HBV core-specific CD8 T cells were detected by staining with MHC class I multimers conjugated with HBV core-derived H-2Kb-restricted peptide C93-100 (C93, MGLKFRQL), as described previously [28 (link)].
For intracellular cytokine staining, splenocytes and liver-associated lymphocytes (LAL) were stimulated with H-2Kb-restricted peptides C93 (HBcAg (ayw), sequence: MGLKFRQL, (JPT Peptide Technologies, Berlin, Germany)) or B8R (MVA, sequence: TSYKFESV (kindly provided by Ingo Drexler, Heinrich Heine Universität Düsseldorf, Germany)] for 5h in the presence of 1 mg/mL Brefeldin A (Sigma-Aldrich, Taufkirchen, Germany). Cells were live/dead-stained with ethidium monoazidebromide (Invitrogen, Karlsruhe, Germany). Surface markers were stained with PB-conjugated anti-CD8 T cell antibody (clone 56.6-7, BD Biosciences, Heidelberg, Germany) and anti-CD4-PE (eBioscience, San Diego, USA). Intracellular cytokine staining (ICS) was performed using a Cytofix/Cytoperm Kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer’s instructions with FITC anti-IFNƴ (clone XMG1.2, eBioscience), PE-Cy7 anti-TNFα (Biolegend) and APC anti-IL2 (eBioscience). Data were acquired on a CytoflexS (Beckmann Coulter) flow cytometer. Analyses were performed using FlowJo-Version9 software (Tree Star, Ashland, OR, USA).
For intracellular cytokine staining, splenocytes and liver-associated lymphocytes (LAL) were stimulated with H-2Kb-restricted peptides C93 (HBcAg (ayw), sequence: MGLKFRQL, (JPT Peptide Technologies, Berlin, Germany)) or B8R (MVA, sequence: TSYKFESV (kindly provided by Ingo Drexler, Heinrich Heine Universität Düsseldorf, Germany)] for 5h in the presence of 1 mg/mL Brefeldin A (Sigma-Aldrich, Taufkirchen, Germany). Cells were live/dead-stained with ethidium monoazidebromide (Invitrogen, Karlsruhe, Germany). Surface markers were stained with PB-conjugated anti-CD8 T cell antibody (clone 56.6-7, BD Biosciences, Heidelberg, Germany) and anti-CD4-PE (eBioscience, San Diego, USA). Intracellular cytokine staining (ICS) was performed using a Cytofix/Cytoperm Kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer’s instructions with FITC anti-IFNƴ (clone XMG1.2, eBioscience), PE-Cy7 anti-TNFα (Biolegend) and APC anti-IL2 (eBioscience). Data were acquired on a CytoflexS (Beckmann Coulter) flow cytometer. Analyses were performed using FlowJo-Version9 software (Tree Star, Ashland, OR, USA).
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