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

In agreement with these data, our histopathological analysis of C57BL/6 mice two days after i.n. inoculation with MVA revealed a marked peribronchiolar and perivascular infiltrate of leukocytes in the infected lung sections compared to lung sections of mock inoculations with PBS ( Figure S1 ). Histopathological inspection at higher magnification clearly showed neutrophil granulocytes and macrophages among the infiltrate ( Figure S2 ). We further characterized the infiltrated leukocytes by bronchoalveolar lavages (BAL) at different time-points after i.n. administration of MVA. During the first 72 hours post infection (h p. i.) the infiltrates mainly consisted of monocytes, dendritic cells (DC), neutrophils, and NK cells ( Figure S3 ), suggesting that the lung environment is highly favorable for antigen presentation and induction of adaptive responses. Indeed, we were able to observe increasing amounts of T and B cells in the BAL fluids of MVA exposed animals at later time points of infection ( Figure 1 ). We detected CD4 and CD8 positive (+) T cells starting 48 h p. i., which increased in numbers to 20.5% (CD4+) and 41.5% (CD8+) of total BAL cells on day 6 p. i. ( Figure 1A ). To monitor VACV-specific CD8+ T cell responses we used the K b -restricted immunodominant determinant TSYKFESV from the VACV B8 protein being referred to as B8R [20] [21] [22] [23] [24] [25] [26] [27] [48] The immunodominance of B8R [20] [21] [22] [23] [24] [25] [26] [27] has been shown to be conserved for various orthopoxviruses including ECTV and VACV, and even in mice lacking IFN-c or perforin [48, 49] . In MVA, the B8R open reading frame lacks some nucleotides compared to the B8R gene sequence of conventional VACV strain Lister/Elstree and encodes for a truncated B8 polypeptide. Importantly, MVA is expected to produce a fully conserved N-terminal part of the B8 protein containing the peptide epitope B8R 20-27 and very similar expression levels of this specific B8R product were found for MVA and conventional VACV strain Elstree ( Figure S4 ). When performing intracellular cytokine staining for interferon gamma (IFN-c) we found proportionally high numbers of activated VACV (B8R 20-27 epitope)-specific CD8+ T cells in BAL liquids by day 5 p. i., but comparatively lower numbers of VACV-specific CD8+ T cells in the spleen. This pattern was also observed by day 7 p. i., with about three times higher numbers of specific CD8+ T cells in BAL than in the spleen ( Figure 1B ). When determining B220+ CD3-B cells in the BAL cell population we could detect the presence of substantial numbers of B cells at day 6 p. i. ( Figure 1C ). Additionally, we also monitored for the presence of MVA-specific