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  • Characterization of CD8 T Cell-Mediated Mutations in the Immunodominant Epitope GP33-41 of Lymphocytic Choriomeningitis Virus.

Characterization of CD8 T Cell-Mediated Mutations in the Immunodominant Epitope GP33-41 of Lymphocytic Choriomeningitis Virus.

Frontiers in immunology (2021-07-02)
Mark Smyth, Kseniya Khamina, Alexandra Popa, Venugopal Gudipati, Benedikt Agerer, Alexander Lercher, Lindsay Kosack, Lukas Endler, Hatoon Baazim, Csilla Viczenczova, Johannes B Huppa, Andreas Bergthaler
ABSTRACT

Cytotoxic T lymphocytes (CTLs) represent key immune effectors of the host response against chronic viruses, due to their cytotoxic response to virus-infected cells. In response to this selection pressure, viruses may accumulate escape mutations that evade CTL-mediated control. To study the emergence of CTL escape mutations, we employed the murine chronic infection model of lymphocytic choriomeningitis virus (LCMV). We developed an amplicon-based next-generation sequencing pipeline to detect low frequency mutations in the viral genome and identified non-synonymous mutations in the immunodominant LCMV CTL epitope, GP33-41, in infected wildtype mice. Infected Rag2-deficient mice lacking CTLs did not contain such viral mutations. By using transgenic mice with T cell receptors specific to GP33-41, we characterized the emergence of viral mutations in this epitope under varying selection pressure. We investigated the two most abundant viral mutations by employing reverse genetically engineered viral mutants encoding the respective mutations. These experiments provided evidence that these mutations prevent activation and expansion of epitope-specific CD8 T cells. Our findings on the mutational dynamics of CTL escape mutations in a widely-studied viral infection model contributes to our understanding of how chronic viruses interact with their host and evade the immune response. This may guide the development of future treatments and vaccines against chronic infections.

MATERIALS
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Product Description

Sigma-Aldrich
Ethidium bromide solution, BioReagent, for molecular biology, 10 mg/mL in H2O
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Tryptose Phosphate Broth solution, sterile-filtered, suitable for cell culture
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Formaldehyde solution, ACS reagent, 37 wt. % in H2O, contains 10-15% Methanol as stabilizer (to prevent polymerization)
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Sodium pyruvate solution, 100 mM, sterile-filtered, BioReagent, suitable for cell culture
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Minimum Essential Medium Eagle, With Earle′s salts, L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
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HEPES solution, 1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture