基于肽矩阵的 HBV 特异性 CD4 T 细胞响应和 HLA-DR 受限 CD4 T 细胞表位的识别分析
Summary
根据乙型肝炎病毒（HBV）衍生肽基质，可在识别乙肝病毒特异性CD4 T细胞表位的同时，对乙肝病毒特异性CD4 T细胞反应进行评估。
Abstract
CD4 T细胞在慢性乙型肝炎的发病机制中起着重要作用。CD4 T细胞作为多功能细胞群，根据它们分泌的细胞因子被归类为独特的功能子集：例如，CD4 T帮手1细胞的IFN-γ，CD4 T帮手2细胞的IL-4和IL-13，CD4 T卵泡辅助细胞的IL-21，CD4 T助剂细胞的IL-17。分析乙型肝炎病毒（HBV）特异性CD4 T细胞，基于细胞因子分泌后，乙肝病毒衍生肽刺激，不仅可以提供有关乙肝病毒特异性CD4 T细胞反应的大小的信息，还可以提供有关HBV特异性CD4 T细胞功能子集的信息。新的方法，如转录学和代谢学分析，可以提供关于HBV特异性CD4 T细胞的更详细的功能信息。这些方法通常需要基于肽主要组织相容性复合II多默的活性HBV特异性CD4 T细胞进行分离，而目前有关HBV特异性CD4 T细胞表位的信息有限。基于乙肝病毒衍生肽基质，开发出一种利用慢性乙肝病毒感染患者的周围血单核细胞样本同时评估HBV特异性CD4 T细胞反应和识别HBV特异性CD4 T细胞表位的方法。
Introduction
目前，有3种主要方法分析抗原特异性T细胞。第一种方法基于T细胞受体和肽（同位素）之间的相互作用。抗原特异性T细胞可直接沾染肽主要组织相容性复合物（MHC）多元体。这种方法的优点是，它可以获得可行的抗原特异性T细胞，适合下游转录学/代谢学分析。这种方法的一个局限性是，它不能提供有关整个T细胞对特定抗原的反应的信息，因为它需要验证表位肽，而目前特定抗原的识别表位数量有限。与乙肝病毒（HBV）特异性CD8T细胞表位相比，目前发现的HBV特异性CD4 T细胞表位较少，使得该方法目前不太适用于HBV特异性CD4 T细胞的分析。
第二种方法是基于抗原肽刺激3后一系列激活诱发的标记物的调高。常用的标记包括CD69，CD25，OX40，CD40L，PD-L1，4-1BB4。目前，该方法已用于分析接种疫苗的5、6、人免疫机能丧失病毒感染患者7例、严重急性呼吸系统综合症冠状病毒2例感染患者8例、9例等抗原特异性T细胞反应。与基于检测的肽-MHC多默尔不同，这种方法不受验证表位的限制，可以获得可行的细胞进行下游分析。这种方法的一个局限性是它不能提供有关抗原特异性T细胞细胞因子特征的信息。此外，一些激活的抗原非特异性细胞表达这些激活诱导标记可能有助于分析中的背景信号，这可能是一个问题，尤其是当目标抗原特异性T细胞很少时。目前，该方法在HBV特异性CD4 T细胞4上的应用有限。该方法能否以可靠的方式用于分析乙肝特异性CD4 T细胞，还有待进一步研究。
第三种方法基于抗原肽刺激后的细胞因子分泌。与激活诱导标记分析一样，此方法不受验证表位的限制。这种方法可以直接揭示抗原特异性T细胞的细胞因子特征。这种方法的灵敏度低于激活诱导标记法，因为它依赖于抗原特异性T细胞的细胞因子分泌，测试的细胞因子数量通常有限。目前，该方法在HBV特异性T细胞分析中得到广泛应用。由于细胞因子分泌HBV特异性T细胞很难通过直接前体内肽刺激10，11检测到，HBV特异性T细胞的细胞因子特征通常分析后，10天的体外肽刺激膨胀12，13，14，15，16。已利用以矩阵形式排列肽池，以方便识别抗原特异性表位17、18。结合肽基质和细胞因子分泌分析，开发出一种评估HBV特异性CD4 T细胞反应，同时识别HBV特异性CD4 T细胞表位的方法。在此协议中，将描述此方法的详细信息。HBV 核心抗原被选为本协议中演示的示例。
Protocol
Representative Results
Discussion
本协议中最关键的步骤如下： 1） 足够高生存能力的 PBMC 开始 PC 扩展：2） 适当的 PBMC 扩展环境;3） 在表位识别之前，完全去除 PBMC 培养中的残留肽池。
此协议中的所有分析都取决于 CD4 T 细胞的强劲增殖。一般来说，10天扩展后的PC数量将是初始数量的2-3倍。PBMC 的细胞数和生存能力是 PBMC 扩展的两个关键因素。如果目的只是分析HBV特异性CD4 T细胞而没有表位识别，则合理减?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学基金（81930061）、重庆市自然科学基金（cstc2019jy-bshX0039、cstc2019jcyj-zdxmX0004）和中国钥匙的支持
传染病专科项目（2018ZX10723203）。
Materials
| Albumin Bovine V (BSA) | Beyotime | ST023 | |
| APC-conjugated Anti-human TNF-α | eBioscience | 17-7349-82 | Keep protected from light |
| Benzonase Nuclease | Sigma-Aldrich | E1014 | Limit cell clumping |
| B lymphoblastoid cell lines (BLCLs) | FRED HUTCHINSON CANCER RESEARCH CENTER | IHW09126 | HLA-DRB1*0803 homozygote |
| B lymphoblastoid cell lines (BLCLs) | FRED HUTCHINSON CANCER RESEARCH CENTER | IHW09121 | HLA-DRB1*1202 homozygote |
| Cell Culture Flask (T75) | Corning | 430641 | |
| Cell Culture Plate (96-well, flat bottom) | Corning | 3599 | Flat bottom |
| Cell Culture Plate (96-well, round bottom) | Corning | 3799 | Round bottom |
| Cell Strainer | Corning | CLS431751 | Pore size 70 μm, white, sterile |
| Centrifuge Tube (15 mL) | KIRGEN | KG2611 | Sterile |
