Differential requirement for CD28 and CD80/86 pathways of costimulation in the long-term control of murine gammaherpesvirus-68

Abstract

The costimulatory molecules CD80 and CD86 (B7-1 and B7-2) are upregulated on mature antigen-presenting cells and interact with positive and negative regulators of CD8 T cell function, CD28 and CD152 (CTLA4) respectively. In this study, we examined the role of CD80 and CD86 in the immune response to murine gammaherpesvirus-68 (MHV-68) using CD80/86-/- mice. As we had previously shown that CD28 (the only known activating receptor for CD80 and 86) is not essential for long-term control of MHV-68, we predicted that CD80 and 86 would also be dispensable for an effective response to this virus. However, surprisingly, we observed that CD80/86-/- mice failed to maintain effective long-term control of MHV-68 and showed viral reactivation in the lungs. We did not observe viral reactivation in mice deficient in either CD80 or CD86 alone, indicating that these molecules play overlapping roles in the long-term control of MHV-68. Antiviral antibody responses were dramatically reduced in CD80/86-/- mice, while CD8 T cell expansion and recruitment to the lungs were not significantly affected. The unexpected disparity in the requirement for CD28 and CD80/86 in the response to MHV-68 suggests that CD28 is not the only positive regulatory receptor for CD80/86.

Figure 1. Differential requirement for CD80/86 and CD28 in the long-term control of MHV-68

Groups of 3–5 mice were infected intranasally with 10⁵PFU MHV-68. At days 16 or 50 after infection, lungs were harvested and virus titers were determined in lung homogenates by plaque assay. Data are expressed as PFU/0.1g of lung tissue from individual mice and are combined from 2 independent experiments. Horizontal bars represent the mean titer for each group. The detection limit of this assay is 10 PFU/0.1g of lung tissue.

Figure 2. CD80 and CD86 play overlapping roles in the long-term control MHV-68

Groups of 3–4 wildtype (WT), CD80−/−, CD86−/− or CD80/86−/− mice were infected with MHV-68 and lung virus titers were determined at days 7 or 50 after infection as described in the legend to Figure 1.

Figure 3. Cell numbers in the bronchoalveolar lavage (BAL) and spleens of MHV-68-infected wildtype and CD80/86−/− mice

Cell numbers in the BAL or spleen were determined at day 16 or 50 after infection with MHV-68. Single cell suspensions were prepared from the spleens of individual mice and viable cell counts were determined by Trypan Blue exclusion. Data are means ± standard deviations for groups of 3–5 mice at each time-point and are derived from 2 independent experiments for WT and CD80/86−/− mice and single experiments for the other groups. WT – wildtype; CII−/− MHC Class II −/−

Figure 4. Lymphocyte subsets in the BAL and spleens of MHV-68-infected wildtype and CD80/86−/− mice

BAL or spleen cells were collected at days 16 and 50 after infection, and stained with PE or FITC-conjugated monoclonal antibodies. The resulting populations were analyzed by flow cytometry using a lymphocyte gate. Data are means ± standard deviations for groups of 3–5 mice at each time-point and are derived from 2 independent experiments for WT and CD80/86−/− mice and single experiments for the other groups. TCR- T cell receptor.

Figure 5. Frequency of virus specific CD8 T cells is unaffected by lack of CD80/86

BAL or spleen cells were collected at days 16 and 50 after infection, and stained with PE-conjugated H-2D^bp56 or H-2K^bp79 MHC peptide tetramers and PerCP-conjugated monoclonal antibody to CD8. The resulting populations were analyzed by flow cytometry using a lymphocyte gate. Data are mean percentages of lymphocytes positive for both CD8 and tetramer staining + standard deviations, groups of 3–5 mice at each time-point and are derived from 2 independent experiments for WT and CD80/86−/− mice and single experiments for the other groups.

Figure 6. CD80 and 86 are essential for the MHV-68 specific antibody response

Serum was collected from groups of 4 wildtype or CD80/86−/− mice 50 days after infection with MHV-68. Virus specific antibody responses were determined by ELISA. Data are expressed as mean A₄₀₅ + standard deviation relative to a positive control serum sample (pooled from MHV-68-infected wildtype mice). There was a highly statistically significant difference between the antibody responses for the two groups (P<0.0001, Student’s t test).