Categorizing B-cell defects using an in-vitro B-cell differentiation assay

Abstract

s ingediend voor het Amsterdam Kindersymposium 2013 61 Categorizing B-cell defects using an in-vitro B-cell differentiation assay Daan J. aan de Kerk (1,2), Machiel H. Jansen (1,2), Mirjam van der Burg (3), Ester M.M. van Leeuwen (2), Taco W. Kuijpers (1) (1) Department of pediatric hematology, immunology and infectious diseases, Emma Children’s Hospital, AMC, Amsterdam (2) Department of Experimental Immunology, AMC, Amsterdam (3) Department of Immunology, Erasmus MC, Rotterdam INTRODUCTION B-cells play an important role in primary immunodefi ciencies, about 65% of cases show altered antibody production. Nevertheless many B-cell defects still have not been characterized in functional terms and at the genetic level. Hence, we need good tools to classify at which diff erentiation stage such a B-cell defect can be located.To identify and further characterize B cell immunodefi ciencies we used an in-vitro assay comparing B-cell proliferation, diff erentiation and function for diff erent known class-switch recombination (CSR) defective patients compared to patients with uncharacterized hypogammaglobulinemia. METHODS An in-vitro assay, mimicking T-cell-dependent and T-cell-independent B-cell activation, was used to investigate the diff erentiation of B-cells. Proliferation was shown by CFSE dilution, diff erentiation into plasmacells by fl owcytometry and somatic hyper mutation (SHM) analysis, function was measured by immunoglobulin (IgG/M/A) production by Elisa. RESULTS B-cells of uncharacterized hypogammaglobulinemia and CSR defective patients showed diff erent blocks within their B-cell diff erentiation. While some of them diff erentiate into memory B-cells and plasmacells in vitro without producing any immunoglobulins, others fail to show any diff erentiation but produce large amount of IgM. Categorizing our fi ndings lead to more specifi c genetic screening for uncharacterized hypogammaglobulinemia, comparing them to known CSR defective patients. CONCLUSION Our fi ndings indicate that (defective) B-cell diff erentiation capacity in-vivo can be tested in-vitro. This also demonstrates that B-cell diff erentiation defects can be further categorized in this way, and then gives a better cue what genetic defect could be at the basis of this dysfunction