No abstract

Several lines of evidence have demonstrated B cell intrinsic activation defects in patients with common variable immunodeficiency (CVID). The rapid increase of intracellular free calcium concentrations after engagement of the BCR represents one crucial element in this activation process. The analysis of 53 patients with CVID for BCR-induced calcium flux identified a subgroup of patients with significantly reduced Ca2+ signals in primary B cells. This subgroup strongly corresponded to the class Ia of the Freiburg classification. Comparison at the level of defined B cell subpopulations revealed reduced Ca2+ signals in all mature B cell populations of patients with CVID class Ia when compared with healthy individuals and other groups of patients with CVID but not in circulating transitional B cells. BCR-induced Ca2+ responses were the lowest in CD21low B cells in patients as well as healthy donors, indicating an additional cell-specific mechanism inhibiting the Ca2+ flux. Although proximal BCR signaling events are unperturbed in patients’ B cells, including normal phospholipase Cγ2 phosphorylation and Ca2+ release from intracellular stores, Ca2+ influx from the extracellular space is significantly impaired. CD22, a negative regulator of calcium signals in B cells, is highly expressed on CD21low B cells from patients with CVID Ia and might be involved in the attenuated Ca2+ response of this B cell subpopulation. These data from patients with CVID suggest that a defect leading to impaired BCR-induced calcium signaling is associated with the expansion of CD21low B cells, hypogammaglobulinemia, autoimmune dysregulation, and lymphadenopathy.

BackgroundFor their application in the area of diagnosis and therapy, single-domain antibodies (sdAbs) offer multiple advantages over conventional antibodies and fragments thereof in terms of size, stability, solubility, immunogenicity, production costs as well as tumor uptake and blood clearance. Thus, sdAbs have been identified as valuable next-generation targeting moieties for molecular imaging and drug delivery in the past years. Since these probes are much less complex than conventional antibody fragments, bacterial expression represents a facile method in order to produce sdAbs in large amounts as soluble and functional proteins.ResultsBy the combined use of high cell density cultivation media with a genetically engineered E. coli mutant strain designed for the cytoplasmic formation of proper disulfide bonds, we achieved high level of intracellular sdAb production (up to 200 mg/L). Due to a carboxyterminal hexahistidine epitope, the soluble recombinant sdAbs could be purified by one-step immobilized metal affinity chromatography to apparent homogeneity and easily radiolabeled with 99mTc within 1 h. The intradomain disulfide bridge being critical for the stability and functionality of the sdAb molecule was shown to be properly formed in ~96% of the purified proteins. In vitro binding studies confirmed the high affinity and specificity of the expressed sdAb 7C12 towards its molecular target.ConclusionsOur study demonstrates an efficient cultivation and expression strategy for the production of substantial amounts of soluble and functional sdAbs, which may be adopted for high-yield production of other more complex proteins with multiple disulfides as well.