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Dendritic cells (DC) play a crucial role in generating and maintaining antiviral immunity. While DC are implicated in the antiviral defense by inducing T cell responses, they can also become infected by Cytomegalovirus (CMV). CMV is not only highly species-specific but also specialized in evading immune protection, and this specialization is in part due to characteristic genes encoded by a given virus. Here, we investigated whether rat CMV can infect XCR1+ DC and if infection of DC alters expression of cell surface markers and migration behavior. We demonstrate that wild-type RCMV and a mutant virus lacking the γ-chemokine ligand xcl1 (Δvxcl1 RCMV) infect splenic rat DC ex vivo and identify viral assembly compartments. Replication-competent RCMV reduced XCR1 and MHCII surface expression. Further, gene expression of infected DC was analyzed by bulk RNA-sequencing (RNA-Seq). RCMV infection reverted a state of DC activation that was induced by DC cultivation. On the functional level, we observed impaired chemotactic activity of infected XCR1+ DC compared to mock-treated cells. We therefore speculate that as a result of RCMV infection, DC exhibit diminished XCR1 expression and are thereby blocked from the lymphocyte crosstalk.

Viral vectors have emerged as a promising alternative to classical vaccines due to their great potential for induction of a potent cellular and humoral immunity. Cytomegalovirus (CMV) is an attractive vaccine vector due to its large genome with many non-essential immunoregulatory genes that can be easily manipulated to modify the immune response. CMV generates a strong antigen-specific CD8 T cell response with a gradual accumulation of these cells in the process called memory inflation. In our previous work, we have constructed a mouse CMV vector expressing NKG2D ligand RAE-1γ in place of its viral inhibitor m152 (RAE-1γMCMV), which proved to be highly attenuated in vivo. Despite attenuation, RAE-1γMCMV induced a substantially stronger CD8 T cell response to vectored antigen than the control vector and provided superior protection against bacterial and tumor challenge. In the present study, we confirmed the enhanced protective capacity of RAE-1γMCMV as a tumor vaccine vector and determined the phenotypical and functional characteristics of memory CD8 T cells induced by the RAE-1γ expressing MCMV. RNAseq data revealed higher transcription of numerous genes associated with effector-like CD8 T cell phenotype in RAE-1γMCMV immunized mice. CD8 T cells primed with RAE-1γMCMV were enriched in TCF1 negative population, with higher expression of KLRG1 and lower expression of CD127, CD27, and Eomes. These phenotypical differences were associated with distinct functional features as cells primed with RAE-1γMCMV showed inferior cytokine-producing abilities but comparable cytotoxic potential. After adoptive transfer into naive hosts, OT-1 cells induced with both RAE-1γMCMV and the control vector were equally efficient in rejecting established tumors, suggesting the context of latent infection and cell numbers as important determinants of enhanced anti-tumor response following RAE-1γMCMV vaccination. Overall, our results shed new light on the phenotypical and functional distinctness of memory CD8 T cells induced with CMV vector expressing cellular ligand for the NKG2D receptor.

The development of a vaccine against human cytomegalovirus (CMV) has been a subject of long-term medical interest. The research during recent years identified CMV as an attractive vaccine vector against infectious diseases and tumors. The immune response to CMV persists over a lifetime and its unique feature is the inflationary T cell response to certain viral epitopes. CMV encodes numerous genes involved in immunoevasion, which are non-essential for virus growth in vitro. The deletion of those genes results in virus attenuation in vivo, which enables us to dramatically manipulate its virulence and the immune response. We have previously shown that the murine CMV (MCMV) expressing RAE-1γ, one of the cellular ligands for the NKG2D receptor, is highly attenuated in vivo but retains the ability to induce a strong CD8+ T cell response. Here, we demonstrate that recombinant MCMV expressing high affinity NKG2D ligand murine UL16 binding protein-like transcript (MULT-1) (MULT-1MCMV) inserted in the place of its viral inhibitor is dramatically attenuated in vivo in a NK cell-dependent manner, both in immunocompetent adult mice and in immunologically immature newborns. MULT-1MCMV was more attenuated than the recombinant virus expressing RAE-1γ. Despite the drastic sensitivity to innate immune control, MULT-1MCMV induced an efficient CD8+ T cell response to viral and vectored antigens. By using in vitro assay, we showed that similar to RAE-1γMCMV, MULT-1 expressing virus provided strong priming of CD8+ T cells. Moreover, MULT-1MCMV was able to induce anti-viral antibodies, which after passing the transplacental barrier protect offspring of immunized mothers from challenge infection. Altogether, this study further supports the concept that CMV expressing NKG2D ligand possesses excellent characteristics to serve as a vaccine or vaccine vector.