title: High-content cytometry and transcriptomic biomarker profiling of human B-cell activation.
abstract: BACKGROUND
  Primary antibody deficiencies represent the most prevalent, although very heterogeneous, group of inborn immunodeficiencies, with a puzzling complexity of cellular and molecular processes involved in disease pathogenesis.
  
  
  OBJECTIVE
  We aimed to study in detail the kinetics of CD40 ligand/IL-21-induced B-cell differentiation to define new biomarker sets for further research into primary antibody deficiencies.
  
  
  METHODS
  We applied high-content screening methods to monitor B-cell activation on the cellular (chip cytometry) and transcriptomic (RNA microarray) levels.
  
  
  RESULTS
  The complete activation process, including stepwise changes in protein and RNA expression patterns, entry into the cell cycle, proliferation and expression of activation-induced cytidine deaminase (AID), DNA repair enzymes, and post-class-switch expression of IgA and IgG, was successfully monitored during in vitro differentiation. We identified a number of unknown pathways engaged during B-cell activation, such as CXCL9/CXCL10 secretion by B cells. Finally, we evaluated a deduced set of biomarkers on a group of 18 patients with putative or proved intrinsic B-cell defects recruited from the European Society for Immunodeficiencies database and successfully predicted 2 AID defects and 1 DNA repair defect. Complete absence of class-switched B cells was a sensitive predictor of AID deficiency and should be further evaluated as a diagnostic biomarker.
  
  
  CONCLUSIONS
  The biomarkers found in this study could be used to further study the complex process of B-cell activation and to understand conditions that lead to the development of primary antibody deficiencies.
authors.affiliation: Department of Pediatric Pneumology, Allergy and Neonatology, Hannover Medical School, Hannover, Germany. Electronic address: hennig.christian@mh-hannover.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Hennig
authors.affiliation: Department of Pediatric Pneumology, Allergy and Neonatology, Hannover Medical School, Hannover, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Ilginus
authors.affiliation: Department of Pediatric Oncology and Hematology, Hannover Medical School, Hannover, Germany; Research Center for Molecular Medicine (CeMM) of the Austrian Academy of Sciences, Vienna, Austria; Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria.
authors.forename: Kaan
authors.initials: K
authors.lastname: Boztug
authors.affiliation: Department of Pediatrics and Molecular Hematopoiesis, Hannover Medical School, Hannover, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Skokowa
authors.affiliation: University of Debrecen and Department of Infectious and Pediatric Immunology, Medical and Health Science Center, Debrecen, Hungary.
authors.forename: Laszlo
authors.initials: L
authors.lastname: Marodi
authors.affiliation: Department of Clinical Immunology, Polish-American Institute of Pediatrics, Jagiellonian University, Medical College, Krakow, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Szaflarska
authors.affiliation: Department of Pediatric Oncology and Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Mareike
authors.initials: M
authors.lastname: Sass
authors.affiliation: Department of Pediatrics, 'Federico II' University, Naples, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Pignata
authors.affiliation: Department of Pediatrics, Uludag University Medical Faculty, Bursa, Turkey.
authors.forename: Sara Sebnem
authors.initials: SS
authors.lastname: Kilic
authors.affiliation: Pediatric Infectious Diseases and Immunodeficiencies Unit, Vall d'Hebron Hospital, Barcelona, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Caragol
authors.affiliation: Department of Pediatric Pneumology, Allergy and Neonatology, Hannover Medical School, Hannover, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Baumann
authors.affiliation: Department of Pediatric Oncology and Hematology, Hannover Medical School, Hannover, Germany; Department of Pediatrics, Ludwig-Maximilians-Universität, Munich, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Klein
authors.affiliation: Department of Pediatrics and Molecular Hematopoiesis, Hannover Medical School, Hannover, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Welte
authors.affiliation: Department of Pediatric Pneumology, Allergy and Neonatology, Hannover Medical School, Hannover, Germany.
authors.forename: Gesine
authors.initials: G
authors.lastname: Hansen
mesh_terms: D000293:Adolescent; D000328:Adult; D001402:B-Lymphocytes / Q000276:immunology*; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D054357:Chemokine CXCL10 / Q000235:genetics / Q000378:metabolism; D054370:Chemokine CXCL9 / Q000235:genetics / Q000378:metabolism; D002648:Child; D005260:Female; D020869:Gene Expression Profiling; D057166:High-Throughput Screening Assays; D006801:Humans; D019044:Image Cytometry; D017578:Immunoglobulin Class Switching; D007153:Immunologic Deficiency Syndromes / Q000175:diagnosis* / Q000276:immunology; D007231:Infant, Newborn; D008213:Lymphocyte Activation / Q000235:genetics*; D008297:Male; D046228:Microarray Analysis; D008875:Middle Aged; D012333:RNA, Messenger / Q000032:analysis; D059467:Transcriptome / Q000276:immunology; D055815:Young Adult
chemical_list: D015415:Biomarkers; D054357:Chemokine CXCL10; D054370:Chemokine CXCL9; D012333:RNA, Messenger
keywords: AID; Activation-induced cytidine deaminase; B-cell immunology; CD40 ligand; CD40L; CSR; CVID; Class-switch recombination; Common variable immunodeficiency; MACS; Magnetic-activated cell sorting; PID; Primary immunodeficiency; chip cytometry; primary antibody deficiency