技术资料

BACKGROUND Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits T-cell activation and is expressed on T-regulatory cells. Human CTLA-4 deficiency results in severe immune dysregulation. Abatacept (CTLA-4 Ig) is approved for the treatment of rheumatoid arthritis (RA) and its mechanism of action is attributed to effects on T-cells. It is known that CTLA-4 modulates the expression of its ligands CD80 and CD86 on antigen presenting cells (APC) by transendocytosis. As B-cells express CD80/CD86 and function as APC,we hypothesize that B-cells are a direct target of abatacept. OBJECTIVES To investigate direct effects of abatacept on human B-lymphocytes in vitro and in RA patients. METHODS The effect of abatacept on healthy donor B-cells' phenotype,activation and CD80/CD86 expression was studied in vitro. Nine abatacept-treated RA patients were studied. Seven of these were followed up to 24 months,and two up to 12 months only and treatment response,immunoglobulins,ACPA,RF concentrations,B-cell phenotype and ACPA-specific switched memory B-cell frequency were assessed. RESULTS B-cell development was unaffected by abatacept. Abatacept treatment resulted in a dose-dependent decrease of CD80/CD86 expression on B-cells in vitro,which was due to dynamin-dependent internalization. RA patients treated with abatacept showed a progressive decrease in plasmablasts and serum IgG. While ACPA-titers only moderately declined,the frequency of ACPA-specific switched memory B-cells significantly decreased. CONCLUSIONS Abatacept directly targets B-cells by reducing CD80/CD86 expression. Impairment of antigen presentation and T-cell activation may result in altered B-cell selection,providing a new therapeutic mechanism and a base for abatacept use in B-cell mediated autoimmunity. View Publication

Missing in Metastasis (MIM),or Metastasis Suppressor 1 (MTSS1),is a highly conserved protein,which links the plasma membrane to the actin cytoskeleton. MIM has been implicated in various cancers,however,its modes of action remain largely enigmatic. Here,we performed an extensive in silico characterisation of MIM to gain better understanding of its function. We detected previously unappreciated functional motifs including adaptor protein (AP) complex interaction site and a C-helix,pointing to a role in endocytosis and regulation of actin dynamics,respectively. We also identified new functional regions,characterised with phosphorylation sites or distinct hydrophilic properties. Strong negative selection during evolution,yielding high conservation of MIM,has been combined with positive selection at key sites. Interestingly,our analysis of intra-molecular co-evolution revealed potential regulatory hotspots that coincided with reduced potentially pathogenic polymorphisms. We explored databases for the mutations and expression levels of MIM in cancer. Experimentally,we focused on chronic lymphocytic leukaemia (CLL),where MIM showed high overall expression,however,downregulation on poor prognosis samples. Finally,we propose strong conservation of MTSS1 also on the transcriptional level and predict novel transcriptional regulators. Our data highlight important targets for future studies on the role of MIM in different tissues and cancers. View Publication

In systemic lupus erythematosus,defective clearance of apoptotic debris and activation of innate cells result in a chronically activated type 1 IFN response,which can be measured in PBMCs of most patients. Metformin,a widely used prescription drug for Type 2 diabetes,has a therapeutic effect in several mouse models of lupus through mechanisms involving inhibition of oxidative phosphorylation and a decrease in CD4+ T cell activation. In this study,we report that in CD4+ T cells from human healthy controls and human systemic lupus erythematosus patients,metformin inhibits the transcription of IFN-stimulated genes (ISGs) after IFN-alpha treatment. Accordingly,metformin inhibited the phosphorylation of pSTAT1 (Y701) and its binding to IFN-stimulated response elements that control ISG expression. These effects were independent of AMPK activation or mTORC1 inhibition but were replicated using inhibitors of the electron transport chain respiratory complexes I,III,and IV. This indicates that mitochondrial respiration is required for ISG expression in CD4+ T cells and provides a novel mechanism by which metformin may exert a therapeutic effect in autoimmune diseases. View Publication

Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation,we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells,cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid,B-lymphoid,and erythroid lineages,but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization,which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice. View Publication

