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Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells
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A method for detecting hepatitis C envelope specific memory B cells from multiple genotypes using cocktail E2

Bing-Ru Wu1, Auda A Eltahla1, Elizabeth Keoshkerian1

  • 1School of Medical Sciences and the Kirby Institute, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia.

Journal of Immunological Methods
|June 22, 2019
PubMed
Summary
This summary is machine-generated.

Researchers developed a new tetramer staining method to detect Hepatitis C virus (HCV)-specific B cells. This sensitive technique aids in understanding B cell responses crucial for clearing HCV infection.

Keywords:
E2-specific B cellsHCVMonoclonal antibodies and hybridoma cellsTetramer

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Area of Science:

  • Immunology
  • Virology
  • Biotechnology

Background:

  • Hepatitis C virus (HCV) rapidly mutates, often causing chronic infection and liver disease.
  • Early neutralizing antibodies correlate with HCV clearance, but HCV-specific B cell characteristics remain poorly understood due to limited detection tools.
  • HCV-specific B cells are present at very low frequencies, hindering their study.

Purpose of the Study:

  • To develop and optimize a sensitive tetramer staining method for detecting Hepatitis C virus (HCV)-specific B cells.
  • To enable detailed investigation of B cell responses in primary HCV infection and their correlation with clinical outcomes.
  • To establish a robust tool for future research on B cell immunity against HCV.

Main Methods:

  • Developed and optimized tetramer staining using a cocktail of recombinant HCV Envelope-2 (rE2) glycoproteins (genotypes 1a and 3a) and streptavidin dyes.
  • Determined optimal weight-to-weight ratios for streptavidin-phycoerythrin (PE) and rE2 proteins for sensitive detection.
  • Validated specificity using HCV E2-specific hybridoma cell lines, peripheral blood mononuclear cells (PBMCs) from HCV-infected individuals, and single-cell sorted B cells.

Main Results:

  • Detected HCV E2-specific B cells (CD19+CD20+CD10-IgD-tetramer+) in 87.8% of 33 subjects with chronic infection or previous clearance.
  • Mean frequency of detected HCV E2-specific B cells was 0.45% (range: 0.012-2.20%).
  • Monoclonal antibodies synthesized from sorted cells showed high reactivity (87.5%) to E2 protein, confirming tetramer specificity.

Conclusions:

  • Developed a sensitive and robust tetramer staining method for detecting low-frequency HCV E2-specific B cells.
  • This method is suitable for future studies investigating B cell responses against the HCV Envelope protein.
  • The findings pave the way for better understanding B cell roles in HCV infection outcomes.