title: Classification of common variable immunodeficiencies using flow cytometry and a memory B-cell functionality assay.
abstract: BACKGROUND
  The population of patients with common variable immunodeficiency (CVID) comprises a heterogeneous group of patients with different causes of hypogammaglobulinemia predisposing to recurrent infections, higher incidence of autoimmunity, and malignancy. Although memory B cells (memBcs) are key players in humoral defense and their numbers are commonly reduced in these patients, their functionality is not part of any current classification.
  
  
  OBJECTIVE
  We established and validated a memBc enzyme-linked immunosorbent spot (ELISpot) assay that reveals the capacity of memBcs to develop into antibody-secreting cells and present an idea for a new classification based on this functional capacity.
  
  
  METHODS
  The memBc ELISpot assay, combined with flow cytometry, was applied to patients with confirmed CVID in comparison with age-matched healthy control subjects.
  
  
  RESULTS
  Ex vivo frequency of IgG-, IgM-, and IgA-secreting plasmablasts was significantly diminished by 27.2-, 2.4-, and 23.3-fold, respectively, compared with that seen in healthy control subjects. Moreover, in vitro differentiation of memBcs into antibody-secreting cells was 6.1-, 2.6-, and 3.7-fold significantly reduced for IgG-, IgM-, and IgA-secreting cells, respectively. Proliferation of memBcs correlates inversely to immunoglobulin-secreting capacity, suggesting compensatory hyperproliferation. Furthermore, patients with no serum IgA can still have a detectable IgA ELISpot assay result in vitro. Most importantly, the large heterogeneity of memBc function in patients with CVID homogenously grouped by means of fluorescence-activated cell sorting allowed additional subclassification based on memBc/plasmablast function.
  
  
  CONCLUSIONS
  These data suggest almost normal memBc/immunoglobulin-secreting plasmablast functionality in some patients if sufficient stimulatory signals are delivered, which might open up opportunities for new therapeutic approaches.
authors.affiliation: Institute for Medical Immunology, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Amelia L
authors.initials: AL
authors.lastname: Rösel
authors.affiliation: Institute for Medical Immunology, Charité University Medicine Berlin, Berlin, Germany; Berlin-Brandenburg Centre for Regenerative Therapies, Berlin, Germany; Out-patients Clinic for Immunodeficiencies, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Carmen
authors.initials: C
authors.lastname: Scheibenbogen
authors.affiliation: Berlin-Brandenburg Centre for Regenerative Therapies, Berlin, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Schliesser
authors.affiliation: Institute for Medical Immunology, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Sollwedel
authors.affiliation: Out-patients Clinic for Immunodeficiencies, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Bodo
authors.initials: B
authors.lastname: Hoffmeister
authors.affiliation: Out-patients Clinic for Immunodeficiencies, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Leif
authors.initials: L
authors.lastname: Hanitsch
authors.affiliation: Clinic for Pediatrics, Department of Pneumology and Immunology, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: von Bernuth
authors.affiliation: Clinic for Pediatrics, Department of Pneumology and Immunology, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Renate
authors.initials: R
authors.lastname: Krüger
authors.affiliation: Centre for Chronic Immunodeficiency, University Clinic and University of Freiburg, Freiburg, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Warnatz
authors.affiliation: Institute for Medical Immunology, Charité University Medicine Berlin, Berlin, Germany; Berlin-Brandenburg Centre for Regenerative Therapies, Berlin, Germany. Electronic address: hans-dieter.volk@charite.de.
authors.forename: Hans-Dieter
authors.initials: HD
authors.lastname: Volk
authors.affiliation: Institute for Medical Immunology, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Sybill
authors.initials: S
authors.lastname: Thomas
mesh_terms: D000328:Adult; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D017074:Common Variable Immunodeficiency / Q000097:blood / Q000145:classification / Q000276:immunology*; D058501:Enzyme-Linked Immunospot Assay; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D007156:Immunologic Memory; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008297:Male; D008875:Middle Aged
chemical_list: D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007075:Immunoglobulin M
keywords: Memory B cell; antibody-secreting cells; common variable immunodeficiency subtyping; enzyme-linked immunosorbent spot assay; flow cytometry