Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.
Toronto Centre for Liver Disease, Toronto General Research Institute, University Health Network, Toronto, ON, Canada.
Methods Mol Biol. 2024;2837:227-240. doi: 10.1007/978-1-0716-4027-2_20.
Virus-specific T cells are critical to mediating viral control; however, Hepatitis B virus (HBV)-specific T cells among chronic Hepatitis B (CHB) patients are functionally exhausted. The inability to consistently measure the ex vivo functionality of HBV-specific T cells has prevented meaningful analysis during antiviral events such as HBeAg seroconversion, hepatic flares, and HBsAg loss. We optimized the traditional IFN-γ ELISpot assay to measure total ex vivo HBV-specific T cell frequencies using CHB PBMCs stimulated with HBV overlapping peptide (OLP) pools. This was then further adapted to assess individual antigen specificity (core, envelop, polymerase, X) and multifunctional HBV-specific T cells using a 3-analyte FluoroSpot assay. This protocol encompasses two major components: (1) PBMC handling/stimulation and (2) assay plate preparation and spot development. By performing this assay, ex vivo CHB patient T cell responses could be assessed longitudinally during immunotherapy or other important clinical events.
病毒特异性 T 细胞对于介导病毒控制至关重要；然而，慢性乙型肝炎 (CHB) 患者中的乙型肝炎病毒 (HBV) 特异性 T 细胞功能耗竭。由于无法在抗病毒事件（如 HBeAg 血清学转换、肝发作和 HBsAg 丢失）期间始终如一地测量 HBV 特异性 T 细胞的体外功能，因此无法进行有意义的分析。我们优化了传统的 IFN-γ ELISpot 测定法，使用 HBV 重叠肽 (OLP) 池刺激 CHB PBMCs 来测量总体外 HBV 特异性 T 细胞频率。然后，使用 3 分析物 FluoroSpot 测定法进一步评估个体抗原特异性（核心、包膜、聚合酶、X）和多功能 HBV 特异性 T 细胞。该方案包括两个主要部分：(1) PBMC 处理/刺激和 (2) 测定板制备和斑点发展。通过进行该测定，可以在免疫治疗或其他重要临床事件期间对体外 CHB 患者 T 细胞反应进行纵向评估。
