In vivo CD8⁺ T-cell activation is heightened in Ctx⁻ mice after LCMV infection. (A) Splenic CD8⁺ T cells producing IFN-γ in vivo were quantitated at the indicated times after LCMV-WE infection by direct ex vivo staining (see “Methods”). Sample dot plots of uninfected and day 8 LCMV-infected WT or prf^−/− mice are shown, with the percentage of CD8⁺ cells that are IFN-γ⁺ displayed (gated on MHC II⁻ cells, 50 000 cells displayed). The numbers of IFN-γ⁺ CD8⁺ cells at day 6 and beyond were significantly different (P < .01) between WT and prf^−/− mice (n = 6-12 per point). (B) Direct ex vivo IFN-γ staining of endogenous, Db-Gp33-specific CD8⁺ T cells was quantitated 8 days after LCMV infection by combining IFN-γ staining with MHC-tetramer staining. As a positive control, LCMV-infected mice were injected with 100 μg of Gp33 peptide 4 hours before animals were killed (⁺Gp33′ or peptide Rx). *P < .001. CD8⁺/CD4⁻ cells are shown in dot plots (n = 4). (C) Splenic CD8⁺ T cells degranulating in vivo were quantitated at the indicated times after infection by direct ex vivo staining (see “Methods”). Numbers of CD107a⁺ cells at days 6, 8, and 10 were significantly different (P < .01) between WT and prf^−/− mice (n = 6-8/point). 50 000 MHC II⁻ cells are shown in the dot plots.