TY - THES A1 - Dirks, Johannes T1 - Charakterisierung der Wechselwirkung zwischen N-Myc und Aurora-A im MYCN-amplifizierten Neuroblastom T1 - Characterization of the Interaction between N-Myc and Aurora-A in MYCN amplified Neuroblastomas N2 - Im Neuroblastom ist die Amplifikation des MYCN-Gens, eines Mitglieds der MYC-Onkogenfamilie, mit einer ungünstigen Prognose assoziiert. Der von dem Gen kodierte Transkriptionsfaktor N-Myc ist für die Proliferation der MYCN-amplifizierten Neuroblastomzelllinien notwendig und seine Depletion oder Destabilisierung führen zum Proliferationsarrest (Otto et al., 2009). Da N-Myc auf Proteinebene durch die Interaktion mit der mitotischen Kinase Aurora-A stabilisiert wird, bewirkt deren Depletion oder die Hemmung der Interaktion der beiden Proteine mittels spezieller Aurora- A-Inhibitoren (z.B. MLN8054 und MLN8237) ebenso eine Hemmung der Proliferation – in vitro und in vivo (Brockmann et al., 2013). Bisher ist jedoch unklar, über welchen Mechanismus Aurora-A die Stabilisierung von N-Myc erreicht, die Kinaseaktivität spielt hierbei jedoch keine Rolle (Otto et al., 2009). Eine Möglichkeit stellt die Rekrutierung von Usps dar, die das angehängte Ubiquitinsignal so modifizieren, dass die Erkennung und der Abbau des Proteins durch das Proteasom verringert werden. In der vorliegenden Arbeit wurde die Wirkung von Usp7 und Usp11 auf die Stabilität von N-Myc untersucht. Für beide konnte in Immunpräzipitationen die Interaktion mit N-Myc gezeigt werden. Ebenso erhöhten beide Proteasen in Überexpressionsexperimenten die vorhandene Menge an NMyc. Die Depletion von Usp7 mittels shRNAs führte in IMR-32 zu einem Arrest in der G1-Phase und zur Differenzierung der Zellen. Gleichzeitig wurden stark erniedrigte mRNA- und Proteinmengen von N-Myc und Aurora-A nachgewiesen. Es konnte jedoch nicht eindeutig gezeigt werden, ob die beobachteten zellulären Effekte durch eine vermehrte proteasomale Degradation von N-Myc begründet sind oder ob dabei die veränderte Regulation weiterer Zielproteine von Usp7 eine Rolle spielt. Die Depletion von Usp11 mit shRNAs bewirkte eine Abnahme der N-Myc-Mengen auf posttranslationaler Ebene. Somit stellen beide Usps vielversprechende Angriffspunkte einer gezielten Therapie in MYCN-amplifizierten Neuroblastomen dar und sollten deshalb Gegenstand weiterführender Untersuchungen sein. Über welche Proteindomäne in N-Myc die Interaktion mit Aurora-A stattfindet ist nicht bekannt. Eine mögliche Pseudosubstratbindungssequenz in Myc-Box I (Idee Richard Bayliss, University of Leicester) wurde in der vorliegenden Arbeit untersucht. Durch Mutation dieser Sequenz sollte die Bindung von Aurora-A unmöglich gemacht werden. Allerdings wurde die erwartete Abnahme der Stärke der Interaktion von Aurora-A und N-Myc durch die Mutation ebensowenig beobachtet wie eine verringerte Stabilität. Die Regulation der Phosphorylierung von N-Myc im Verlauf des Zellzyklus wurde durch die Mutation beeinträchtigt. Wie diese Veränderung exakt zu begründen ist bedarf weiterer Experimente N2 - Neuroblastomas with an amplification of the MYCN-gene, a member of the MYC-oncogene family, are associated with a poor prognosis. The transcription factor encoded by this gene, N-Myc, is essential for the proliferation of MYCN-amplified neuroblastoma cell lines and its depletion or destabilization leads to an arrest of proliferation (Otto et al., 2009). Since N-Myc is stabilized by the interaction with the mitotic kinase Aurora-A, the depletion of the kinase or the inhibition of the interaction with N-Myc using a special class of Aurora-A inhibitors (e.g. MLN8054 and MLN8237) inhibits proliferation – in vitro and in vivo (Brockmann et al., 2013). Up to date it is not known by which mechanism Aurora-A is able to stabilize N-Myc preventing it from Fbxw7-mediated proteasomal degradation, interestingly the Aurora-A kinase activity is not necessary (Otto et al., 2009). One possible explanation is the recruitment of Usps, which modify the attached ubiquitin signal and therefore reduce the recognition and degradation of the protein by the proteasome. In this thesis the influence of Usp7 and Usp11 on N-Myc stability was studied. For both the interaction with N-Myc was shown in immune precipitations. Furthermore the overexpression of both proteases increased the amount of N-Myc protein in transfection experiments. The depletion of Usp7 via shRNAs caused the arrest of IMR-32 cells in G1-phase and the differentiation of the cells. Simultaneously strongly reduced amounts of N-Myc and Aurora-A mRNA and proteins were observed. However it could not be shown that the observed effects were mediated by an increased proteasomal degradation of N-Myc and not via the changed regulation of other targets of Usp7. The depletion of Usp11 led to a decrease of the N-Myc amounts, whereas mRNA-levels were unaffected. Thus both Usps are promising targets for a targeted therapy of MYCN-amplified neuroblastomas