Research-based flow cytometry assays for pathogenic assessment in the human B-cell biology of gene variants revealed in the diagnosis of inborn errors of immunity: a Bruton's tyrosine kinase case-study.

Introduction: Inborn errors of immunity (IEI) are an expanding group of rare diseases whose field has been boosted by next-generation sequencing (NGS), revealing several new entities, accelerating routine diagnoses, expanding the number of atypical presentations and generating uncertainties regarding the pathogenic relevance of several novel variants. Methods: Research laboratories that diagnose and provide support for IEI require accurate, reproducible and sustainable phenotypic, cellular and molecular functional assays to explore the pathogenic consequences of human leukocyte gene variants and contribute to their assessment. We have implemented a set of advanced flow cytometry-based assays to better dissect human B-cell biology in a translational research laboratory. We illustrate the utility of these techniques for the in-depth characterization of a novel (c.1685G>A, p.R562Q) de novo gene variant predicted as probably pathogenic but with no previous insights into the protein and cellular effects, located in the tyrosine kinase domain of the Bruton's tyrosine kinase (BTK) gene, in an apparently healthy 14-year-old male patient referred to our clinic for an incidental finding of low immunoglobulin (Ig) M levels with no history of recurrent infections. Results and discussion: A phenotypic analysis of bone marrow (BM) revealed a slightly high percentage of pre-B-I subset in BM, with no blockage at this stage, as typically observed in classical X-linked agammaglobulinemia (XLA) patients. The phenotypic analysis in peripheral blood also revealed reduced absolute numbers of B cells, all pre-germinal center maturation stages, together with reduced but detectable numbers of different memory and plasma cell isotypes. The R562Q variant allows Btk expression and normal activation of anti-IgM-induced phosphorylation of Y551 but diminished autophosphorylation at Y223 after anti IgM and CXCL12 stimulation. Lastly, we explored the potential impact of the variant protein for downstream Btk signaling in B cells. Within the canonical nuclear factor kappa B (NF-κB) activation pathway, normal IκBα degradation occurs after CD40L stimulation in patient and control cells. In contrast, disturbed IκBα degradation and reduced calcium ion (Ca2+) influx occurs on anti-IgM stimulation in the patient's B cells, suggesting an enzymatic impairment of the mutated tyrosine kinase domain.

Defective Bcl-2 expression in memory B cells from common variable immunodeficiency patients.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and different degrees of B cell compartment alteration. Memory B cell differentiation requires the orchestrated activation of several intracellular signaling pathways that lead to the activation of a number of factors, such as nuclear factor kappa B (NF-κB) which, in turn, promote transcriptional programs required for long-term survival. The aim of this study was to determine if disrupted B cell differentiation, survival and activation in B cells in CVID patients could be related to defects in intracellular signaling pathways. For this purpose, we selected intracellular readouts that reflected the strength of homeostatic signaling pathways in resting cells, as the protein expression levels of the Bcl-2 family which transcription is promoted by NF-κB. We found reduced Bcl-2 protein levels in memory B cells from CVID patients. We further explored the possible alteration of this crucial prosurvival signaling pathway in CVID patients by analysing the expression levels of mRNAs from anti-apoptotic proteins in naive B cells, mimicking T cell-dependent activation in vitro with CD40L and interleukin (IL)-21. BCL-XL mRNA levels were decreased, together with reduced levels of AICDA, after naive B-cell activation in CVID patients. The data suggested a molecular mechanism for this tendency towards apoptosis in B cells from CVID patients. Lower Bcl-2 protein levels in memory B cells could compromise their long-term survival, and a possible less activity of NF-κB in naive B cells, may condition an inabilityto increase BCL-XL mRNA levels, thus not promoting survival in the germinal centers.

Mutations in PIK3R1 can lead to APDS2, SHORT syndrome or a combination of the two.

Mutations in PIK3R1 gene have been associated to two different conditions: a primary immunodeficiency, called APDS2, of recent description and SHORT syndrome. 47 patients with APDS2 have been reported to date, only one of them sharing both PIK3R1-related phenotypes. Here we describe two more patients affected by APDS2 and SHORT syndrome, which highlights that this association may not be so infrequent. We recommend that patients with mutations in PIK3R1 gene should be assessed by both clinical immunologists and clinical geneticists.

Hypercalcemia, hypervitaminosis A and 3-epi-25-OH-D3 levels after consumption of an "over the counter" vitamin D remedy. a case report.

Intoxication from vitamin D supplements has been rarely reported but, nowadays, it occurs more frequently. 3-epi-25-OH-D(3) is highly prevalent in adults and it is considered of biological relevance. We report a case of vitamin D toxicity with hypercalcemia, acute renal failure and hypervitaminosis A after consuming an over-the-counter vitamin D supplement. Our data suggest that the contribution of 3-epi-25-OH-D(3) is not altered during vitamin D toxicity, although the serum levels of 25-OH-D(3) and 3-epi-25-OH-D(3) may display a different rate of clearance. The patient also displayed hypervitaminosis A unrelated to diet, possibly caused by renal failure related to the hypercalcemia induced by vitamin D toxicity. Because of the increasing use of over-the-counter vitamin D supplements and the potential iatrogenic hypercalcemia related to hypervitaminosis A, the present case highlights the importance of evaluating both the use of (non-) prescribed medication and vitamin A status during vitamin D toxicity.

