Perturbation of Lytic and Latent Gammaherpesvirus Infection in the Absence of the Inhibitory Receptor CEACAM1

Control of gammaherpesvirus infections requires a complex, well orchestrated immune response regulated by positive and negative co-signaling molecules. While the impact of co-stimulatory molecules has been addressed in various studies, the role of co-inhibitory receptors has not been tested. The ITIM-bearing CEACAM1 is an inhibitory receptor expressed by a variety of immune cells, including B, T and NK cells. Using Ceacam1−/− mice, we analyzed the in vivo function of CEACAM1 during acute and latent murine gammaherpesvirus 68 (MHV-68) infection. During acute lytic replication, we observed lower virus titers in the lungs of Ceacam1−/− mice than in WT mice. In contrast, during latency amplification, Ceacam1−/− mice displayed increased splenomegaly and a higher latent viral load in the spleen. Analysis of the immune response revealed increased virus-specific antibody levels in Ceacam1−/− mice, while the magnitude of the T cell-mediated antiviral immune response was reduced. These findings suggest that inhibitory receptors can modulate the efficacy of immune responses against gammaherpesvirus infections

A Gammaherpesviral Internal Repeat Contributes to Latency Amplification

BACKGROUND: Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. CONCLUSIONS/SIGNIFICANCE: These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression

<i>Ceacam1^−/−</i> mice develop a significant weaker MHV-68-specific CD8⁺ T cell immune response during infection than WT mice.

<p>Mice were infected i.n. with 5×10⁴ PFU of MHV-68. Spleen cells (3×10⁶/mL) obtained on days 17 and 42 p.i. were restimulated with MHV-68 ORF6- and ORF61-deduced peptides prior to intracellular staining for IFN-γ. Double-positive (CD8⁺ IFN-γ⁺) cells were peptide-specific T cells. A) Dotplots of a representative experiment (ORF6 at day 17) with controls (without addition of peptides or after stimulation with anti-CD3 mAb). The mean numbers of double-positive CD8⁺ IFN-γ⁺ T cells per 10⁵ CD8⁺ spleen cells±SD of three individual mice (upper panel) and the mean number of double-positive CD8⁺ IFN-γ⁺ T cells per spleen±SD of three individual mice (lower panel) are shown. The p values for the differences between <i>Ceacam1^−/−</i> and WT mice are indicated.</p

Splenomegaly and composition of spleen cells during MHV-68 infection differ in <i>Ceacam1^−/−</i> and WT mice.

<p>A) Splenomegaly. Mice were infected i.n. with 5×10⁴ PFU of MHV-68. At the indicated time points after infection, spleen weights were determined. Data shown in panel A are means±SD of 5 mice (day 6), 11 mice (day 17), 8 mice (day 42) and 3 mice (day 300). Data from uninfected (naive) mice are shown for comparison (n = 24 for WT and n = 13 for <i>Ceacam1^−/−</i> mice). B) Mice were infected i.n. with 5×10⁴ PFU of MHV-68. At days 6, 17 and 42 after infection, the immunophenotype of spleen cells was determined by three color flow cytometry. In each experiment, three mice were used per group, and the experiments were repeated twice (day 6 p.i.) and three times (days 17 and 42 p.i.), respectively. For determination of the percentage of CD3 and CD19 positive cells, only cells within the lymphocyte forward/sideward scatter gate were accepted. For all other markers (indicated by the blue box), only CD3⁺ cells were accepted. CD62L^medium cells are not depicted. Asterisks denote statistically significant differences between WT and <i>Ceacam1^−/−</i> mice (*: p < 0.05; **: p < 0.01). C) The absolute cell numbers were calculated as follows: number of cells = number of lymphocytes per spleen×living cells (%) / 100.</p

<i>Ex vivo</i> virus reactivation and viral genomic load are increased in <i>Ceacam1^−/−</i> mice.

<p>A) E<i>x vivo</i> reactivation of splenocytes. Mice were infected i.n. with 5×10⁴ PFU of MHV-68. At the indicated time points after infection, spleens were harvested and single splenocyte suspensions were prepared and analyzed in the <i>ex vivo</i> reactivation assay. Data shown in panel A are means±SEM pooled from three independent experiments. In each experiment, splenocytes from 3 to 5 mice per group were pooled. The dashed line in panel A indicates the point of 63.2 % Poisson distribution, determined by nonlinear regression, which was used to calculate the frequency of cells reactivating lytic MHV-68 replication. To calculate significance, frequencies of reactivation events were statistically analyzed by paired t-test over all cell dilutions. The statistical significance of the difference between WT and <i>Ceacam1^−/−</i> mice is p = 0.02. B) Viral genomic load. Mice were infected i.n. with 5×10⁴ PFU of MHV-68. At days 17, 42 and 300 after infection, spleens were harvested, single splenocyte suspensions were prepared and used for DNA isolation for real time PCR analysis. The data are presented as viral genome copy numbers (as determined by quantitation of the copy number of the MHV-68 gB gene) relative to the copy number of the murine ribosomal protein L8 gene (<i>Rpl8)</i>. Data shown are means±SD of 14 mice per group at day 17, 8 mice per group at day 42 and 2–3 mice per group at day 300.</p