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Results WP3: In Vitro Models of PAD

BAFF as the survival factor for peripheral B cells

Interaction of the ligand BAFF and its receptor (BAFFR, which is exclusively expressed on B cells) is one of the most important survival signals, if not the most important survival signal to B cells. As antibody deficiencies often lack of sufficient B cell numbers in the periphery, and also B cell differentiation defects have been demonstrated in patients with antibody deficiencies (B cell differentiation is tightly linked to proliferation during ongoing immune responses), the study of this ligand-receptor pair was a paramount interest. We analysed BAFF serum levels in CVID and BTK-deficient patients, 2 patients. Highest BAFF serum concentrations were found in BTK patients (10-100 ng/mL) followed by a group of 21 CVID patients with <2% of B cells and >10 ng/mL BAFF. The analysis revealed that BAFF serum concentrations correlate inversely with B cell numbers but not with autoimmunity or B cell neoplasia. Sequencing of the BAFF genes in selected patients did not reveal mutations. BAFF-R expression was studied by FACS in >80 patients. About 15% expressed significantly reduced BAFF-R levels. Expression went back to normal in EBV lines indicating regulatory changes in CVID patients. We developed several readout systems for BAFF-R function including analysis of the alternative NF-kB activation and p100 processing by western blotting and in vitro survival assays for human primary B cells and for murine B cells from BAFF-R deficient mice which were transduced with various BAFF-R mutants. Analysis of BAFF-R receptor structure revealed that BAFF-R associates in multimeric receptor complexes. Different from wildtype, the BAFF-R variant P21R shows impaired complex formation, ligand binding and NF-kB activation. Presently we are studying the impacts of BAFF-R multimerization on the association with downstream signalling components such as TRAF6, TRAF2 and TRAF3.

CD21low B Cell Populations In Primary Human Antibody Deficiencies

The so called CD21-low B cells population has been primarily described in patients with CVID, but has recently also been shown to be increased in cohorts of autoimmune diseases. Interestingly, CVID patients also suffer from autoimmune phenomena. CD21(low) B cells were shown to be profoundly anergic, and defects of BCR-mediated calcium signalling and of T cells have been described in CVID. We found that also the classical naive B cells from many CVID patients proliferated poorly. The B cells of some CVID patients had a reduced capacity to divide reminiscent of the proliferative arrest associated with replicative senescence which is known to be influenced by telomere length. Thus, we investigated whether lymphocyte dysfunction in CVID was related to telomere-dependent replicative senescence, and found that both the B and the T cells from other CVID patietns. Telomere lengths in B and T cells were significantly correlated, indicating that the rate of telomere attrition in lymphocytes is an individual characteristic of CVID patients. Our findings suggest that telomere-dependent replicative senescence contributes to the immune dysfunction of some CVID patients, and may provide an important clue for a better understanding of the pathogenesis of CVID.

IFN Signature in CD21-low B Cells

The percentage of CD21low B cells was shown to be significantly expanded in HCV-viremic patients supporting the idea of an association of CD21low B cells to IFN rich environment. Detailed examination of IFN signature in CD21low B cells of CVID patients demonstrated increased expression of MXA, OASI, while other targets like IFIT1 showed higher expression in naive B cells of patients. The IFN signature was B cell specific in CVID, but IFN itself failed failed to induce the phenotype of CD21low B cells in vitro.

Plasma Cells In Antibody Deficiencies

Our research activities focused on the role of epigenetic and protein modifying histone acetylase/deacetylase activities during plasma cell development. We found that inhibition of HDAC by valproic acid and other iHDAC blocks IL21R signalling by affecting STAT5 phosphorilation. The VPA-mediated inhibition of IL21R signalling impairs plasma cell formation and antibody secretion.

IgM Memory B Cells In Antibody Deficiencies

In order to demonstrate the developmental connection between IgM memory and plasma cells, we first determined the presence of sIgA and/or IgM in jejunal biopsies of two groups of CVID patients, with or without circulating IgM memory B cells and in splenectomised patients. By confocal microscopy we analysed IgA plasma cells and sIgA on the epithelial cells in jejunal biopsies. After splenectomy the reduction of memory B cells in the peripheral blood is associated to a significant diminution of IgA plasma cells and the disruption of the film of sIgA on the luminal side of epithelial cells. In CVID the absence of sIgA in the gut and IgA in the serum are associated to a significant reduction of memory B cells in the peripheral blood and high risk of respiratory infections. CVID patients with sIgA at mucosal sites had a normal number of circulating memory B cells, detectable serum IgA and have a low risk of respiratory infections. Memory B cells are indispensable for the production of sIgA at mucosal sites. The disruption of the sIgA film in the gut impairs the local defence against invading pathogens.

