title: T-helper signals restore B-cell receptor signaling in autoreactive anergic B cells by upregulating CD45 phosphatase activity.
abstract: We recently identified a human B-cell population that is naturally autoreactive and tolerized by functional anergy (BND cells).
  
  
  
  We sought to identify the molecular mechanism of how anergic autoreactive BND cells escape functional anergy and whether this process is altered in patients with lupus.
  
  
  
  Isolated peripheral blood naive and BND cells were cultured with various stimuli, and their activation status was determined by using an intracellular Ca(2+) mobilization assay. Lyn kinase and Syk activities were assessed by using phospho-flow analysis. CD45 phosphatase activity was determined by using a novel flow-based assay, which takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid, an analog of phosphotyrosine, which can be incorporated into peptides. Real-time quantitative PCR was used to quantitate LYN, SYK, and CD45 mRNA.
  
  
  
  T-helper signals reversed the state of anergy, allowing BND cells to fully respond to antigenic stimulation by restoring signaling through the B-cell receptor (BCR). The mechanism was dependent on increased activity of the tyrosine phosphatase CD45 and CD45-dependent activation of Lyn and Syk. CD45 phosphatase activity was increased by T-cell help both in BND and naive B cells. Furthermore, we found that BND cells obtained from patients with systemic lupus erythematosus exhibited increased CD45 activity and BCR-signaling capacity, thus being less tolerized than BND cells from healthy control subjects.
  
  
  
  Our findings suggest that CD45 is a key regulator of BCR-signaling thresholds mediated by T-cell help. This raises the possibility that BND cells could represent precursors of autoantibody-secreting plasma cells and suggests a role for these autoreactive B cells in contributing to autoimmunity if not properly controlled.
authors.affiliation: Centre for Immune Regulation, Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway. Electronic address: peter.szodoray@medisin.uio.no.
authors.forename: Peter
authors.initials: P
authors.lastname: Szodoray
authors.affiliation: Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, Calif.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Stanford
authors.affiliation: Department of Rheumatology, Oslo University Hospital-Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
authors.forename: Øyvind
authors.initials: Ø
authors.lastname: Molberg
authors.affiliation: Centre for Immune Regulation, Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
authors.forename: Ludvig A
authors.initials: LA
authors.lastname: Munthe
authors.affiliation: Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, Calif.
authors.forename: Nunzio
authors.initials: N
authors.lastname: Bottini
authors.affiliation: Centre for Immune Regulation, Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway.
authors.forename: Britt
authors.initials: B
authors.lastname: Nakken
mesh_terms: D000328:Adult; D001402:B-Lymphocytes / Q000276:immunology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D017635:Clonal Anergy; D006801:Humans; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D012333:RNA, Messenger / Q000378:metabolism; D011947:Receptors, Antigen, B-Cell / Q000276:immunology*; D015398:Signal Transduction; D000072377:Syk Kinase / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology*; D015854:Up-Regulation; D019061:src-Family Kinases / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D011947:Receptors, Antigen, B-Cell; C000606896:SYK protein, human; D000072377:Syk Kinase; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human; D002118:Calcium
keywords: B-cell anergy; CD45 phosphatase activity; T-cell help; break of tolerance; systemic lupus erythematosus