Studies have suggested involvement of interleukin 17 (IL-17) in autoimmune diseases,although its effect on B cell biology has not been clearly established. Here we demonstrate that IL-17 alone or in combination with B cell-activating factor controlled the survival and proliferation of human B cells and their differentiation into immunoglobulin-secreting cells. This effect was mediated mainly through the nuclear factor-kappaB-regulated transcription factor Twist-1. In support of the relevance of our observations and the potential involvement of IL-17 in B cell biology,we found that the serum of patients with systemic lupus erythematosus had higher concentrations of IL-17 than did the serum of healthy people and that IL-17 abundance correlated with the disease severity of systemic lupus erythematosus. View Publication

The development of mature blood cells from hematopoietic stem cells requires coordinated activities of transcriptional networks. Transcriptional repressor growth factor independence 1 (Gfi-1) is required for the development of B cells,T cells,neutrophils,and for the maintenance of hematopoietic stem cell function. However,the mechanisms by which Gfi-1 regulates hematopoiesis and how Gfi-1 integrates into transcriptional networks remain unclear. Here,we provide evidence that Id2 is a transcriptional target of Gfi-1,and repression of Id2 by Gfi-1 is required for B-cell and myeloid development. Gfi-1 binds to 3 conserved regions in the Id2 promoter and represses Id2 promoter activity in transient reporter assays. Increased Id2 expression was observed in multipotent progenitors,myeloid progenitors,T-cell progenitors,and B-cell progenitors in Gfi-1(-/-) mice. Knockdown of Id2 expression or heterozygosity at the Id2 locus partially rescues the B-cell and myeloid development but not the T-cell development in Gfi-1(-/-) mice. These studies demonstrate a role of Id2 in mediating Gfi-1 functions in B-cell and myeloid development and provide a direct link between Gfi-1 and the B-cell transcriptional network by its ability to repress Id2 expression. View Publication

The B cell-activating factor of the TNF family receptor (BAFF-R),encoded by the TNFRSF13C gene,is critically important for transitional B cell survival to maturity. Thus,ligation of BAFF-R by BAFF delivers a potent survival signal. Reports implicating the BAFF/BAFF-R signaling axis in the pathogenesis of autoimmune human diseases and B lineage malignancies have largely prompted studies focusing on BAFF expression; however,there is an equally critical need to better understand BAFF-R expression. Initial BAFF-R expression,although characterized in murine B cells,has not yet been reported in human B lymphopoiesis. In this study,we first demonstrate that BAFF-R expression is absent from early precursors and is acquired by bone marrow B cells newly expressing the BCR. We next focused on identifying the specific genomic region that controls BAFF-R expression in mature B cells (i.e.,the TNFRSF13C promoter). To accomplish this,we used in silico tools examining interspecies genomic conservation in conjunction with reporter constructs transfected into malignant B and plasma cell lines. DNase protection assays using nuclear extracts from BAFF-R-expressing cells suggested potential regulatory sites,which allowed the generation of EMSA probes that bound NFs specific to BAFF-R-expressing cells. With a more stringent analysis of interspecies homology,these assays identified a site at which a single nucleotide substitution could distinctly impact promoter activity. Finally,chromatin immunoprecipitation assays revealed the in vivo binding of the specific transcription factor c-Rel to the most proximal genomic region,and c-Rel small interfering RNA transfections in BAFF-R-expressing lines demonstrated a coincident knockdown of both c-Rel and BAFF-R mRNA. View Publication

Mast cells are tissue-resident immune cells that play a key role in inflammation and allergy. Here we show that interaction of mast cells with antibody-targeted cells induces the polarized exocytosis of their granules resulting in a sustained exposure of effector enzymes,such as tryptase and chymase,at the cell-cell contact site. This previously unidentified mast cell effector mechanism,which we name the antibody-dependent degranulatory synapse (ADDS),is triggered by both IgE- and IgG-targeted cells. ADDSs take place within an area of cortical actin cytoskeleton clearance in the absence of microtubule organizing centre and Golgi apparatus repositioning towards the stimulating cell. Remarkably,IgG-mediated degranulatory synapses also occur upon contact with opsonized Toxoplasma gondii tachyzoites resulting in tryptase-dependent parasite death. Our results broaden current views of mast cell degranulation by revealing that human mast cells form degranulatory synapses with antibody-targeted cells and pathogens for dedicated secretion and defence. View Publication

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and,thereby,leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies,IGHV1-2(∗)02-rearranging mice,which also express a VRC01-antibody precursor light chain,can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages. View Publication

Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen,we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study,we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore,we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition,deficiency in Kin17 affects the functionality of multiple DSB repair pathways,namely homologous recombination,non-homologous end-joining,and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR. View Publication