Maja Mitrović

Virus Progeny of Murine Cytomegalovirus Bacterial Artificial Chromosome pSM3fr Show Reduced Growth in Salivary Glands due to a Fixed Mutation of MCK-2

ABSTRACT Murine cytomegalovirus (MCMV) Smith strain has been cloned as a bacterial artificial chromosome (BAC) named pSM3fr and used for analysis of virus gene functions in vitro and in vivo. When sequencing the complete BAC genome, we identified a frameshift mutation within the open reading frame (ORF) encoding MCMV chemokine homologue MCK-2. This mutation would result in a truncated MCK-2 protein. When mice were infected with pSM3fr-derived virus, we observed reduced virus production in salivary glands, which could be reverted by repair of the frameshift mutation. When looking for the source of the mutation, we consistently found that virus stocks of cell culture-passaged MCMV Smith strain are mixtures of viruses with or without the MCK-2 mutation. We conclude that the MCK-2 mutation in the pSM3fr BAC is the result of clonal selection during the BAC cloning procedure.

ST2 deletion enhances innate and acquired immunity to murine mammary carcinoma

ST2 is a member of the IL‐1 receptor family and IL‐33 was recently identified as its natural ligand. The IL‐33/ST2 pathway regulates Th1/Th2 immune responses in autoimmune and inflammatory conditions, but the role of ST2 signaling in tumor growth and metastasis has not been investigated. We aimed to investigate whether ST2 gene deletion affects tumor appearance, growth, and metastasis, and antitumor immunity in an experimental metastatic breast cancer model. Deletion of ST2 in BALB/c mice bearing mammary carcinoma attenuated tumor growth and metastasis, which was accompanied by increased serum levels of IL‐17, IFN‐γ, and TNF‐α and decreased IL‐4. Tumor‐bearing ST2−/− mice had significantly higher percentages of activated CD27highCD11bhigh NK cells, CD69+ and KLRG− NK cells and higher cytotoxic activity of splenocytes, NK cells, and CD8+ T cells in vitro. A significantly higher number of NK cells expressing IFN‐γ were found in ST2−/− mice compared with WT recipients. In vivo depletion of CD8+ or NK cells revealed a key role for NK cells in enhanced antitumor immunity in ST2−/− mice. We report for the first time that suppressed breast cancer progression and metastasis in mice lacking ST2 corresponds mainly with enhanced cytotoxic activity of NK cells, and increased systemic Th1/Th17 cytokines.

Recombinant mouse cytomegalovirus expressing a ligand for the NKG2D receptor is attenuated and has improved vaccine properties

Human CMV (HCMV) is a major cause of morbidity and mortality in both congenitally infected and immunocompromised individuals. Development of an effective HCMV vaccine would help protect these vulnerable groups. NK group 2, member D (NKG2D) is a potent activating receptor expressed by cells of the innate and adaptive immune systems. Its importance in HCMV immune surveillance is indicated by the elaborative evasion mechanisms evolved by the virus to avoid NKG2D. In order to study this signaling pathway, we engineered a recombinant mouse CMV expressing the high-affinity NKG2D ligand RAE-1γ (RAE-1γMCMV). Expression of RAE-1γ by MCMV resulted in profound virus attenuation in vivo and lower latent viral DNA loads. RAE-1γMCMV infection was efficiently controlled by immunodeficient hosts, including mice lacking type I interferon receptors or immunosuppressed by sublethal γ-irradiation. Features of MCMV infection in neonates were also diminished. Despite tight innate immune control, RAE-1γMCMV infection elicited strong and long-lasting protective immunity. Maternal RAE-1γMCMV immunization protected neonatal mice from MCMV disease via placental transfer of antiviral Abs. Despite strong selective pressure, the RAE-1γ transgene did not exhibit sequence variation following infection. Together, our results indicate that use of a recombinant virus encoding the ligand for an activating NK cell receptor could be a powerful approach to developing a safe and immunogenic HCMV vaccine.

