An interferon regulatory factor element controls the major immune modulator of murine cytomegalovirus

Immune modulation by herpesviruses, such as cytomegalovirus, is critical for the establishment of acute and persistent infection confronting a vigorous antiviral immune response of the host. Therefore, the action of immune-modulatory proteins has long been the subject of research, with the final goal to identify new strategies for antiviral therapy.rnIn the case of murine cytomegalovirus (mCMV), the viral m152 protein has been identified to play a major role in targeting components of both the innate and the adaptive immune system in terms of infected host-cell recognition in the effector phase of the antiviral immune response. On the one hand, it inhibits cell surface expression of RAE-1 and thereby prevents ligation of the activating natural killer (NK)-cell receptor NKG2D. On the other hand, it decreases cell surface expression of peptide-loaded MHC class I molecules thereby preventing antigen presentation to CD8 T cells. Ultimately, the outcome of CMV infection is determined by the interplay between viral and cellular factors.rnIn this context, the work presented here has revealed a novel and intriguing connection between viral m152 and cellular interferon (IFN), a key cytokine of the immune system: rnthe m152 promoter region contains an interferon regulatory factor element (IRFE) perfectly matching the consensus sequence of cellular IRFEs.rnThe biological relevance of this regulatory element was first suggested by sequence comparisons revealing its evolutionary conservation among various established laboratory strains of mCMV and more recent low-passage wild-derived virus isolates. Moreover, search of the mCMV genome revealed only three IRFE sites in the complete sequence. Importantly, the functionality of the IRFE in the m152 promoter was confirmed with the use of a mutant virus, representing a functional deletion of the IRFE, and its corresponding revertant virus. In particular, m152 gene expression was found to be inhibited in an IRFE-dependent manner in infected cells. Essentially, this inhibition proved to have a severe impact on the immune-modulatory function of m152, first demonstrated by a restored direct antigen presentation on infected cells for CD8 T-cell activation. Even more importantly, this effect of IRFE-mediated IFN signaling was validated in vivo by showing that the protective antiviral capacity of adoptively-transferred, antigen-specific CD8 T cells is also significantly restored by the IRFE-dependent inhibition of m152. Somewhat curious and surprising, the decrease in m152 protein simultaneously prevented an enhanced activation of NK cells in acute-infected mice, apparently independent of the RAE-1/NKG2D ligand/receptor interaction but rather due to reduced ‘missing-self’ recognition.rnTaken together, this work presents a so far unknown mechanism of IFN signaling to control mCMV immune modulation in acute infection.rnrn

Detrimental effects of exuberant and restrained immune responses against the central nervous system in the context of multiple sclerosis and glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and most aggressive astrocytic tumor of the central nervous system (CNS) in adults. The standard treatment consisting of surgery, followed by a combinatorial radio- and chemotherapy, is only palliative and prolongs patient median survival to 12 to 15 months. The tumor subpopulation of stem cell-like glioma-initiating cells (GICs) shows resistance against radiation as well as chemotherapy, and has been suggested to be responsible for relapses of more aggressive tumors after therapy. The efficacy of immunotherapies, which exploit the immune system to specifically recognize and eliminate malignant cells, is limited due to strong immunosuppressive activities of the GICs and the generation of a specialized protective microenvironment. The molecular mechanisms underlying the therapy resistance of GICs are largely unknown. rnThe first aim of this study was to identify immune evasion mechanisms in GICs triggered by radiation. A model was used in which patient-derived GICs were treated in vitro with fractionated ionizing radiation (2.5 Gy in 7 consecutive passages) to select for a more radio-resistant phenotype. In the model cell line 1080, this selection process resulted in increased proliferative but diminished migratory capacities in comparison to untreated control GICs. Furthermore, radio-selected GICs downregulated various proteins involved in antigen processing and presentation, resulting in decreased expression of MHC class I molecules on the cellular surface and diminished recognition potential by cytotoxic CD8+ T cells. Thus, sub-lethal fractionated radiation can promote immune evasion and hamper the success of adjuvant immunotherapy. Among several immune-associated proteins, interferon-induced transmembrane protein 3 (IFITM3) was found to be upregulated in radio-selected GICs. While high expression of IFITM3 was associated with a worse overall survival of GBM patients (TCGA database) and increased proliferation and migration of differentiated glioma cell lines, a strong contribution of IFITM3 to proliferation in vitro as well as tumor growth and invasiveness in a xenograft model could not be observed. rnMultiple sclerosis (MS) is the most common autoimmune disease of the CNS in young adults of the Western World, which leads to progressive disability in genetically susceptible individuals, possibly triggered by environmental factors. It is assumed that self-reactive, myelin-specific T helper cell 1 (Th1) and Th17 cells, which have escaped the control mechanisms of the immune system, are critical in the pathogenesis of the human disease and its animal model experimental autoimmune encephalomyelitis (EAE). It was observed that in vitro differentiated interleukin 17 (IL-17) producing Th17 cells co-expressed the Th1-phenotypic cytokine Interferon-gamma (IFN-γ) in combination with the two respective lineage-associated transcription factors RORγt and T-bet after re-isolation from the CNS of diseased mice. Pathogenic molecular mechanisms that render a CD4+ T cell encephalitogenic have scarcely been investigated up to date. rnIn the second part of the thesis, whole transcriptional changes occurring in in vitro differentiated Th17 cells in the course of EAE were analyzed. Evaluation of signaling networks revealed an overrepresentation of genes involved in communication between the innate and adaptive immune system and metabolic alterations including cholesterol biosynthesis. The transcription factors Cebpa, Fos, Klf4, Nfatc1 and Spi1, associated with thymocyte development and naïve T cells were upregulated in encephalitogenic CNS-isolated CD4+ T cells, proposing a contribution to T cell plasticity. Correlation of the murine T-cell gene expression dataset to putative MS risk genes, which were selected based on their proximity (± 500 kb; ensembl database, release 75) to the MS risk single nucleotide polymorphisms (SNPs) proposed by the most recent multiple sclerosis GWAS in 2011, revealed that 67.3% of the MS risk genes were differentially expressed in EAE. Expression patterns of Bach2, Il2ra, Irf8, Mertk, Odf3b, Plek, Rgs1, Slc30a7, and Thada were confirmed in independent experiments, suggesting a contribution to T cell pathogenicity. Functional analysis of Nfatc1 revealed that Nfatc1-deficient CD4+ T cells were restrained in their ability to induce clinical signs of EAE. Nfatc1-deficiency allowed proper T cell activation, but diminished their potential to fully differentiate into Th17 cells and to express high amounts of lineage cytokines. As the inducible Nfatc1/αA transcript is distinct from the other family members, it could represent an interesting target for therapeutic intervention in MS.rn