Critical Role of Perforin-dependent CD8+ T Cell Immunity for Rapid Protective Vaccination in a Murine Model for Human Smallpox

Kremer, Melanie; Suezer, Yasemin; Volz, Asisa; Frenz, Theresa; Majzoub, Monir; Hanschmann, Kay-Martin; Lehmann, Michael H.; Kalinke, Ulrich; Sutter, Gerd

Analysis of antigen-specific CD8+ T cells by intracellular cytokine staining Splenocytes or BAL cells from vaccinated C57BL/6 mice were stimulated for 5 h with VACV-specific peptide B8R 20-27 (TSYK-FESV) [48] or control peptide LacZ 876 (TPHPARIGL) [93] purchased from Thermo Fisher Scientific GmbH (Ulm, Germany) in the presence of GolgiStop TM (BD Biosciences, Heidelberg, Germany). Cells were blocked with anti-CD16/CD32-Fc-Block (BD Biosciences) and surface markers were stained with PacBlueconjugated anti-CD8 (BD Biosciences) and APC-conjugated anti-CD62L (BD Biosciences, Heidelberg, Germany) in the presence of Fc-Block (BD biosciences, Heidelberg, Germany) and washed twice with PBS containing 3% FCS. Intracellular cytokine staining for IFN-c production was performed with FITC anti-IFN-c (BD Biosciences, Heidelberg, Germany) using the Cytofix/Cytoperm kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer's recommendations. Data were acquired in a BD LSRII flow cytometer and analyzed with BD FACSDiva 6.0 software (BD Biosciences, Heidelberg, Germany).