Cytomegalovirus immunoevasin reveals the physiological role of “missing self” recognition in natural killer cell dependent virus control in vivo

Natural killer cell recognition of “missing self” contributes meaningfully to control of mouse cytomegalovirus infection in vivo.

Deletion of galectin-3 in the host attenuates metastasis of murine melanoma by modulating tumor adhesion and NK cell activity

Galectin-3, a β galactoside–binding lectin, plays an important role in the processes relevant to tumorigenesis such as malignant cell transformation, invasion and metastasis. We have investigated whether deletion of Galectin-3 in the host affects the metastasis of B16F1 malignant melanoma. Galectin-3-deficient (Gal-3−/−) mice are more resistant to metastatic malignant melanoma as evaluated by number and size of metastatic colonies in the lung. In vitro assays showed lower number of attached malignant cells in the tissue section derived from Gal-3−/− mice. Furthermore, lack of Galectin-3 correlates with higher serum levels of IFN-γ and IL-17 in tumor bearing hosts. Interestingly, spleens of Gal-3−/− mice have lower number of Foxp3+ T cells after injection of B16F1 melanoma cells. Finally, we found that while CD8+ T cell and adherent cell cytotoxicity were similar, there was greater cytotoxic activity of splenic NK cells of Gal-3−/− mice compared with “wild-type” (Gal-3+/+) mice. Despite the reduction in total number of CD3ε−NK1.1+, Gal-3−/− mice constitutively have a significantly higher percentage of effective cytotoxic CD27highCD11bhigh NK cells as well as the percentage of immature CD27highCD11blow NK cells. In contrast, CD27lowCD11bhigh less functionally exhausted NK cells and NK cells bearing inhibitory KLRG1 receptor were more numerous in Gal-3+/+ mice. It appears that lack of Galectin-3 affects tumor metastasis by at least two independent mechanisms: by a decrease in binding of melanoma cells onto target tissue and by enhanced NK-mediated anti-tumor response suggesting that Galectin-3 may be considered as therapeutic target.

Innate immunity regulates adaptive immune response: lessons learned from studying the interplay between NK and CD8+ T cells during MCMV infection

Natural killer (NK) cells play a crucial role in early immune response against cytomegalovirus infection. A large and mounting body of data indicate that these cells are involved in the regulation of the adaptive immune response as well. By using mouse cytomegalovirus (MCMV) as a model, several groups provided novel insights into the role of NK cells in the development and kinetics of antiviral CD8+ T cell response. Depending on infection conditions, virus strain and the genetic background of mice used, NK cells are either positive or negative regulators of the CD8+ T cell response. At present, there is no unique explanation for the observed differences between various experimental systems used. In this review we discuss the mechanisms involved in the interplay between NK and CD8+ T cells in the early control of MCMV infection.

Natural killer cells are required for extramedullary hematopoiesis following murine cytomegalovirus infection.

The immune response against a variety of pathogens can lead to activation of blood formation at ectopic sites, a process termed extramedullary hematopoiesis (EMH). The underlying mechanisms of EMH have been enigmatic. Investigating splenic EMH in mice infected with murine cytomegalovirus (MCMV), we find that, while cells of the adaptive immune system were dispensable for EMH, natural killer (NK) cells were essential. EMH required recognition of infected cells via activating NK cell receptors Ly49H or NKG2D, and correspondingly, viral interference with NK cell recognition abolished EMH. Surprisingly, development of EMH was not induced by NK cell-derived cytokines but was dependent on perforin-mediated cytotoxicity in order to control virus spread. Spreading virus reduced the numbers of F4/80(+) macrophages that were crucial for inflammatory EMH. Hence, whereas MCMV suppresses inflammation-induced EMH, NK cells confine virus spread, thereby protecting extramedullary hematopoietic niches and facilitating EMH.