| Centrifuge Tube (50 mL) | Corning | 430829 | Sterile |
| Centrifuge, Refrigerated | Eppendorf | 5804R | |
| Centrifuge, Refrigerated | Thermo | ST16R | |
| Centrifuge, Refrigerated | Thermo | Legend Micro 21R | |
| Cytofix/Cytoperm Kit (Transcription Factor Buffer Set) | BD Biosciences | 562574 | Prepare solution before use |
| Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich | D2650 | Keep at room temperature to prevent crystallization |
| Dulbecco’s Phosphate Buffered Saline | Prepare ddH2O (1000 ml) containing NaCl (8000 mg), KCl (200 mg), KH2PO4 (200 mg), and Na2HPO4.7H2O (2160 mg). Adjust PH to 7.4. Sterilize through autoclave. | ||
| Ficoll-Paque Premium | GE Healthcare | 17-5442-03 | |
| Filter Tips (0.5-10) | Kirgen | KG5131 | Sterile |
| Filter Tips (100-1000) | Kirgen | KG5333 | Sterile |
| Filter Tips (1-200) | Kirgen | KG5233 | Sterile |
| FITC-conjugated Anti-human CD4 | BioLegend | 300506 | Keep protected from light |
| Fixable Viability Dye eFluor780 | eBioscience | 65-0865-14 | Keep protected from light |
| GolgiStop Protein Transport Inhibitor (Containing Monensin) | BD Biosciences | 554724 | Protein Transport Inhibitor |
| Haemocytometer | Brand | 718620 | |
| HBV Core Antigen Derived Peptides | ChinaPeptides | ||
| HEPES | Gibco | 15630080 | 100 ml |
| Human Serum AB | Gemini Bio-Products | 100-51 | 100 ml |
| Ionomycin | Sigma-Aldrich | I0634 | |
| KCl | Sangon Biotech | A100395-0500 | |
| KH2PO4 | Sangon Biotech | A100781-0500 | |
| LSRFortessa Flow Cytometer | BD | ||
| L-glutamine | Gibco | 25030081 | 100 ml |
| Microcentrifuge Tube (1.5 mL) | Corning | MCT-150-C | Autoclaved sterilization before using |
| Microplate Shakers | Scientific Industries | MicroPlate Genie | |
| Mitomycin C | Roche | 10107409001 | |
| Na2HPO4.7H2O | Sangon Biotech | A100348-0500 | |
| NaCl | Sangon Biotech | A100241-0500 | |
| PCR Tubes (0.2 mL) | Kirgen | KG2331 | |
| PE/Cy7-conjugated Anti-human CD8 | BioLegend | 300914 | Keep protected from light |
| PE-conjugated Anti-human IFN-γ | eBioscience | 12-7319-42 | Keep protected from light |
| Penicillin Streptomycin | Gibco | 15140122 | 100 ml |
| PerCP-Cy5.5-conjugated Anti-human CD3 | eBioscience | 45-0037-42 | Keep protected from light |
| Phorbol 12-myristate 13-acetate (PMA) | Sigma-Aldrich | P1585 | |
| Recombinant Human IL-2 | PeproTech | 200-02 | |
| Recombinant Human IL-7 | PeproTech | 200-07 | |
| RPMI Medium 1640 | Gibco | C11875500BT | 500 ml |
| Sodium pyruvate,100mM | Gibco | 15360070 | |
| Trypan Blue Stain (0.4%) | Gibco | 15250-061 | |
| Ultra-LEAF Purified Anti-human HLA-DR | BioLegend | 307648 | |
| Wizard Genomic DNA Purification Kit | Promega | A1125 |
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