使用培养的ELISPOT方法鉴定HBcAg特异性T细胞与慢性HBV感染中的

StephenW

Identification of the Association Between HBcAg-specific T Cell and Viral Control in Chronic HBV Infection Using a Cultured ELISPOT Assay
Chengcong Chen  1   2 , Xiaotao Jiang  3   4 , Xuan Liu  5 , Ling Guo  1 , Weibin Wang  1 , Shuqin Gu  1 , Chunhua Wen  1 , Xuan Yi  1 , Libo Tang  1 , Yongyin Li  1
Affiliations
Affiliations

    1
    State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China.
    2
    Department of Radiation Oncology, Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China.
    3
    Department of Immunology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.
    4
    Guangdong Provincial Key Laboratory of Proteomic, Guangzhou, China.
    5
    Department of Pediatrics, Nanfang Hospital, Southern Medical University, Guangzhou, China.

    PMID: 32620046 DOI: 10.1002/JLB.5MA0620-023RR

Abstract

Hepatitis B virus (HBV)-specific T cells play a critical role in determining the outcome of HBV infection. However, T cell response induced by predominant Ag in chronic infection is hardly detectable owing to the lack of a suitable assay. We herein established an optimized method to enumerate HBV-specific T cells and assessed the association between HBV surface Ag (HBsAg) and HBV DNA. Sixty chronic HBV infection patients were enrolled. HBV-specific T cells were expanded by using overlapping peptide pools covering the entire sequence of HBV genotypes B and C. IFN-γ-producing HBV-specific T cells were detected by a cultured enzyme-linked immunospot (ELISPOT) assay, ex vivo ELISPOT assay, or flow cytometry staining. The association between HBV-specific T cells and serum levels of HBsAg and HBV DNA were analyzed. Cultured ELISPOT assay had a higher sensitivity than ex vivo ELISPOT in the detection of HBV-specific T cells. Moreover, consistent results were acquired by flow cytometry analysis and cultured ELISPOT assay, but the latter required only a limited number of cells for detection. Interestingly, HBV core peptide pool induced a robust HBV-specific T cell response in patients with lower levels of HBV DNA and HBsAg. Specifically, the frequency of HBV core Ag-specific IFN-γ+ spot-forming cells was inversely correlated with serum levels of HBV DNA and HBsAg. An optimized cultured ELISPOT assay reveals the association between HBV core Ag-induced T cell response and HBV control; this method may favor the investigation of HBV-specific T cell in chronic HBV infection.

Keywords: ELISPOT; HBV; HBcAg; T cells.

©2020 Society for Leukocyte Biology.

StephenW

使用培养的ELISPOT方法鉴定HBcAg特异性T细胞与慢性HBV感染中的病毒控制之间的关联
陈成聪1 2，姜小涛3 4，宣柳5，郭国1，王卫斌1，书琴古1，春华文1，宣义1，唐荔波1，李永银1
隶属关系
隶属关系

    1个
    南方医科大学附属南方医院传染病科，器官衰竭研究国家重点实验室，广东省病毒性肝炎研究重点实验室，广州。
    2
    广州医科大学附属肿瘤医院放射肿瘤科，广州。
    3
    南方医科大学基础医学院免疫学教研室，广州
    4
    广东省蛋白质组学重点实验室，广州。
    5
    南方医科大学附属南方医院儿科，广州

    PMID：32620046 DOI：10.1002 / JLB.5MA0620-023RR

抽象

乙肝病毒（HBV）特异性T细胞在决定HBV感染的结局中起关键作用。然而，由于缺乏合适的检测方法，在慢性感染中主要由Ag诱导的T细胞反应很难检测到。我们在本文中建立了枚举HBV特异性T细胞的优化方法，并评估了HBV表面Ag（HBsAg）和HBV DNA之间的关联。招募了60名慢性HBV感染患者。通过使用覆盖整个HBV基因型B和C的整个序列的重叠肽库扩增HBV特异性T细胞。通过培养的酶联免疫斑点法（ELISPOT），离体ELISPOT检测出产生IFN-γ的HBV特异性T细胞。分析或流式细胞仪染色。分析了HBV特异性T细胞与血清HBsAg和HBV DNA的相关性。培养的ELISPOT分析在检测HBV特异性T细胞方面比离体ELISPOT具有更高的灵敏度。此外，通过流式细胞仪分析和培养的ELISPOT分析获得了一致的结果，但是后者仅需要有限数量的细胞进行检测。有趣的是，HBV核心肽库在HBV DNA和HBsAg水平较低的患者中引起了强烈的HBV特异性T细胞应答。具体而言，HBV核心银特异性IFN-γ+点形成细胞的频率与血清HBV DNA和HBsAg呈负相关。优化的培养ELISPOT分析揭示了HBV核心银诱导的T细胞反应与HBV控制之间的联系。这种方法可能有助于研究慢性HBV感染中HBV特异性T细胞。

关键字：ELISPOT；乙肝病毒乙肝表面抗原T细胞。

©2020年白细胞生物学学会。