and the underlying mechanism should be the object of further research. Furthermore the N-Myc domain binding to Aurora-A still remains to be idientified. A possible pseudosubstrate binding site in Myc-Box I (idea of Richard Bayliss, University of Leicester) was investigated in this thesis. To inhibit the possible binding of Aurora-A to this site, two lysines in Myc-Box I were mutated to glutamate (KK51EE). Nonetheless neither the expected decrease of the intensity of interaction of N-Myc and Aurora-A was observed nor was a decrease of the stability of N-Myc. The regulation of the phosphorylation of N-Myc during the cell cycle was changed through the mutagenesis however. It must be clarified in further experiments, what the reasons for this change are. KW - Neuroblastom KW - N-Myc KW - Aurora-A Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186600 ER - TY - JOUR A1 - Dirks, Johannes A1 - Fischer, Jonas A1 - Haase, Gabriele A1 - Holl-Wieden, Annette A1 - Hofmann, Christine A1 - Girschick, Hermann A1 - Morbach, Henner T1 - CD21\(^{lo/−}\)CD27\(^−\)IgM\(^−\) Double-Negative B Cells Accumulate in the Joints of Patients With Antinuclear Antibody-Positive Juvenile Idiopathic Arthritis JF - Frontiers in Pediatrics N2 - Juvenile idiopathic arthritis (JIA) encompasses a heterogeneous group of diseases. The appearance of antinuclear antibodies (ANAs) in almost half of the patients suggests B cell dysregulation as a distinct pathomechanism in these patients. Additionally, ANAs were considered potential biomarkers encompassing a clinically homogenous subgroup of JIA patients. However, in ANA+ JIA patients, the site of dysregulated B cell activation as well as the B cell subsets involved in this process is still unknown. Hence, in this cross-sectional study, we aimed in an explorative approach at characterizing potential divergences in B cell differentiation in ANA+ JIA patients by assessing the distribution of peripheral blood (PB) and synovial fluid (SF) B cell subpopulations using flow cytometry. The frequency of transitional as well as switched-memory B cells was higher in PB of JIA patients than in healthy controls. There were no differences in the distribution of B cell subsets between ANA- and ANA+ patients in PB. However, the composition of SF B cells was different between ANA- and ANA+ patients with increased frequencies of CD21\(^{lo/−}\)CD27\(^−\)IgM\(^−\) “double negative” (DN) B cells in the latter. DN B cells might be a characteristic subset expanding in the joints of ANA+ JIA patients and are potentially involved in the antinuclear immune response in these patients. The results of our explorative study might foster further research dissecting the pathogenesis of ANA+ JIA patients. KW - juvenile idiopathic arthritis KW - B cells KW - antinuclear antibodies KW - synovial fluid KW - double negative B cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236286 SN - 2296-2360 VL - 9 ER - TY - JOUR A1 - Fischer, Jonas A1 - Dirks, Johannes A1 - Klaussner, Julia A1 - Haase, Gabriele A1 - Holl-Wieden, Annette A1 - Hofmann, Christine A1 - Hackenberg, Stephan A1 - Girschick, Hermann A1 - Morbach, Henner T1 - Effect of clonally expanded PD-1\(^h\)\(^i\)\(^g\)\(^h\) CXCR5-CD4+ peripheral T Helper cells on B cell differentiation in the joints of patients with antinuclear antibody-positive juvenile idiopathic arthritis JF - Arthritis & Rheumatology N2 - Objective Antinuclear antibody (ANA)–positive juvenile idiopathic arthritis (JIA) is characterized by synovial B cell hyperactivity, but the precise role of CD4+ T cells in promoting local B cell activation is unknown. This study was undertaken to determine the phenotype and function of synovial CD4+ T cells that promote aberrant B cell activation in JIA. Methods Flow cytometry was performed to compare the phenotype and cytokine patterns of PD-1\(^h\)\(^i\)\(^g\)\(^h\)CD4+ T cells in the synovial fluid (SF) of patients with JIA and T follicular helper cells in the tonsils of control individuals. TCRVB next-generation sequencing was used to analyze T cell subsets for signs of clonal expansion. The functional impact of these T cell subsets on B cells was examined in cocultures in vitro. Results Multidimensional flow cytometry revealed the expansion of interleukin-21 (IL-21) and interferon-γ (IFNγ)–coexpressing PD-1\(^h\)\(^i\)\(^g\)\(^h\)CXCR5–HLA–DR+CD4+ T cells that accumulate in the joints of ANA-positive JIA patients. These T cells exhibited signs of clonal expansion with restricted T cell receptor clonotypes. The phenotype resembled peripheral T helper (Tph) cells with an extrafollicular chemokine receptor pattern and high T-bet and B lymphocyte–induced maturation protein 1 expression, but low B cell lymphoma 6 expression. SF Tph cells, by provision of IL-21 and IFNy, skewed B cell differentiation toward a CD21\(^l\)\(^o\)\(^w\)\(^/\)\(^-\)CD11c+ phenotype in vitro. Additionally, SF Tph cell frequencies correlated