Protection against polyoma virus-induced tumors is perforin-independent

Abstract

CD8 T cells are necessary for controlling tumors induced by mouse polyoma virus (PyV), but the effector mechanism(s) responsible have not been determined. We examined the PyV tumorigenicity in C57BL/6 mice mutated in Fas or carrying targeted disruptions in the perforin gene or in both TNF receptor type I and type II genes. Surprisingly, none of these mice developed tumors. Perforin/Fas double-deficient radiation bone marrow chimeric mice were also resistant to PyV-induced tumors. Anti-PyV CD8 T cells in perforin-deficient mice were found not to differ from wild type mice with respect to phenotype, capacity to produce cytokines or maintenance of memory T cells, indicating that perforin does not modulate the PyV-specific CD8 T cell response. In addition, virus was cleared and persisted to similar extents in wild type and perforin-deficient mice. In summary, perforin/granzyme exocytosis is not an essential effector pathway for protection against PyV infection or tumorigenesis.

Fig. 1

PyV clearance in wild type and PKO mice. (A) Spleens from wild type (C57BL/6) and PKO mice at the indicated days postinfection were harvested, homogenized, and titered for PyV by plaque assay. Each value represents the mean ± SEM PFU/mg from 3 individual mice and is representative of 2 experiments. *, below detection limit of 1 PFU/mg. (B) and (C) Levels of PyV DNA in the indicated organs of C57BL/6 or PKO mice during acute (B, day 8 postinfection) or persistent (C, > 40 days postinfection) phase of infection were quantitated by Taqman real-time PCR. Student’s t-test values between C57BL/6 and PKO: acute spleen p = 0.29, acute lung p = 0.30, acute heart p = 0.62; persistent spleen p = 0.31, persistent lung p < 0.001, persistent heart p = 0.03. Mean ± SEM values for 3 mice per group are shown and data are representative of 2 experiments.

Fig. 2

Visualization of the PyV-specific CD8 T cell response in PKO mice. (A) Spleen cells from PKO (■) or C57BL/6 (◆) mice at the indicated day after infection were stained ex vivo with anti-CD8α and D^b LT359 tetramers and analyzed by flow cytometry. (B) Spleen cells from PKO (■) or C57BL/6 (◆) mice at the indicated day after infection were stained ex vivo with anti-CD8α, D^b LT359 tetramers, and anti-CD127, anti-CD122, or anti-CD27 and analyzed by flow cytometry. Data are representative of 3 experiments with 3 mice per experiment.

Fig. 3

PyV-specific CD8 T cells in PKO and wild type mice are functionally similar. (A) Spleen cells from C57BL/6 or PKO mice at day 8 postinfection were stained ex vivo with anti-CD8α, D^b LT359 tetramers, and intracellularly for anti-granzyme B and analyzed by flow cytometry. Plots are gated on CD8 T cells and numbers indicate the percentage of cells in the indicated quadrant. Data are representative of 3 experiments with 3 mice per experiment. (B) Spleen cells from C57BL/6 and PKO mice at day 8 postinfection were stimulated with or without 10 μm LT359 peptide for 5 hours in the presence of BFA, then surface-stained with anti-CD8α and intracellularly stained for IFN-γ or TNF-α, and analyzed by flow cytometry. Plots are gated on live lymphocytes. Axes of the plots are log of fluorescent intensity. Data are representative of 2-3 experiments with 3 mice per experiment.

Fig. 4

Dependence of PyV-specific in vivo CTL activity on perforin and Fas. (A) LT359 peptide-pulsed and unpulsed naïve C57BL/6, lpr, or TNFR^−/− spleen cells were injected into C57BL/6 mice 7 days after PyV infection. Peptide-pulsed and unpulsed naïve spleen cells were injected into PKO mice 7 days postinfection. Peptide-pulsed and unpulsed lpr spleen cells were injected into PKO mice 7 days postinfection. (B) LT359 peptide-pulsed and unpulsed naïve C57BL/6 spleen cells were injected into C57BL/6 or PKO mice during the acute or persistent phase of the immune response. For both (A) and (B), values represent mean ± SEM percent specific lysis of peptide-pulsed spleen cells at 4 hours of 3 recipient mice in 2 separate experiments.