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

Spleens were isolated from euthanized C57BL/6 mice in prewarmed RPMI medium enriched with 10% fetal calf serum. Single cell suspensions were obtained by passing cells through a nylon mesh (Nybolt PA-150/38, Franz Eckert GmBH, Germany) and erythrocytes were lysed by treatment with Red blood cell lysis buffer (Sigma Aldrich, Taufkirchen, Germany). Subsequently, cells were washed with medium and passed through a 70 mm filter (Filcon, BD Biosciences, Heidelberg, Germany) and again washed with medium. For isolation of untouched T cells the spleen cell suspension was magnetically labeled using the Pan T Cell Isolation Kit (Miltenyi, Bergisch Gladbach, Germany) and isolated using an autoMacs TM separator according to the manufacturer's protocol. Purity of the isolated T cells was confirmed by flow cytometry. Rag-1 2/2 mice were injected intravenously with 2610 7 CD3+ cells per mouse two days before immunization and the engraftment was confirmed by FACS analysis of blood samples on days 4, 9, 11, 15, 21 and 37 after administration.