Calcitonin gene-related peptide modulates interleukin-13 in circulating cutaneous lymphocyte-associated antigen-positive T cells in patients with atopic dermatitis.

BACKGROUND: Neuropeptides (NPs) may play an important role in the pathogenesis of atopic dermatitis (AD) by regulating immune responses and contributing to the cross-talk between the immune and nervous systems. OBJECTIVES: To assess the ability of NPs to influence interleukin (IL)-13 and interferon (IFN)-gamma production and the expression of the activation marker HLA-DR in skin memory T cells [cutaneous lymphocyte-associated antigen (CLA)+ T cells] from patients with AD with severe, chronic lesions and intense pruritus, and from nonatopic controls. METHODS: Cells were cultured in the presence and absence of different NPs, calcitonin gene-related peptide (CGRP), somatostatin (SOM) and substance P (SP). IL-13 and IFN-gamma production and HLA-DR expression were measured in both CLA+ and CLA- T-cell subsets by flow cytometry. RESULTS: CGRP increased IL-13 production in peripheral blood mononuclear cells from patients with AD (P < 0.05), with no changes detected in the presence of SOM or SP. These patients with AD had a lower expression of CGRP receptor compared with controls (P < 0.05). Memory T cells incubated with CGRP also showed an increase in IL-13 (P < 0.05) and HLA-DR (P < 0.05) in CLA+ T cells from patients with AD compared with controls, but not in CLA- T cells. Patients with a higher production of IL-13 were those with higher total IgE and percentage of skin area involved. Furthermore, the IL-13/IFN-gamma ratio was increased in patients with AD after cells were cultured with CGRP (P < 0.05). CONCLUSIONS: Our results suggest an immunomodulatory role of CGRP towards a Th2 pattern in CLA+ T cells, which may contribute to exacerbating clinical symptoms in patients with AD.

Lymphocyte proliferation response in patients with delayed hypersensitivity reactions to heparins.

BACKGROUND: Heparins can induce delayed-type hypersensitivity (DTH) reactions mediated by specific T lymphocytes. However, the interaction between heparins and lymphocytes has not been sufficiently studied. OBJECTIVES: To analyse the lymphocyte response to heparins using different types of antigen-presenting cells in patients with DTH reactions to these drugs. METHODS: We studied seven patients with DTH reactions to heparins diagnosed by delayed reading of intradermal skin testing (n = 5) or drug provocation test (n = 2) and nine tolerant controls. Biopsies were obtained from intradermal testing or during the acute reaction. Peripheral blood mononuclear cells were used to obtain T and B lymphocytes, monocytes and monocyte-derived dendritic cells (DC). T-lymphocyte proliferation was assayed by means of (3)H-thymidine incorporation. RESULTS: Skin testing showed a high degree of cross-reactivity within low molecular weight heparins with good tolerance to sodium heparin, fondaparinux and lepirudin in most cases. The proliferative response was positive in six patients to most of the heparins tested with both monocytes and B cells (the classical lymphocyte transformation test) or immature DC as antigen-presenting cells, giving a higher response with DC. At a second evaluation 1 year later, the proliferative response was found only with DC, and mainly to the culprit drug. CONCLUSIONS: A model using DC in the lymphocyte proliferation test is a more appropriate way to assess the immunological response in DTH to heparins; additionally, it can detect a response over a longer time. These findings may be useful for the diagnostic evaluation of drug reactions.

T cell assessment in allergic drug reactions during the acute phase according to the time of occurrence.

Allergic drug reactions can be classified as immediate, accelerated or delayed. This classification usually correlates with the mechanism involved: immediate reactions are IgE mediated and delayed reactions are T cell dependent. We analyzed lymphocyte involvement in patients with these reactions by determining cell subpopulations, activation state and skin homing receptor expression (CLA) in blood and skin. Patients with immediate, accelerated and delayed reactions were evaluated during the acute phase and after resolution. Controls taking drugs were included. Phenotypic immunofluorescence analysis was done by flow cytometry in peripheral blood, and by immunohistochemistry in skin for delayed reactions. Forty-six patients were included, 17 with immediate reactions, 10 accelerated and 19 delayed. At the acute phase CLA was significantly increased in delayed reactions and HLA-DR in all three types of reaction. In the severest delayed reactions, Steven-Johnson/Lyell syndromes, the CD4 subsets were increased in peripheral blood and skin compared to maculopapular exanthemas and urticaria and HLA-DR when compared with urticaria. In maculopapular exanthemas CLA was significantly increased in peripheral blood and skin compared to urticaria and the severe reactions. We found that T-cells are implicated, besides delayed reactions, in immediate and accelerated reactions. In delayed reactions there is a parallelism between results found in skin and peripheral blood with a higher participation of CD4+ cells the more severe the reaction.