Impact of B cell specific transcription factors, especially EBF1, on tumour cells of classical Hodgkin Lymphoma

Besonderes Charakteristikum des von B-Zellen abstammenden Hodgkin-Lymphoms (HL) ist die heterogene Ausprägung verschiedenster Gene. Diese umfasst neben einem Verlust der B-Zellsignatur die aberrante Ausprägung von Genen anderer hämatopoetischen Linien. Die Rolle dieses ungewöhnlichen Expressionsmusters für die Pathogenese des HL ist bisher unbekannt. Um einen Einblick in die möglichen Mechanismen dieser Dedifferenzierung zu erhalten wurde EBF1, einer der wichtigsten B-Zelltranskriptionsfaktoren der B-Zelle, der in HL-Linien im Allgemeinen nicht oder nur schwach ausgeprägt ist, reexprimiert. Im Rahmen der vorliegenden Arbeit konnte die Expression von EBF1 in HL-Linien erfolgreich etabliert werden. Die funktionellen Experimente mit den transduzierten Linien haben gezeigt, dass EBF1 in der Lage ist verschiedene positiv regulierte B-Zellzielgene wie CD19, CD79A und CD79B in HL-Linien wieder auszuprägen. Für andere B-Zellzielgene wie PAX5 und Foxo1 trifft das nicht zu. Ebenso konnten negativ regulierte Zielgene anderer Linien wie ID2 und Notch1 nicht in ihrer Ausprägung unterdrückt werden. Im Gegenteil, in der Linie L-1236 war ein Anstieg der Notch1-Expression zu beobachten. Eine globale B-Zellsignatur konnte durch die Reexpression von EBF1 nicht wieder hergestellt werden, wie Analysen über Genchips gezeigt haben. Der in der Literatur beschriebene Einfluss von EBF1 auf DNA-Methylierungen in nicht malignen Zellen konnte für einzelne untersuchte CpGs in den Promotoren von CD19, CD79B und PAX5α bestätigt werden. Ein Nachweis eines negativen Einflusses der EBF1-Reexpression auf die allgemeine Fitness der Zellen konnte für die Linie L-1236 erbracht werden. Eine Mutationsanalyse der EBF1 cDNA in den HL-Linien L-1236, L-428, KM-H2, HDLM-2, U-HO1 und SUP-HD1 detektiert eine Protein-verkürzende Mutation in einem der KM-H2-Allele. Mutationen in EBF1 sind somit vermutlich nicht der Grund für fehlendes EBF1-Protein. Die Daten zeigen, dass EBF1 in der Lage ist die B-Zellsignatur teilweise wieder herzustellen. Es beeinflusst das DNA-Methylierungsmuster geringfügig und kann in der Linie L-1236 die Fitness der Zellen reduzieren. EBF1 trägt somit zum Verlust des B-Zellphänotyps bei.One characteristic feature of classical Hodgkin Lymphoma (cHL), which is of B-cell origin, is the heterogeneous expression of different genes. The tumour cells, called HRS cells, lose most of their typical B-cell signature and express markers of other haematopoietic lineages. So far it is unknown if this dedifferentiated expression pattern takes part in the pathogenesis of cHL. To better understand possible mechanisms concerning this phenotype early B-cell factor (EBF1), one of the three major transcription factors in B-cells, was reintroduced in cHL cell lines. Naturally EBF1 is lowly expressed or even absent in cHL cell lines. In this study reexpression of EBF1 was successfully established in cHL cell lines. It could be shown that positively regulated EBF1 target genes - such as CD19, CD79A and CD79B - are reexpressed upon EBF1 transduction. Other positively regulated EBF1 target genes - namely PAX5 and FOXO1 - did not change their expression. Likewise the expression level of negatively regulated target genes ID2 and Notch1, which are originally part of other haematopoietic lineages, are not repressed. The cell line L-1236 actually shows an increased expression of Notch1. Microarray analysis shows that a global B-cell signature could not be reconstituted in EBF1 transduced cells. The literature describes an influence of EBF1 on DNA methylation, which could be confirmed for single CpGs in the promoter region of CD19, CD79B and PAX5α upon reexpression of EBF1 in cHL cell lines. It could be shown that L-1236 loses part of its fitness if EBF1 is reexpressed. A mutational analysis of EBF1 cDNA in the cell lines L-1236, L-428, KM-H2, HDLM-2, U-HO1 und SUP-HD1 detected a heterozygous mutation in KM-H2. This mutation leads to a truncated protein, which might have an influence on transcriptional activity of EBF1 in KM-H2, but mutational inactivation of EBF1 does not seem to be a general feature of cHL cell lines. The presented data show that EBF1 has the ability to reintroduce part of the B-cell signature in cHL cell lines. EBF1 has a slight influence on the DNA methylation pattern of its target genes and is capable of reducing the fitness of L-1236 cells. Loss of EBF1 therefore partially contributes to the lost B-cell phenotype

