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.

Phenotypic and Functional Comparison of Class Switch Recombination Deficiencies with a Subgroup of Common Variable Immunodeficiencies

“…On the other hand, patients who suffered from defects in AICDA showed normal numbers of non-switched B cells and even some memory sIgD − CD27 + B cells that had not undergone any class switching, i.e., these cells did not show any sIgG or sIgA expression and expressed sIgM only. Similar to patients with an AICDA gene defect, the individual that had been identified with an UNG gene defect [15], contained non-switched sIgM + B cell population in the absence of sIgD − CD27 + B cells, indicating a lack of switched sIgG + and sIgA + memory B cells (Table 3). …”

“…Using our previously described experimental conditions for B cell activation [15], we have to date functionally screened over 40 PAD patients who were suspected or diagnosed with CVID by their clinicians because of their clinical presentation, low serum IgG and IgA, and a lack of humoral response to polysaccharides at presentation.…”

“…The capacity of the B cells to proliferate and differentiate upon in vitro activation in a 6-day culture was tested with CpG in the presence of a small B cell activating dose of IL-2 (to which purified T cells do not show proliferation and cytokine induction and acts by direct B cell activation of the IL-2 receptor) [15, 21]. T cell-dependent B cell stimulation was mimicked by the combination of antibodies against sIgM to trigger the B cell antigen-receptor (BCR) on the majority of circulating B cells in the blood, together with costimulatory CD40 activation and Tfh cell-associated IL-21 (αIgM/αCD40/IL-21) [22].…”

“…Supernatants were tested for secreted IgM and IgG with an in-house ELISA using polyclonal rabbit anti-human IgG and IgM reagents and a serum protein calibrator all from Dako (Heverlee, Belgium), as described before [15]…”

“…We have previously used a comprehensive culture system to characterize CVID in functional terms by the capacity of the patient’s B cells to proliferate and differentiate into Ig-secreting plasmablasts (PBs) [15]. In this way, we have identified a subgroup of CVID patients, which was highly reminiscent of CSR-like B cell defects.…”