Analysis of immune synapses by τau-STED imaging and 3D-quantitative colocalization of lytic granule markers

Methods in cell biology · 2023-02-26 · 3 citations

Novel Compound Heterozygous ZAP70 R37G A507T Mutations in Infant with Severe Immunodeficiency

Journal of Clinical Immunology · 2023-12-22 · 2 citations

Extended-Synaptotagmin-1 and -2 control T cell signaling and function

EMBO Reports · 2023-12-19 · 4 citations

Upon T-cell activation, the levels of the secondary messenger diacylglycerol (DAG) at the plasma membrane need to be controlled to ensure appropriate T-cell receptor signaling and T-cell functions. Extended-Synaptotagmins (E-Syts) are a family of inter-organelle lipid transport proteins that bridge the endoplasmic reticulum and the plasma membrane. In this study, we identify a novel regulatory mechanism of DAG-mediated signaling for T-cell effector functions based on E-Syt proteins. We demonstrate that E-Syts downmodulate T-cell receptor signaling, T-cell-mediated cytotoxicity, degranulation, and cytokine production by reducing plasma membrane levels of DAG. Mechanistically, E-Syt2 predominantly modulates DAG levels at the plasma membrane in resting-state T cells, while E-Syt1 and E-Syt2 negatively control T-cell receptor signaling upon stimulation. These results reveal a previously underappreciated role of E-Syts in regulating DAG dynamics in T-cell signaling.

Apoptotic vesicles ameliorate lupus and arthritis via phosphatidylserine-mediated modulation of T cell receptor signaling

Bioactive Materials · 2022-08-09 · 57 citations

Mesenchymal stem cells (MSCs) influence T cells in health, disease and therapy through messengers of intercellular communication including extracellular vesicles (EVs). Apoptosis is a mode of cell death that tends to promote immune tolerance, and a large number of apoptotic vesicles (apoVs) are generated from MSCs during apoptosis. In an effort to characterize these apoVs and explore their immunomodulatory potential, here we show that after replenishing them systemically, the apoV deficiency in Fas mutant mice and pathological lymphoproliferation were rescued, leading to the amelioration of inflammation and lupus activity. ApoVs directly interacted with CD4+ T cells and inhibited CD25 expression and IL-2 production in a dose-dependent manner. A broad range of Th1/2/17 subsets and cytokines including IFNγ, IL17A and IL-10 were suppressed while Foxp3+ cells were maintained. Mechanistically, exposed phosphatidylserine (PtdSer/PS) on apoVs mediated the interaction with T cells to disrupt proximal T cell receptor signaling transduction. Remarkably, administration of apoVs prevented Th17 differentiation and memory formation, and ameliorated inflammation and joint erosion in murine arthritis. Collectively, our findings unveil a previously unrecognized crosstalk between MSC apoVs and CD4+ T cells and suggest a promising therapeutic use of apoVs for autoimmune diseases.

<i>RHOG</i>: Rac1-ing up another HLH gene

Blood · 2021-04-15 · 1 citations

Variants in <i>STXBP3</i> are Associated with Very Early Onset Inflammatory Bowel Disease, Bilateral Sensorineural Hearing Loss and Immune Dysregulation

Journal of Crohn s and Colitis · 2021-04-22 · 16 citations

BACKGROUND AND AIMS: Very early onset inflammatory bowel disease [VEOIBD] is characterized by intestinal inflammation affecting infants and children less than 6 years of age. To date, over 60 monogenic aetiologies of VEOIBD have been identified, many characterized by highly penetrant recessive or dominant variants in underlying immune and/or epithelial pathways. We sought to identify the genetic cause of VEOIBD in a subset of patients with a unique clinical presentation. METHODS: Whole exome sequencing was performed on five families with ten patients who presented with a similar constellation of symptoms including medically refractory infantile-onset IBD, bilateral sensorineural hearing loss and, in the majority, recurrent infections. Genetic aetiologies of VEOIBD were assessed and Sanger sequencing was performed to confirm novel genetic findings. Western analysis on peripheral blood mononuclear cells and functional studies with epithelial cell lines were employed. RESULTS: In each of the ten patients, we identified damaging heterozygous or biallelic variants in the Syntaxin-Binding Protein 3 gene [STXBP3], a protein known to regulate intracellular vesicular trafficking in the syntaxin-binding protein family of molecules, but not associated to date with either VEOIBD or sensorineural hearing loss. These mutations interfere with either intron splicing or protein stability and lead to reduced STXBP3 protein expression. Knock-down of STXBP3 in CaCo2 cells resulted in defects in cell polarity. CONCLUSION: Overall, we describe a novel genetic syndrome and identify a critical role for STXBP3 in VEOIBD, sensorineural hearing loss and immune dysregulation.