TNFAIP3 (A20) is a tumor suppressor gene in Hodgkin lymphoma and primary mediastinal B cell lymphoma

Proliferation and survival of Hodgkin and Reed/Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), are dependent on constitutive activation of nuclear factor κB (NF-κB). NF-κB activation through various stimuli is negatively regulated by the zinc finger protein A20. To determine whether A20 contributes to the pathogenesis of cHL, we sequenced TNFAIP3, encoding A20, in HL cell lines and laser-microdissected HRS cells from cHL biopsies. We detected somatic mutations in 16 out of 36 cHLs (44%), including missense mutations in 2 out of 16 Epstein-Barr virus–positive (EBV+) cHLs and a missense mutation, nonsense mutations, and frameshift-causing insertions or deletions in 14 out of 20 EBV− cHLs. In most mutated cases, both TNFAIP3 alleles were inactivated, including frequent chromosomal deletions of TNFAIP3. Reconstitution of wild-type TNFAIP3 in A20-deficient cHL cell lines revealed a significant decrease in transcripts of selected NF-κB target genes and caused cytotoxicity. Extending the mutation analysis to primary mediastinal B cell lymphoma (PMBL), another lymphoma with constitutive NF-κB activity, revealed destructive mutations in 5 out of 14 PMBLs (36%). This report identifies TNFAIP3 (A20), a key regulator of NF-κB activity, as a novel tumor suppressor gene in cHL and PMBL. The significantly higher frequency of TNFAIP3 mutations in EBV− than EBV+ cHL suggests complementing functions of TNFAIP3 inactivation and EBV infection in cHL pathogenesis

Adoptive transfer of allergen-expressing B cells prevents IgE-mediated allergy

IntroductionProphylactic strategies to prevent the development of allergies by establishing tolerance remain an unmet medical need. We previously reported that the transfer of autologous hematopoietic stem cells (HSC) expressing the major timothy grass pollen allergen, Phl p 5, on their cell surface induced allergen-specific tolerance in mice. In this study, we investigated the ability of allergen-expressing immune cells (dendritic cells, CD4+ T cells, CD8+ T cells, and CD19+ B cells) to induce allergen-specific tolerance in naive mice and identified CD19+ B cells as promising candidates for allergen-specific cell therapy.MethodsFor this purpose, CD19+ B cells were isolated from Phl p 5-transgenic BALB/c mice and transferred to naive BALB/c mice, pre-treated with a short course of rapamycin and an anti-CD40L antibody. Subsequently, the mice were subcutaneously sensitized three times at 4-week intervals to Phl p 5 and Bet v 1 as an unrelated control allergen. Allergen-expressing cells were followed in the blood to monitor molecular chimerism, and sera were analyzed for Phl p 5- and Bet v 1-specific IgE and IgG1 levels by RBL assay and ELISA, respectively. In vivo allergen-induced lung inflammation was measured by whole-body plethysmography, and mast cell degranulation was determined by skin testing.ResultsThe transfer of purified Phl p 5-expressing CD19+ B cells to naive BALB/c mice induced B cell chimerism for up to three months and prevented the development of Phl p 5-specific IgE and IgG1 antibody responses for a follow-up period of 26 weeks. Since Bet v 1 but not Phl p 5-specific antibodies were detected, the induction of tolerance was specific for Phl p 5. Whole-body plethysmography revealed preserved lung function in CD19+ B cell-treated mice in contrast to sensitized mice, and there was no Phl p 5-induced mast cell degranulation in treated mice.DiscussionThus, we demonstrated that the transfer of Phl p 5-expressing CD19+ B cells induces allergen-specific tolerance in a mouse model of grass pollen allergy. This approach could be further translated into a prophylactic regimen for the prevention of IgE-mediated allergy in humans

