Identification of B cell defects using age-defined reference ranges for in vivo and in vitro B cell differentiation

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 defect

Transition of adolescents with inflammatory bowel disease from pediatric to adult care

Pediatric Inflammatory Bowel Disease (IBD) patients eventually need to transition to adult settings. Transition is of interest in many chronic illnesses with childhood onset. Pediatric providers should understand adult providers' expectations to better prepare their patients. Using a recent North American survey, we explored the insights of adult gastroenterologists in the Netherlands. The survey was sent to 288 Dutch adult gastroenterologists. Respondents indicated the importance of various transition issues, and reported which problems occurred often in their practice. They also evaluated importance and personal competence regarding uniquely adolescent medical issues. A response rate of 47% was achieved. Patients' ability to discuss IBD impact on overall daily life (60%), knowledge of their medications (53%) and impact of substance use on their health (53%) were often problematic. Patients' ability to attend the visit alone (12%) or identify health care providers (9%) were infrequently problematic. While transfer of accurate medical history by pediatricians was ranked of highest importance, it was rarely problematic (14%). Academic and younger physicians reported a higher frequency of problems. Dutch gastroenterologists reported less problems than their American colleagues. Many respondents deemed medical (94%) and developmental (89%) issues in adolescence important. However, fewer respondents reported competency regarding those issues (61% and 34%, respectively). Conclusion: Pediatric providers should focus patient education on areas recognized as important and problematic by adult providers. Adolescent medical and developmental issues should be incorporated in specialist training. Dutch providers report less problems, perhaps due the proximity of pediatric and adult health facilities

High-throughput compound screen reveals mTOR inhibitors as potential therapeutics to reduce (auto)antibody production by human plasma cells

Antibody production by the B cell compartment is a crucial part of the adaptive immune response. Dysregulated antibody production in the form of autoantibodies can cause autoimmune disease. To date, B-cell depletion with anti-CD20 antibodies is commonly applied in autoimmunity, but pre-existing plasma cells are not eliminated in this way. Alternative ways of more selective inhibition of antibody production would add to the treatment of these autoimmune diseases. To explore novel therapeutic targets in signaling pathways essential for plasmablast formation and/or immunoglobulin production, we performed a compound screen of almost 200 protein kinase inhibitors in a robust B-cell differentiation culture system. This study yielded 35 small cell-permeable compounds with a reproducible inhibitory effect on B-cell activation and plasmablast formation, among which was the clinically applied mammalian target of rapamycin (mTOR) inhibitor rapamycin. Two additional compounds targeting the phosphoinositide 3-kinase-AKT-mTOR pathway (BKM120 and WYE-354) did not affect proliferation and plasmablast formation, but specifically reduced the immunoglobulin production. With this compound screen we successfully applied a method to investigate therapeutic targets for B-cell differentiation and identified compounds in the phosphoinositide 3-kinase-AKT-mTOR pathway that could specifically inhibit immunoglobulin production only. These drugs may well be explored to be of value in current B-cell-depleting treatment regimens in autoimmune disorders

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

Primary antibody deficiencies (PADs) are the most common immunodeficiency in humans, characterized by low levels of immunoglobulins and inadequate antibody responses upon immunization. These PADs may result from an early block in B cell development with a complete absence of peripheral B cells and lack of immunoglobulins. In the presence of circulating B cells, some PADs are genetically caused by a class switch recombination (CSR) defect, but in the most common PAD, common variable immunodeficiency (CVID), very few gene defects have as yet been characterized despite various phenotypic classifications. Using a functional read-out, we previously identified a functional subgroup of CVID patients with plasmablasts (PBs) producing IgM only. We have now further characterized such CVID patients by a direct functional comparison with patients having genetically well-characterized CSR defects in CD40L, activation-induced cytidine deaminase (AID) and uracil N-glycosylase activity (UNG). The CSR-like CVID patients showed a failure in B cell activation patterns similar to the classical AID/UNG defects in three out of five CVID patients and distinct more individual defects in the two other CVID cases when tested for cellular activation and PB differentiation. Thus, functional categorization of B cell activation and differentiation pathways extends the expected variation in CVID to CSR-like defects of as yet unknown genetic etiolog