Craig D. Platt

Mature dendritic cells use endocytic receptors to capture and present antigens.

In response to inflammatory stimuli, dendritic cells (DCs) trigger the process of maturation, a terminal differentiation program required to initiate T-lymphocyte responses. A hallmark of maturation is down-regulation of endocytosis, which is widely assumed to restrict the ability of mature DCs to capture and present antigens encountered after the initial stimulus. We found that mature DCs continue to accumulate antigens, especially by receptor-mediated endocytosis and phagocytosis. Internalized antigens are transported normally to late endosomes and lysosomes, loaded onto MHC class II molecules (MHCII), and then presented efficiently to T cells. This occurs despite the fact that maturation results in the general depletion of MHCII from late endocytic compartments, with MHCII enrichment being typically thought to be a required feature of antigen processing and peptide loading compartments. Internalized antigens can also be cross-presented on MHC class I molecules, without any reduction in efficiency relative to immature DCs. Thus, although mature DCs markedly down-regulate their capacity for macropinocytosis, they continue to capture, process, and present antigens internalized via endocytic receptors, suggesting that they may continuously initiate responses to newly encountered antigens during the course of an infection.

Exaggerated follicular helper T-cell responses in patients with LRBA deficiency caused by failure of CTLA4-mediated regulation

Background: LPS‐responsive beige‐like anchor protein (LRBA) and cytotoxic T lymphocyte–associated antigen 4 (CTLA4) deficiencies give rise to overlapping phenotypes of immune dysregulation and autoimmunity, with dramatically increased frequencies of circulating follicular helper T (cTFH) cells. Objective: We sought to determine the mechanisms of cTFH cell dysregulation in patients with LRBA deficiency and the utility of monitoring cTFH cells as a correlate of clinical response to CTLA4‐Ig therapy. Methods: cTFH cells and other lymphocyte subpopulations were characterized. Functional analyses included in vitro follicular helper T (TFH) cell differentiation and cTFH/naive B‐cell cocultures. Serum soluble IL‐2 receptor &agr; chain levels and in vitro immunoglobulin production by cultured B cells were quantified by using ELISA. Results: cTFH cell frequencies in patients with LRBA or CTLA4 deficiency sharply decreased with CTLA4‐Ig therapy in parallel with other markers of immune dysregulation, including soluble IL‐2 receptor &agr; chain, CD45RO+CD4+ effector T cells, and autoantibodies, and this was predictive of favorable clinical responses. cTFH cells in patients with LRBA deficiency were biased toward a TH1‐like cell phenotype, which was partially reversed by CTLA4‐Ig therapy. LRBA‐sufficient but not LRBA‐deficient regulatory T cells suppressed in vitro TFH cell differentiation in a CTLA4‐dependent manner. LRBA‐deficient TFH cells supported in vitro antibody production by naive LRBA‐sufficient B cells. Conclusions: cTFH cell dysregulation in patients with LRBA deficiency reflects impaired control of TFH cell differentiation because of profoundly decreased CTLA4 expression on regulatory T cells and probably contributes to autoimmunity in patients with this disease. Serial monitoring of cTFH cell frequencies is highly useful in gauging the clinical response of LRBA‐deficient patients to CTLA4‐Ig therapy.

Combined immunodeficiency with EBV positive B cell lymphoma and epidermodysplasia verruciformis due to a novel homozygous mutation in RASGRP1

RASGRP1 is a guanine-nucleotide-exchange factor essential for MAP-kinase mediated signaling in lymphocytes. We report the second case of RASGRP1 deficiency in a patient with a homozygous nonsense mutation in the catalytic domain of the protein. The patient had epidermodysplasia verruciformis, suggesting a clinically important intrinsic T cell function defect. Like the previously described patient, our proband also presented with CD4+ T cell lymphopenia, impaired T cell proliferation to mitogens and antigens, reduced NK cell function, and EBV-associated lymphoma. The severity of the disease and the development of EBV lymphoma in both patients suggest that hematopoietic stem cell transplantation should be performed rapidly in patients with RASGRP1 deficiency.

Leucine-rich repeat containing 8A (LRRC8A)–dependent volume-regulated anion channel activity is dispensable for T-cell development and function

Background: Leucine‐rich repeat containing 8A (LRRC8A) is an ubiquitously expressed transmembrane protein with 17 leucine‐rich repeats (LRRs) at its C‐terminal end and is an essential component of the volume‐regulated anion channel (VRAC), which controls cellular volume. A heterozygous mutation in LRRC8A that truncates the 2 terminal LRRs was reported in a patient with agammaglobulinemia and absent B cells and was demonstrated to exert a dominant negative effect on T‐ and B‐cell development in mice. Lrrc8a−/− mice have severely defective T‐cell development and function. It is not known whether the T‐ and B‐cell defects caused by LRRC8A deficiency are caused by loss of VRAC activity. Objective: We sought to determine whether VRAC activity is required for normal T‐cell development and function. Methods: VRAC activity was examined by using patch‐clamp analysis. Flow cytometry was used to examine T‐cell development. T‐cell proliferation, cytokine secretion, and antibody titers were measured by using standard techniques. Results: We demonstrate that the spontaneous mouse mutant ébouriffé (ebo/ebo) harbors a homozygous 2‐bp frameshift mutation in Lrrc8a that truncates the 15 terminal LRRs of LRRC8A. The Lrrc8aebo mutation does not affect protein expression but drastically diminishes VRAC activity in T cells. ebo/ebo mice share features with Lrrc8a−/− mice that include curly hair, infertility, reduced longevity, and kidney abnormalities. However, in contrast to Lrrc8a−/− mice, ebo/ebo mice have normal T‐cell development and function and intact antibody response to T‐dependent antigen. Conclusion: LRRC8A‐dependent VRAC activity is dispensable for T‐cell development and function.

