLs, in a T-75 flask. On day 12 of the PBMC expansion, count the number of viable BLCLs, and transfer them to 15 milliliter centrifuge tubes.Centrifuge the cells at 350 times G for 10 minutes and remove the supernatant. Re-suspend the cell pellet and complete culture medium. Then aliquot the BLCLs to a 96-Well round bottom plate at 4x10 to the fourth cells per well, and 80 microliters of complete culture medium.Add 10 micrograms per milliliter of a single peptide, and set two background controls. Peptide pulsing with HLADR blocking, and DMSO pulsing. Incubate the plates at 37 degrees Celsius and 5%carbon dioxide, for two hours.Add 100 micrograms per milliliter Mitomycin C and incubate the plate at 37 degrees Celsius and 5%carbon dioxide for one hour. Wash the plate three times with 200 microliters of RPMI 1640 to remove unpulsed peptide and Mitomycin C, then re-suspend the cells in 120 microliters of complete culture medium, using vibrator. On day 12 of the PBMC expansion, transfer the PBMCs to a 96-Well round bottom plate.Centrifuge the cells in the plate, remove the supernatant, and wash them twice with 200 microliters of RPMI 1640, as previously demonstrated. Re-suspend the PBMCs in each well with 210 microliters of complete culture medium. For the PBMC Wells chosen for epitope identification, aliquot 70 microliters of the cell suspension and mix with peptide pulsed BLCLs in three Wells, including two background controls.Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for six hours. After one hour of incubation, add 1.37 micrograms per milliliter monensin to the culture and bring the final volume in each well to 200 microliters with complete culture medium. In this representative example, the TNF Alpha and Interferon-gamma secreting CD4 T cell responses for peptide pool Core11 are lower than two times the background values, hence, considered negative.Meanwhile, the responses for peptide pool Core09 are higher than two times the background, and considered positive. The gray background indicates Wells with a positive CD4 T cell response and candidate peptides for epitope identification are indicated in red, Core01 has the highest response rate, judging from both TNF Alpha and Interferon-gamma secreting CD4T cells in column peptide pools. Peptides C1 to 15, C31 to 45, C61 to 75, and C91 to 105 in this peptide pool, are set as candidate peptides as the row of peptide pools containing those peptides also show positive results.PBMCs expanded with peptide pools Core07, 08, 09 and 10, are used for epitope identification of these peptides. Similarly, Core09 has the highest response rate in row peptide pools. All the peptides in this pool are set as candidate peptides and PBMCs expanded with peptide pools:Core01, 02, 03, 04, 05 and 06 are used for epitope identification of these peptides.For peptide pool Core08 expanded PBMCs, after stimulation with peptide C31 to 45 pulsed BLCLs, the frequencies of TNF Alpha and Interferon-gamma secreting CD4 T cells are two times higher than background controls. Thus, peptide C31 to 45 is a verified HLADR restricted CD4 T cell epitope. It was there, additional expanded PBMCs remaining after epitope identification.Fine marking of the identified epitopes can be performed using a panel of shortened peptides.

Research

JoVE Journal

Immunology and Infection

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