Optimized Ex Vivo Fluorospot Assay to Measure Multifunctional HBV-Specific T Cell Frequencies in Chronic Hepatitis B Patients

Conan Chua; Mireia García-López; Adam J Gehring

doi:10.1007/978-1-0716-4027-2_20

Optimized Ex Vivo Fluorospot Assay to Measure Multifunctional HBV-Specific T Cell Frequencies in Chronic Hepatitis B Patients

Methods Mol Biol. 2024:2837:227-240. doi: 10.1007/978-1-0716-4027-2_20.

Authors

Conan Chua^{1

2}, Mireia García-López³, Adam J Gehring^{4

5

6}

Affiliations

¹ Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.
² Toronto Centre for Liver Disease, Toronto General Research Institute, University Health Network, Toronto, ON, Canada.
³ Liver Unit, Hospital Clínic, University of Barcelona, IDIBAPS, CIBERehd, Barcelona, Spain.
⁴ Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada. [email protected].
⁵ Toronto Centre for Liver Disease, Toronto General Research Institute, University Health Network, Toronto, ON, Canada. [email protected].
⁶ Department of Immunology, University of Toronto, Toronto, ON, Canada. [email protected].

PMID: 39044089
DOI: 10.1007/978-1-0716-4027-2_20

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.

Keywords: ELISpot; HBV; Human; Immune monitoring; Immunotherapy.

MeSH terms

Enzyme-Linked Immunospot Assay* / methods
Hepatitis B virus* / immunology
Hepatitis B, Chronic* / immunology
Hepatitis B, Chronic* / virology
Humans
Interferon-gamma / immunology
Interferon-gamma / metabolism
Leukocytes, Mononuclear / immunology
Leukocytes, Mononuclear / metabolism
Leukocytes, Mononuclear / virology
T-Lymphocytes* / immunology

Substances

Interferon-gamma