(A) WT, B7.1^−/−, B7.2^−/−, B7.1/2^−/−, CD28^−/− and WT mice treated with blocking B7.1 and B7.2 antibodies (αB7.1/2) were infected with MCMV and 8 days later the virus-specific CD8 T cell response was analyzed by intracellular cytokine staining after restimulation with the indicated class I epitopes. Graphs show the total number of IFN-γ⁺ CD8 T cells in the spleen. (B) 1 × 10⁴ congenic (CD45.1) WT OVA-specific TCR transgenic CD8 T cells (OT-I) or CD28^−/− OT-I cells or (C) 1 × 103 CD45.1 WT OT-I or B7.1/2^−/− OT-I cells were adoptively transferred into WT (CD45.2) mice that were subsequently infected with MCMV-OVA. The number of transgenic T cells was determined in the spleen 8 days following transfer. Statistical significance was determined by Student’s t test (** indicates p<0.005). Fold difference is indicated. All bar graphs in this figure are shown as means with standard errors and each symbol (○) represents an individual mouse. Data shown are representative of two independent experiments.

WT and B7.1/2^−/− mice were infected with MCMV. (A) Flow plots show the cell surface phenotype of M45- and M38-tetramer positive (Tet+) CD8 T cells at day 8 post-infection. CD8 T cells were analyzed for tetramer reactivity, CD44, CD62L and CD127 expression. (B) The cell surface phenotype of M45-, M38- and IE3-Tet+ positive CD8 T cells was analyzed at day 40 post-infection. Plots were gated on tetramer positive CD8 T cells and analyzed for CD27 and CD62L expression. (C) At day 100 post-infection, MCMV-specific CD8 T cells were analyzed for tetramer binding or intracellular expression of IFN-γ after restimulation with the indicated class I epitopes. Graph shows the percentage of Tet+ versus IFN-γ+ CD8 T cell population at day 100 post-infection. (D) At day 100 post-infection, M45- and IE3-Tet+ CD8 T cells in the spleen were analyzed for expression of PD-1 and CD27. Data shown are representative of two independent experiments.

WT and B7.1/2^−/− mice were infected with MCMV. (A) At day 8 post-infection BrdU staining was performed after in vivo labeling with BrdU (1 mg/ml in drinking water) for 7 days in combination with intracellular cytokine staining after restimulating splenocytes with the indicated class I epitopes. Plots are gated on CD8 T cells and show BrdU expression versus IFN-γ reactivity. (B) At day 40 post-infection the intracellular expression of the cell cycle marker Ki67 was analyzed on M45-, M38- or IE3-tetramer positive (Tet+) CD8 T cells by flow cytometry. Histograms are gated on splenic CD8 Tet+ cells. Data shown are representative of two independent experiments.

WT and B7.1/2^−/− mice were infected with MCMV and 8 days later the virus-specific CD8 T cell response was analyzed by intracellular cytokine staining after restimulation with the indicated class I epitopes. (A and B) Representative flow plots showing the percentage of CD8⁺ T cells producing TNF and IFN-γ in (A) spleen, (B) liver or lung. (C) Graphs depict the total number of IFN-γ⁺ CD8 T cells in the spleen. Numbers are shown as means with standard errors (n=5-7 per group) and each symbol (○) represents an individual mouse. (D) Groups of WT and B7.1/2^−/− MCMV-infected mice received anti-CD25 mAb (clone PC61) to deplete Tregs. Graphs show the total number of IFN-γ⁺ CD8 T cells in the spleen at day 8 post-infection. All bar graphs are shown as means with standard errors (n=5 per group). Statistical significance was determined by Student’s t test (* indicates p<0.05 and ** p<0.005). Data shown are representative of two independent experiments.

(A) WT and B7.1/2^−/− mice were infected with MCMV. Graphs depict the time course analysis of the total numbers of IFN-γ producing MCMV-specific CD8 T cells in the spleen at day 8, 15, 35 and 100 after infection. IFN-γ production was determined by intracellular cytokine staining after restimulation with the MHC class I-restricted peptides M45, M38, m139 and IE3. Data are shown as means with standard errors (5-8 mice per time point). (B) Time course analysis of viral titers in the salivary glands at day 15, 35 and 100 post-infection. Data are shown as means with standard errors (5-8 mice per time point). Bar graph shows MCMV DNA levels in the lung at day 15 post-infection as means with standard errors and each symbol (○) represents an individual mouse. (C) MCMV-infected WT mice were treated with blocking B7.1 and B7.2 antibodies (αB7.1/2) or with control IgG from day 12 to day 20 or from day 20 to 30 post-infection. Graphs show the total number of IFN-γ⁺ CD8 T cells in the spleen at day 20 (αB7.1/2 blockade day 12-20) or day 30 (αB7.1/2 blockade day 20-30) post-infection after restimulation with the class I peptide epitopes M45, m139, M38 and IE3. Bar graphs are shown as means with standard errors (n=5 mice). Statistical significance of viral titers was determined with the Mann-Whitney test and statistical significance of T cell responses was determined with the two-tailed Student’s t test. All data are the average for 4-8 mice per time point. Differences between groups were considered significant at p-values <0.05 (* indicates p<0.05 and ** p<0.005). Data shown are representative of two independent experiments.

WT and B7.1/2^−/− mice were infected with MCMV. At day 100 post-infection, MCMV-specific CD8 T cells were analyzed for intracellular expression of IFN-γ, TNF and IL-2 after restimulation with the indicated class I epitopes. Flow cytometry plots are gated on IFN-γ⁺ CD8 T cells and show the percentage of TNF and/or IL-2 production within this population. Graphs show the percentage of IL-2⁺ or TNF⁺ cells within the IFN-γ⁺ CD8 T cell population. Similar results were found at day 15 and day 40 post-infection. Statistical significance was determined by Student’s t test (* indicates p<0.05 and ** p<0.005). Bar graphs are shown as means with standard errors (n=5 per group). Data shown are representative of two independent experiments.

Peptide-pulsed target cells were differentially labeled with two separate fluorescent dyes in order to track 4 separate populations (no peptide, M45-, M38- and IE3-peptide pulsed), and were transferred into uninfected mice (naïve control) or into WT and B7.1/2^−/− mice infected with MCMV for 8 or 40 days. The top panels represent a typical analysis of target cells recovered from the spleen after transfer (shown is day 8). Numbers indicate the percent killing of labeled targets after normalization (see methods). Graphs summarize the killing efficiency and show the averaged values. 4-7 mice were used per group and each experiment was repeated twice. Statistical significance was determined by Student’s t test (* indicates p<0.05 and ** p<0.005). Data shown are representative of two independent experiments.