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

Approximately 10 5 cells were stained in 50 ml PBS supplemented with 3% FCS using monoclonal antibodies obtained from BD Biosciences (Heidelberg, Germany). Monocytes, neutrophils, dendritic cells, NK cells, B cells, and T cells were detected using APC-labeled CD11c, PerCP-Cy5.5-labeled CD11b, PE-Cy7-labeled Gr-1, PE-labeled CD49b, PE-Cy7labeled NK1.1, APC-Cy7-labeled B220, PerCp-Cy5.5-labeled CD3, PE-labeled CD4 and FITC-labeled CD8 antibodies. To ensure specificity of staining, all staining tests contained an isotype-matched control antibody. Stained cells were fixed with PBS supplemented with 0.5% paraformaldehyde and analyzed with BD LSRII and BD FACSDiva 6.0 software (BD Biosciences, Heidelberg, Germany). vaccinated (m) mice served as controls. In all experiments weight loss of individual mice was monitored daily (n = 2 to 6 per group). +indicate the individual time of death. Error bars indicate SEMs, and the numbers of surviving/total animals are given in parentheses. Data are representative of two similar experiments. Statistical significance of differences between groups is indicated by * for p-value,0.05, ** for p-value,0.01 and *** for pvalue,0.001. doi:10.1371/journal.ppat.1002557.g008