Molekulare Charakterisierung der Il-9-Genexpression in T-Helfer-Typ-9-Zellen

CD4+ T-Zellen können in verschiedene T-Helferzellsubpopulationen differenzieren. Dabei hängt es von verschiedensten Milieubedingungen ab, welche Subpopulation sich ausprägt, damit die CD4+ T-Zelle durch die Sekretion verschiedenster Zytokine ihre Funktion im Immunsystem wahrnehmen kann.rnBei der Th9-Subpopulation handelt es sich um einen IL-9-produzierenden Phänotyp, welcher sich in der Anwesenheit von TGF-ß und IL-4 entwickelt39. Als treibender Transkriptionsfaktor für diese Subpopulation wurde das Protein IRF4 beschrieben45. Da dieser Transkriptionsfaktor auch für die Differenzierung weiterer Subpopulationen, wie Th2- und Th17-Zellen von Bedeutung ist30,121, stellte sich die Frage, welcher Interaktionspartner von IRF4 darüber entscheidet, welcher Subtyp sich entwickelt. Deshalb wurde in dieser Arbeit der Transkriptionsfaktor NFATc2 als möglicher Interaktionspartner für IRF4 am murinen Il9 Promotor untersucht. Allerdings zeigten Reportergen¬analysen, dass NFATc2 die IL-9-Produktion in Th9-Zellen inhibiert anstatt sie zu fördern. Th9-Zellen aus NFATc2-defizienten Tieren zeigen folglich im Vergleich zu wildtypischen Th9-Zellen sowohl nach Primär- als auch nach Restimulation eine verstärkte IL-9-Produktion. Der Faktor NFATc2 kann somit als transkriptioneller Aktivator für die IL-9-Expression in Th9-Zellen ausgeschlossen werden. In vivo wurden diese Beobachtungen dadurch untermauert, dass NFATc2-defiziente Tiere im Rahmen des Asthma bronchiale zu einer verstärkten pulmonalen Inflammation neigen und auch einen erhöhten Atemwegswiderstand nach Methacholin-Provokation aufweisen. Diese asthmatischen Symptome konnten durch Applikation eines neutralisierenden Antikörpers für IL-9 wesentlich gemildert werden. In einem B16F10-Melanommodell konnten NFATc2-defiziente Tiere gegenüber dem Wildtyp eine verbesserte anti-Tumorantwort ausprägen. Nach Gabe eines IL-9-neutralisierenden Antikörpers, wurde dieser Effekt wiederum gemildert.rnZusammenfassend lässt sich sagen, dass IRF4 nicht mit NFATc2 am murinen Il9 Promotor interagiert, um die IL-9-Expression in Th9-Zellen zu fördern. Eine NFATc2-Defizienz resultiert sogar in einer gesteigerten IL-9-Produktion, womit ein inhibitorischer Einfluss von NFATc2 in Bezug auf die IL-9-Expression in Th9-Zellen nachgewiesen werden konnte.rn

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