Optimized Ex Vivo Fluorospot Assay to Measure Multifunctional HBV-Specific T Cell Frequencies in Chronic Hepatitis B Patients.
Methods Mol Biol
; 2837: 227-240, 2024.
Article
em En
| MEDLINE
| ID: mdl-39044089
ABSTRACT
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Linfócitos T
/
Vírus da Hepatite B
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Hepatite B Crônica
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ELISPOT
Limite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Ano de publicação:
2024
Tipo de documento:
Article