Classification of common variable immunodeficiencies using flow cytometry and a memory B-cell functionality assay

• Published in: Journal of allergy and clinical immunology Vol. 135; no. 1; pp. 198 - 208.e5
• Main Authors: Rösel, Amelia L., Scheibenbogen, Carmen, Schliesser, Ulrike, Sollwedel, André, Hoffmeister, Bodo, Hanitsch, Leif, von Bernuth, Horst, Krüger, Renate, Warnatz, Klaus, Volk, Hans-Dieter, Thomas, Sybill
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2015; Elsevier Limited
• Subjects: Adult; Age; Allergy and Immunology; antibody-secreting cells; B-Lymphocytes - cytology; B-Lymphocytes - immunology; Cell Differentiation; Cells, Cultured; Classification; Common Variable Immunodeficiency - blood; Common Variable Immunodeficiency - classification; Common Variable Immunodeficiency - immunology; common variable immunodeficiency subtyping; enzyme-linked immunosorbent spot assay; Enzyme-Linked Immunospot Assay; Female; Flow Cytometry; Humans; Immunoglobulin A - blood; Immunoglobulin G - blood; Immunoglobulin M - blood; Immunologic Memory; Leukocytes, Mononuclear - cytology; Male; Medical treatment; Memory B cell; Middle Aged; Patients; enzyme-linked immunosorbent spot assay; Ig-sPb; Memory B cell; common variable immunodeficiency subtyping; FACS; ELISpot; ASC; CVID; PB; PE; memBc; flow cytometry; HC; antibody-secreting cells; Healthy control; Immunoglobulin-secreting plasmablast; Common variable immunodeficiency; Enzyme-linked immunosorbent spot; Pacific Blue; Fluorescence-activated cell sorting; Antibody-secreting cell; Phycoerythrin
• ISSN: 0091-6749; 1097-6825; 1097-6825
• DOI: 10.1016/j.jaci.2014.06.022

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. 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. The memBc ELISpot assay, combined with flow cytometry, was applied to patients with confirmed CVID in comparison with age-matched healthy control subjects. 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. 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.