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The role of AID and NF-κB for B cell development and lymphomagenesis.

München, Technische Universität München, Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, Diss., 2011, 109 S.
Verlagsversion Volltext
Peripheral B cells that encounter a cognate antigen and become activated via CD40 binding start to form germinal centres where they undergo affinity maturation. The affinity maturation process somatic hypermutation (SHM) depends on the activation-induced cytidine deaminase protein (AID) that inserts mutations into the immunoglobulin (Ig) locus, enhancing the genomic variety of antibodies. AID recruitment to the Ig locus and AID mutations must be strictly regulated to ensure genomic stability. However, the specific targeting of AID has not been clarified. In the scope of this project, cis- elements included in the Ig locus and potential trans- factors involved in the specific AID recruitment were identified. To this end a chicken DT40 cell line was used that solely undergoes somatic hypermutation. The assay for mutation was a GFP2 reporter system in the Ig locus that accumulates AID-induced mutations. The compact size of the DT40 Igl locus of 10 kb facilitates a successive deletion analysis. A deletion of the complete Igl locus demonstrated that elements essential for SHM are located in the Ig sequence. Via several staggered deletion analyses, the Igl enhancer was identified as the core sequence essential for AID recruitment and in particular a 200 bp sequence located at the 5’ end of the enhancer. This hypermutation core element (‘HyCorE’) was sufficient for AID recruitment. Multimerization of the element enhanced the mutation frequency. An identification of ‘HyCorE’ homologes in closely-related species and their efficient recruitment of AID confirmed the exclusive role of this sequence. An in silico analysis of the 200 bp core element identified binding sites for E2A, NF-kB, MEF-2, SP1 and Pax5. This is the first time demonstration of a sequence of 200 bp that is sufficient to induce hypermutation and carries binding sites for trans-acting factors for a putative AID recruitment complex. CD40 signalling, that is essential for germinal centre formation and initiation of SHM, was known to be deregulated in several lymphomas. The LMP1/CD40 mouse model, established in our lab, allowed a detailed analysis of constitutive CD40 signalling. The LMP1/CD40 mouse expresses a chimeric protein consisting of the self-activated LMP1 transmembrane domain and the CD40 intracellular domain. It has previously been shown that a constitutive CD40 signal in vivo initiates B cell expansion and promotes B cell tumourigenesis. Analysis of signalling pathways in LMP1/CD40 expressing B cells revealed that the MAPK ERK and JNK and the non-canonical NF-kB pathway are activated. To analyze, whether the canonical NF- kB pathway influences B cell expansion and lymphomagenesis, mice with a NEMO or IKK2 null deletion were crossed with LMP1/CD40 mice. The disruption of the canonical NF-kB pathway blocked the LMP1/CD40 induced B cell expansion, reduced the LMP1/CD40 mediated survival and inhibited proliferation. Interestingly, the depletion of NEMO or IKK2 in LMP1/CD40 mice did not influence the translocation of the NF-kB components, but led to diminished pERK and pJNK levels, indicating a cross-talk between the NF-kB and MAPK pathways. Previous publications demonstrated a connection between NEMO/IKK2 and ERK via Tpl2/MEK1, resulting in a specific activation of p65 (Phospho-Ser276). Indeed p65(Ser276) was specifically phosphorylated in LMP1/CD40 mice, but not in NEMO or IKK2 depleted LMP1/CD40 or in wild type mice. These data imply that the canonical NF-kB pathway contributes to LMP1/CD40 mediated B cell expansion via the Tpl2/ERK/pp65(Ser276) regulation mechanism. This result elucidates a completely new role of the canonical NF-kB pathway for CD40 signalling in B cells and creates a foundation for detailed signalling analyses.
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Publikationstyp Sonstiges: Hochschulschrift
Typ der Hochschulschrift Dissertationsschrift
Quellenangaben Band: , Heft: , Seiten: 109 S. Artikelnummer: , Supplement: ,
Hochschule Technische Universität München
Hochschulort München
Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt
Institut(e) Molekulare Endokrinologie und Metabolismus (MEM)
Research Unit Analytical Pathology (AAP)