title: Identification of B cell defects using age-defined reference ranges for in vivo and in vitro B cell differentiation.
abstract: Primary immunodeficiencies consist to a large extent of B cell defects, as indicated by inadequate Ab levels or response upon immunization. Many B cell defects have not yet been well characterized. Our objective was to create reliable in vivo and in vitro assays to routinely analyze human B cell differentiation, proliferation, and Ig production and to define reference ranges for different age categories. The in vitro assays were applied to classify the developmental and/or functional B cell defects in patients previously diagnosed with common variable immunodeficiency. Apart from standard immunophenotyping of circulating human B cell subsets, an in vitro CFSE dilution assay was used for the assessment of proliferative capacity comparing T cell-dependent and T cell-independent B cell activation. Plasmablast/plasma cell differentiation was assessed by staining for CD20, CD38, and CD138, and measurement of in vitro Ig secretion. At young age, B cells proliferate upon in vitro activation, but neither differentiate nor produce IgG. These latter functions reached adult levels at 5 and 10 y of age for T cell-dependent versus T cell-independent stimulations, respectively. The capacity of B cells to differentiate into plasmablasts and to produce IgG appeared to be contained within the switched memory B cell pool. Using these assays, we could categorize common variable immunodeficiency patients into subgroups and identified a class-switch recombination defect caused by an UNG mutation in one of the patients. We defined age-related reference ranges for human B cell differentiation. Our findings indicate that in vivo B cell functionality can be tested in vitro and helps to diagnose suspected B cell defects.
authors.affiliation: Department of Pediatric Hematology, Immunology, and Infectious Diseases, Emma Children's Hospital, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands. d.j.aandekerk@amc.nl
authors.forename: Daan J
authors.initials: DJ
authors.lastname: aan de Kerk
authors.forename: Machiel H
authors.initials: MH
authors.lastname: Jansen
authors.forename: Ineke J M
authors.initials: IJ
authors.lastname: ten Berge
authors.forename: Ester M M
authors.initials: EM
authors.lastname: van Leeuwen
authors.forename: Taco W
authors.initials: TW
authors.lastname: Kuijpers
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000032:analysis; D000328:Adult; D000906:Antibodies / Q000097:blood; D018951:Antigens, CD20 / Q000032:analysis; D016175:B-Lymphocyte Subsets / Q000145:classification / Q000166:cytology / Q000276:immunology*; D001402:B-Lymphocytes / Q000145:classification / Q000166:cytology / Q000276:immunology*; D002454:Cell Differentiation / Q000276:immunology; D049109:Cell Proliferation; D002478:Cells, Cultured; D017074:Common Variable Immunodeficiency / Q000145:classification / Q000276:immunology*; D005260:Female; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis; D016130:Immunophenotyping; D008213:Lymphocyte Activation / Q000276:immunology*; D008297:Male; D012016:Reference Values; D053668:Syndecan-1 / Q000032:analysis; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000906:Antibodies; D018951:Antigens, CD20; D007136:Immunoglobulins; D053668:Syndecan-1; D051997:ADP-ribosyl Cyclase 1