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.
Department of Radiation Oncology, Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China.
J Leukoc Biol. 2021 Feb;109(2):455-465. doi: 10.1002/JLB.5MA0620-023RR. Epub 2020 Jul 3.
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.
乙型肝炎病毒（HBV）特异性 T 细胞在决定 HBV 感染结局方面起着关键作用。然而，由于缺乏合适的检测方法，慢性感染中主要抗原诱导的 T 细胞反应几乎无法检测到。我们在此建立了一种优化的方法来计数 HBV 特异性 T 细胞，并评估 HBV 表面抗原（HBsAg）与 HBV DNA 之间的关联。共纳入 60 例慢性 HBV 感染患者。使用覆盖 HBV 基因型 B 和 C 整个序列的重叠肽池来扩增 HBV 特异性 T 细胞。通过培养酶联免疫斑点（ELISPOT）检测、体外 ELISPOT 检测或流式细胞术染色检测 IFN-γ产生的 HBV 特异性 T 细胞。分析 HBV 特异性 T 细胞与血清 HBsAg 和 HBV DNA 水平之间的关联。与体外 ELISPOT 相比，培养 ELISPOT 在检测 HBV 特异性 T 细胞方面具有更高的灵敏度。此外，通过流式细胞术分析和培养 ELISPOT 检测获得了一致的结果，但后者仅需有限数量的细胞进行检测。有趣的是，在 HBV DNA 和 HBsAg 水平较低的患者中，HBV 核心肽池诱导了强烈的 HBV 特异性 T 细胞反应。具体而言，HBV 核心 Ag 特异性 IFN-γ斑点形成细胞的频率与血清 HBV DNA 和 HBsAg 水平呈负相关。优化的培养 ELISPOT 检测揭示了 HBV 核心 Ag 诱导的 T 细胞反应与 HBV 控制之间的关联；这种方法可能有利于慢性 HBV 感染中 HBV 特异性 T 细胞的研究。