Clinical, immunologic, and genetic spectrum of 696 patients with combined immunodeficiency

Background: Combined immunodeficiencies (CIDs) are diseases of defective adaptive immunity with diverse clinical phenotypes. Although CIDs are more prevalent in the Middle East than Western countries, the resources for genetic diagnosis are limited. Objectives: This study aims to characterize the categories of patients with CIDs in Iran clinically and genetically. Methods: Clinical and laboratory data were obtained from 696 patients with CIDs. Patients were subdivided into those with syndromic (344 patients) and nonsyndromic (352 patients) CIDs. Targeted DNA sequencing was performed on 243 (34.9%) patients. Results: The overall diagnostic yield of the 243 sequenced patients was 77.8% (189 patients). The clinical diagnosis of hyper‐IgE syndrome (P < .001), onset of disease at greater than 5 years (P = .02), and absence of multiple affected family members (P = .04) were significantly more frequent in the patients without a genetic diagnosis. An autosomal recessive disease was found in 62.9% of patients, reflecting the high rate of consanguinity in this cohort. Mutations impairing VDJ recombination and DNA repair were the most common underlying causes of CIDs. However, in patients with syndromic CIDs, autosomal recessive mutations in ataxia‐telangiectasia mutated (ATM), autosomal dominant mutations in signal transducer and activator of transcription 3 (STAT3), and microdeletions in 22q11.21 were the most commonly affected genomic loci. Patients with syndromic CIDs had a significantly lower 5‐year survival rate rather than those with nonsyndromic CIDs. Conclusions: This study provides proof of principle for the application of targeted next‐generation sequencing panels in countries with limited diagnostic resources. The effect of genetic diagnosis on clinical care requires continued improvements in therapeutic resources for these patients.

The LRRC8A mediated "swell activated" chloride conductance is dispensable for vacuolar homeostasis in neutrophils

The dialysis of human and mouse neutrophils in patch clamp experiments in the conventional whole-cell mode induces the emergence of a chloride (Cl-) current that appeared to be primarily regulated by cytoplasmic ionic strength. The characteristics of this current resembled that of the classical, and ubiquitous volume-sensitive outwardly rectifying Cl- current: strong outward rectification, selectivity sequence of the Eisenman1 type, insensitivity to external pH and strong inhibition by tamoxifen, DCPIB and WW781. We show that this current is essentially supported by the leucine rich repeat containing 8 A (LRRC8A); the naturally occurring LRRC8A truncation mutant in ebo/ebo mice drastically reduced Cl- conductance in neutrophils. Remarkably, the residual component presents a distinct pharmacology, but appears equally potentiated by reduced ionic strength. We have investigated the role of the LRRC8A-supported current in the ionic homeostasis of the phagosomal compartment. The vacuolar pH, measured using SNARF-1 labeled Candida albicans, normally rises because of NADPH oxidase activity, and this elevation is blocked by certain Cl- channel inhibitors. However, the pH rise remains intact in neutrophils from the ebo/ebo mice which also demonstrate preserved phagocytic and respiratory burst capacities and normal-sized vacuoles. Thus, the LRRC8A-dependent conductance of neutrophils largely accounts for their “swell activated” Cl- current, but is not required for homeostasis of the phagosomal killing compartment.

A quality improvement initiative to increase access to food challenges

Food challenges are critical to determine if an individual has food allergy when history and testing are inconclusive or the allergy may have been outgrown.(1) Passing a challenge may have nutritional, psychological and financial benefits.(1-3) We and others have shown that challenges can be safely carried out in a clinic setting.(4-9) This article is protected by copyright. All rights reserved.

Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice

Ion channel-controlled cell volume regulation is of fundamental significance to the physiological function of sperm. In addition to volume regulation, LRRC8A-dependent volume-regulated anion channel (VRAC) activity is involved in cell cycle progression, insulin signaling, and cisplatin resistance. Nevertheless, the contribution of LRRC8A and its dependent VRAC activity in the germ cell lineage remain unknown. By utilizing a spontaneous Lrrc8a mouse mutation (c.1325delTG, p.F443*) and genetically engineered mouse models, we demonstrate that LRRC8A-dependent VRAC activity is essential for male germ cell development and fertility. Lrrc8a-null male germ cells undergo progressive degeneration independent of the apoptotic pathway during postnatal testicular development. Lrrc8a-deficient mouse sperm exhibit multiple morphological abnormalities of the flagella (MMAF), a feature commonly observed in the sperm of infertile human patients. Importantly, we identified a human patient with a rare LRRC8A hypomorphic mutation (c.1634G>A, p.Arg545His) possibly linked to Sertoli cell-only syndrome (SCOS), a male sterility disorder characterized by the loss of germ cells. Thus, LRRC8A is a critical factor required for germ cell development and volume regulation in the mouse, and it might serve as a novel diagnostic and therapeutic target for SCOS patients.