Identifying Precursors Of IgM Memory B Cells

The expansion of specific B cell subsets expressing low levels of CD21 in patients with PAD is an expression of an underlying pathogenic dysregulation. These B cells subsets are being used to define subgroups of patients for specific functional as well as molecular characterisation. We demonstrated that, upon exposure to CpG, a fraction (26%) of transitional B cells proliferate (P) and differentiate either to IgM-secreting plasma cells (19.2%) or to cells phenotypically identical to IgM memory B cells (41.3%). The remaining non-proliferating (NP) B cells down-regulate the expression of CD24 and CD38, thus resembling mature B cells. We have also demonstrated that natural antibodies, including immunoglobulins with anti-pneumococcal specificity, can be generated "in vitro" from CpG-stimulated transitional B cells. The progenitors of IgM memory B cells are recent bone marrow emigrant transitional B cells, which through the blood arrive to the spleen, an optimal anatomical site where blood-borne pathogens and cellular debris are filtered by resident macrophages. Here, transitional B cells diversify their antigen receptors in the presence of bacterial components, e.g. CpG DNA. CpG induces somatic mutations in the Ig heavy chains (VH) regions in P B cells, as opposed to the prevalence of germline sequence in the NP cells. The mutations are confined almost exclusively to the framework regions. Moreover, the incidence of mutations is VH-specific, being VH1 and VH4 the most mutated, and VH5 the least mutated family. In summary, early recognition of bacterial DNA induces proliferation, SHM, antibody secretion or survival in transitional B cells. IgM memory B cells in one year-old children mirror the mutation pattern seen in CpG-stimulated transitional B cells.

The Role of TACI Mutations for Antibody Deficiencies

Coding variants in TACI, aka tumour necrosis factor receptor superfamily member 13B (TNFRSF13B) have been implicated in common variable immunodeficiency (CVID), but the functional effects of such mutations in relation to the development of the disease have not been entirely established. To examine the potential contribution of TNFRSF13B variants to CVID, we studied the mutant alleles in vitro. In summary we found that each mutation acts via a different pathomechanism: I. frameshift mutations, nonsense or missense, leading to loss of protein expression (examples ins204A, S194X, L171R); II. expressed missense mutant that fails to bind ligand (example C104R, I87N); III. mutant with intracellular retention of mutated protein (examples C104R and L171R); IV. failure of trimeric assembly as proposed by Lee JJ Blood. 2009;114:2254-62 (example A181E); V. mutants with no effect at all = non pathogenic variants (examples W40R, H81N, K188M). Most importantly, we were able to show that having a pathogenic mutation in TACI increases the risk for developing CVID by 4. Presence of a heterozygous mutation was associated with antibody deficiency (P<.001, relative risk 3.6). Heterozygosity for the most common mutation, C104R, was associated with disease (P<.001, relative risk 4.2). Furthermore, heterozygosity for C104R was associated with low numbers of IgD-CD27+ B cells (P=.019), benign lymphoproliferation (P<.001) and autoimmune complications (P=.001). These associations indicate that C104R heterozygosity increases the risk for common variable immunodeficiency disorders and influences clinical presentation (Salzer et al., Blood. 2009;113:1967-1976).

Class-switch Recombination Defects and Mismatch Repair Defects

This consortium described PMS2-deficiency as causative of a partial Class-Switch-Recombination (CSR) defect. The group went on studying other MisMatch-Repair-Deficiencies. Seven further PMS2-, two MLH1- and seven MSH6-deficient patients could be diagnosed. All of the patients presented with a partial CSR defect. Interestingly enough, the group provided evidence that PMS2 is involved in the CSR-induced generation of DNA double strand breaks (DSB) in switch regions, likely because of its endonuclease domain. The group observed that both double-strand-break (DSB) generation and repair is impaired in PMS2-deficiency, we asked the question whether impaired DSB generation could influence their repair. For this purpose, we studied S#-S# junctions in B cells in which DSB occurrence is very likely (AID+/-AID Cter#/+) or certainly impaired (UNG-/-). We showed a progressive bias towards microhomology usage for switch junction repair from AID+/- to AID Cter#/- and to UNG-/-. Our results strongly suggest for the first time that proper induction of CSR-DSB either recruits NHEJ, or excludes other repair pathways (Alternative End Joining). In other hyper-IgM patients, we identified a transcription elongation factor, the expression of which was strongly diminished in EBV B cell lines from four patients. Although the RNA level and the genomic sequence was found normal, this molecule appears indirectly down-regulated in these patients. The role of this molecule in CSR is being studied by shRNA in CSR-induced CH12 cell line.