Vom Seelengefährt zum Glorienleib

Das Konzept des Seelengefährts, das bereits von Platon und zuvor von Parmenides konzipiert wurde, ist für die philosophische Selbstverständigung von der Antike bis in die frühe Auklärung und darüber hinaus die moderne Esoterik von zentraler Bedeutung. Sein veranschaulichendes Potenzial reicht von der Erkundung der Möglichkeiten der Kommunikation zwischen eigentlich klar voneinander geschiedenen ontologischen Räumen – dem des Unstofflich-Göttlichen einerseits und dem des Stofflich-Irdischen bzw. menschlich Seelischen andererseits – bis hin zu naturphilosophischen Entwürfen zur Erklärung des Phänomens der Elektrizität. Immer wieder wurde es von so unterschiedlichen Denkern wie z. B. Plutarch, Bernardus Silvestris, Kepler, Suarez oder Christian Wolf herangezogen, um das Problem der Einwirkung des Intelligiblen und Göttlichen auf die menschliche Seele oder die irdische Natur zu erklären. Es motivierte Dichter wie Traherne oder Donne zu literarischen Inszenierungen sympathetisch-aitherischer Leiblichkeit und deren eschatologischen Implikationen. Diesem zentralen und spannungsvollen Themenspektrum in seiner historischen wie thematischen Kontinuität nähern sich die Autor*innen dieses Bandes aus interdisziplinär verschränkter Perspektive, die zudem eine innovative, künstlerische Auseinandersetzung mit den verschiedenen Aspekten des Licht(-leib)es umfasst

One learns wisdom by suffering. Knowledge, experience and learning in dealing with risks. Contributions from science and practice to the 27th meeting of the working group natural hazards/ natural risks

Nach größeren überregionalen und auch kleineren regionalen Schadenereignissen wird wiederholt diskutiert, welche Mechanismen und Maßnahmen erforderlich sind, um die Gesellschaft widerstandsfähiger gegen Naturgefahren (und auch andere Gefahren) zu machen. Diese öffentlich, auf politischer und wissenschaftlicher Ebene geführte Auseinandersetzung führt stets zu einem gewissen Aktionismus, zu unmittelbaren Bekundungen von monetärer Abgeltung der Schäden, und zu Bekenntnissen im Sinne einer erforderlichen nachhaltigeren Schutzstrategie basierend auf dem Risikoansatz. lm Laufe von Wochen und Monaten nach den Ereignissen verebbt diese Diskussion allerdings regelmäßig, nur vereinzelt werden grundlegende Transformationsprozesse im Umgang mit Naturgefahren angestoßen. Vor diesem Hintergrund stellte das 27. Treffen des Arbeitskreises Naturgefahren/ Naturrisiken die Fragen nach dem Zusammenhang zwischen der Rolle von Wissen, Erfahrung und Lernen im Umgang mit gegenwärtigen aber auch zukünftigen Naturgefahren in den Mittelpunkt. Die vorliegende Ausgabe der Schriftenreihe ‚Integrative Risk and Security Research‘ bietet nun, im Rahmen von Fach- sowie Diskussionsbeiträgen, Einblicke in die dort diskutierten Perspektiven und deckt dabei eine Vielzahl von Gefahren und Risiken ab, wie z.B. Überschwemmung, Hitze, Frost- und Brandschaden in der Landwirtschaft, ein mögliches Versagen des Stromnetzes, Erdbeben, Sturmfluten und Lawinen.After large-scale but also small-scale disasters mechanisms and procedures promoting resilience get in the focus of political and scientific discussions and become a prominent part of their agenda. However, after a certain time the discussion subsides regularly and overarching transformation processes are barely accomplished. The 27th meeting of the working group natural hazards/ natural risks tackled this phenomenon by discussing the role of knowledge, experience and learning in dealing with present and future natural hazards and risks. This volume of the publication series ‘Integrative Risk and Security Research’ presents thus, via peer reviewed research articles as well as opinion papers, insights in the discussed perspectives and covers a wide range of hazards and risks such as floods, droughts, frost, blackouts, storm tides and avalanches

Coupling of a Major Allergen to the Surface of Immune Cells for Use in Prophylactic Cell Therapy for the Prevention of IgE-Mediated Allergy

Up to a third of the world’s population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols

The IgE-reactive autoantigen Hom s 2 induces damage of respiratory epithelial cells and keratinocytes via induction of IFN-gamma

Hom s 2, the alpha-chain of the nascent polypeptide-associated complex, is an intracellular autoantigen that has been identified with IgE autoantibodies from atopic dermatitis patients. We investigated the humoral and cellular immune response to purified recombinant Hom s 2 (rHom s 2). rHom s 2 exhibited IgE reactivity comparable to exogenous allergens, but did not induce relevant basophil cell degranulation. The latter may be attributed to the fact that patients recognized single epitopes on Hom s 2 as revealed by IgE epitope mapping with rHom s 2 fragments. In contrast to exogenous allergens, rHom s 2 had the intrinsic ability to induce the release of IFN-gamma in cultured peripheral blood mononuclear cells from atopic as well as non-atopic individuals. IFN-gamma-containing culture supernatants from Hom s 2-stimulated peripheral blood mononuclear cells caused disintegration of respiratory epithelial cell layers and apoptosis of skin keratinocytes, which could be inhibited with a neutralizing anti-IFN-gamma antibody. Our data demonstrate that the Hom s 2 autoantigen can cause IFN-gamma-mediated cell damage