B7-mediated costimulation is required to control MCMV replication. (A) WT and B7.1/2^−/− mice were infected with MCMV, and 3 days later PFU were determined in the spleen, lung, and liver. (B) At day 15 postinfection PFU in the liver and salivary glands of MCMV-infected WT and B7.1/2^−/− mice were determined. Dashed line represents detection limit (d.l.). (C) MCMV-infected WT and B7.1/2^−/− mice were antibody depleted of CD8 or CD4 T cells (αCD8/CD4) or treated with blocking B7.1 and B7.2 MAbs (αB7.1/2). Graphs depict MCMV PFU present in salivary glands 35 days postinfection. All bar graphs display means with standard errors, and each symbol (○) represents an individual mouse. Data shown are representative of two independent experiments, and statistical significance was analyzed using Mann-Whitney tests (*, P < 0.05; **, P < 0.005).

Both B7.1 and B7.2 promote the expansion of MCMV-specific CD4 T cells. (A) Graphs depict the total number of IFN-γ⁺ CD4 T cells in the spleen of MCMV-infected WT, B7.1^−/−, B7.2^−/−, and B7.1/2^−/− and CD28^−/− mice or WT mice treated with blocking B7.1 and B7.2 antibodies (αB7.1/2). (B) Congenically marked (CD90.1) WT and CD28^−/− OVA-specific TCR transgenic CD4 T cells (OT-II) were adoptively transferred into 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 (**, P < 0.005). Fold difference is indicated. Bar graphs are shown as means with standard errors, and each symbol (○) represents an individual mouse. Data shown are representative of two independent experiments.

Dispensable role of B7-CD28 for CD4 T cells at later times of MCMV infection. MCMV-infected WT mice were treated with blocking B7.1 and B7.2 antibodies (αB7.1/2) from day 12 to day 20 postinfection. Graph shows the total number of IFN-γ⁺ CD4 T cells in the spleen at day 20 after restimulation with m09-, M25-, and m142-derived peptide epitopes. Bar graphs are shown as means with standard errors, and each symbol (○) represents an individual mouse. Experiment was performed twice with similar results.

Optimal MCMV-specific CD4 T cell responses are dependent on B7-mediated costimulation. WT and B7.1/2^−/− mice were infected with MCMV. (A) Flow cytometric plots represent the percentage of gated splenic CD4 T cells of WT or B7.1/2^−/− mice producing IFN-γ and TNF after restimulation with the MHC class II restricted peptide epitopes derived from the M25, m139, m141, m142, and m18 proteins at day 8 postinfection. Restimulation with the m09 peptide epitope was performed at day 40 postinfection and with vehicle-stimulated splenocytes at day 8 after infection (no peptide) (B) Flow plots show gated CD4 T cells that were isolated from livers and lungs at day 8 postinfection and restimulated with the M25 peptide epitope. Data shown are representative of two independent experiments.

Impact of CTLA-4 blockade on the expansion of MCMV-specific CD4 T cells in WT and B7.1/2^−/− mice. (A) MCMV-infected WT or B7.1/2^−/− mice were treated with blocking αCTLA-4 or left untreated, and 8 days later MCMV-specific CD4 T cells present in the spleen were analyzed after restimulation with class II peptide epitopes. Dot plots depict the frequency of IFN-γ-producing M25-specific CD4 T cells from spleens, and numbers represent the percentage of IFN-γ⁺ CD4⁺ cells within the total CD4 T cell population. (B) Same conditions as in panel A, and the total numbers of M25-, m139-, m141-, or m142-specific CD4 T cells producing IFN-γ⁺ are depicted. The data are representative of two independent experiments each with four or five mice per group. Numbers are shown as means with standard errors. Statistical significance was determined by Student's t test.

Restriction of B7.1 and B7.2 expression by MCMV suppresses CD4 T cell responses and enhances viral replication. (A) Bone marrow-derived dendritic cells (DCs) were treated with lipopolysaccharide (LPS) for 24 h and then infected with MCMV_GFP-WT or MCMV_GFP-Δm138/m147.5 at an MOI of ∼0.3. Cell surface expression of B7.1 and B7.2 was determined 24 h later. Thick gray histograms represent GFP⁺ DCs, thin black histograms are GFP⁻ DCs in the same culture, and shaded histograms are isotype control antibody staining. (B) Bone marrow-derived DCs were infected with MCMV_GFP-WT or MCMV_GFP-Δm138/m147.5, and 48 h later GFP⁺ DCs were adoptively transferred into WT or B7.1/2^−/− mice. Eight days later, the numbers of MCMV epitope-specific CD4 T cells (left panel) or CD8 T cells (right panel) were determined after peptide restimulation of splenocytes and intracellular cytokine staining. (C) WT and B7.1/2^−/− mice were infected with MCMV_C3X-WT or MCMV_C3X-Δm138/m147.5, and 8 days later the numbers of IFN-γ⁺ MCMV-specific CD4 T cells in spleens were determined. (D) MCMV PFU were determined in the spleen, lung, and liver at day 3 postinfection. (E) PFU were determined from livers of WT B6 mice infected with MCMV_C3X-WT or MCMV_C3X-Δm138/m147.5 at day 8 and at day 14 from salivary glands of infected BALB/c mice. Dotted line represents detection limit (d.l.). The data are representative of two independent experiments each with three to six mice per group. Bar graphs are shown as means with standard errors, and each symbol (○) represents an individual mouse. 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 t test. Differences between groups were considered significant at P values of <0.05 (*, P < 0.05; **, P < 0.005).