with the appearance of SF CD21\(^l\)\(^o\)\(^w\)\(^/\)\(^-\)CD11c+CD27–IgM– double-negative (DN) B cells in situ. KW - medicine Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256607 VL - 74 IS - 1 ER - TY - JOUR A1 - Fortmann, Mats Ingmar A1 - Dirks, Johannes A1 - Goedicke-Fritz, Sybelle A1 - Liese, Johannes A1 - Zemlin, Michael A1 - Morbach, Henner A1 - Härtel, Christoph T1 - Immunization of preterm infants: current evidence and future strategies to individualized approaches JF - Seminars in Immunopathology N2 - Preterm infants are at particularly high risk for infectious diseases. As this vulnerability extends beyond the neonatal period into childhood and adolescence, preterm infants benefit greatly from infection-preventive measures such as immunizations. However, there is an ongoing discussion about vaccine safety and efficacy due to preterm infants’ distinct immunological features. A significant proportion of infants remains un- or under-immunized when discharged from primary hospital stay. Educating health care professionals and parents, promoting maternal immunization and evaluating the potential of new vaccination tools are important means to reduce the overall burden from infectious diseases in preterm infants. In this narrative review, we summarize the current knowledge about vaccinations in premature infants. We discuss the specificities of early life immunity and memory function, including the role of polyreactive B cells, restricted B cell receptor diversity and heterologous immunity mediated by a cross-reactive T cell repertoire. Recently, mechanistic studies indicated that tissue-resident memory (Trm) cell populations including T cells, B cells and macrophages are already established in the fetus. Their role in human early life immunity, however, is not yet understood. Tissue-resident memory T cells, for example, are diminished in airway tissues in neonates as compared to older children or adults. Hence, the ability to make specific recall responses after secondary infectious stimulus is hampered, a phenomenon that is transcriptionally regulated by enhanced expression of T-bet. Furthermore, the microbiome establishment is a dominant factor to shape resident immunity at mucosal surfaces, but it is often disturbed in the context of preterm birth. The proposed function of Trm T cells to remember benign interactions with the microbiome might therefore be reduced which would contribute to an increased risk for sustained inflammation. An improved understanding of Trm interactions may determine novel targets of vaccination, e.g., modulation of T-bet responses and facilitate more individualized approaches to protect preterm babies in the future. KW - preterm infants KW - immunization KW - vaccination KW - safety KW - mechanisms KW - resident memory T cells Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324261 VL - 44 IS - 6 ER - TY - JOUR A1 - Dirks, Johannes A1 - Haase, Gabriele A1 - Cantaert, Tineke A1 - Frey, Lea A1 - Klaas, Moritz A1 - Rickert, Christian H. A1 - Girschick, Hermann A1 - Meffre, Eric A1 - Morbach, Henner T1 - A novel AICDA splice-site mutation in two siblings with HIGM2 permits somatic hypermutation but abrogates mutational targeting JF - Journal of Clinical Immunology N2 - Hyper-IgM syndrome type 2 (HIGM2) is a B cell intrinsic primary immunodeficiency caused by mutations in AICDA encoding activation-induced cytidine deaminase (AID) which impair immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM). Whereas autosomal-recessive AID-deficiency (AR-AID) affects both CSR and SHM, the autosomal-dominant form (AD-AID) due to C-terminal heterozygous variants completely abolishes CSR but only partially affects SHM. AR-AID patients display enhanced germinal center (GC) reactions and autoimmune manifestations, which are not present in AD-AID, suggesting that SHM but not CSR regulates GC reactions and peripheral B cell tolerance. Herein, we describe two siblings with HIGM2 due to a novel homozygous AICDA mutation (c.428-1G > T) which disrupts the splice acceptor site of exon 4 and results in the sole expression of a truncated AID variant that lacks 10 highly conserved amino acids encoded by exon 4 (AID-ΔE4a). AID-ΔE4a patients suffered from defective CSR and enhanced GC reactions and were therefore indistinguishable from other AR-AID patients. However, the AID-ΔE4a variant only partially affected SHM as observed in AD-AID patients. In addition, AID-ΔE4a but not AD-AID patients revealed impaired targeting of mutational hotspot motives and distorted mutational patterns. Hence, qualitative defects in AID function and altered SHM rather than global decreased SHM activity may account for the disease phenotype in these patients. KW - hyper-IgM syndrome type 2 (HIGM2) KW - AICDA KW - AID-ΔE4a KW - AD-AID KW - mutational targeting KW - somatic hypermutation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324253 VL - 42 IS - 4 ER -