Colonoids From Patients With Pediatric Inflammatory Bowel Disease Exhibit Decreased Growth Associated With Inflammation Severity and Durable Upregulation of Antigen Presentation Genes

Inflammatory Bowel Diseases · 2020-06-19 · 21 citations

BACKGROUND: Defining epithelial cell contributions to inflammatory bowel disease (IBD) is essential for the development of much needed therapies for barrier repair. Children with very early onset (VEO)-IBD have more extensive, severe, and refractory disease than older children and adults with IBD and, in some cases, have defective barrier function. We therefore evaluated functional and transcriptomic differences between pediatric IBD (VEO and older onset) and non-IBD epithelium using 3-dimensional, biopsy-derived organoids. METHODS: We measured growth efficiency relative to histopathological and clinical parameters in patient enteroid (ileum) and colonoid (colon) lines. We performed RNA-sequencing on patient colonoids and subsequent flow cytometry after multiple passages to evaluate changes that persisted in culture. RESULTS: Enteroids and colonoids from pediatric patients with IBD exhibited decreased growth associated with histological inflammation compared with non-IBD controls. We observed increased LYZ expression in colonoids from pediatric IBD patients, which has been reported previously in adult patients with IBD. We also observed upregulation of antigen presentation genes HLA-DRB1 and HLA-DRA, which persisted after prolonged passaging in patients with pediatric IBD. CONCLUSIONS: We present the first functional evaluation of enteroids and colonoids from patients with VEO-IBD and older onset pediatric IBD, a subset of which exhibits poor growth. Enhanced, persistent epithelial antigen presentation gene expression in patient colonoids supports the notion that epithelial cell-intrinsic differences may contribute to IBD pathogenesis.

STXBP2-R190C Variant in a Patient With Neonatal Hemophagocytic Lymphohistiocytosis (HLH) and G6PD Deficiency Reveals a Critical Role of STXBP2 Domain 2 on Granule Exocytosis

Frontiers in Immunology · 2020-10-08 · 15 citations

Neonatal hemophagocytic lymphohistiocytosis (HLH) is a medical emergency that can be associated with significant morbidity and mortality. Often these patients present with familial HLH (f-HLH) which is caused by gene mutations interfering with the cytolytic pathway of Cytotoxic T-lymphocytes (CTLs) and Natural Killer (NK) cells. Here we describe a male newborn who met HLH-diagnostic criteria, presented with profound cholestasis and carried a maternally inherited heterozygous mutation in Syntaxin-Binding Protein-2 [STXBP2, c.568C&gt;T (p.Arg190Cys)] in addition to a severe pathogenic variant in Glucose 6-Phosphate Dehydrogenase [G6PD, Hemizygous c.1153T&gt;C (Cys385Arg)]. Although mutations in STXBP2 gene are associated with f-HLH type 5, the clinical and biological relevance of the p.Arg190Cys mutation identified in this patient was uncertain. To assess its role in disease pathogenesis, we performed functional assays and biochemical and microscopic studies. We found that p.Arg190Cys mutation did not alter the expression or subcellular localization of STXBP2 or STX11, neither impaired the STXBP2/STX11 interaction. In contrast, forced expression of the mutated protein into normal CTLs strongly inhibited degranulation and reduced the cytolytic activity out-competing the effect of endogenous wild-type STXBP2. Interestingly, Arginine 190 is located in a structurally conserved region of STXBP2 where other f-HLH-5 mutations have been identified. Collectively, data strongly suggest that STXBP2-R190C is a deleterious variant that acts in a dominant-negative manner by probably stabilizing non-productive interactions between STXBP2/STX11 complex and other still unknown factors such as the membrane surface or Munc13-4 protein and thus impairing the release of cytolytic granules. In addition to the contribution of STXBP2-R190C to f-HLH, the accompanied G6PD mutation may have compounded the clinical symptoms; however the extent by which G6PD deficiency has contributed to HLH in our patient remains unclear

883 – Integration of Clinical, Immunologic and Genomic Data in Children with Very-Early Onset Inflammatory Bowel Disease Leads to Identification of Candidates for Curative Therapy with Hematopoeitic Stem Cell Transplantation

Gastroenterology · 2019-05-01

25 MUTATIONS IN STXBP3 CONTRIBUTE TO VERY EARLY ONSET OF IBD, IMMUNODEFICIENCY AND HEARING LOSS

Gastroenterology · 2018-01-01 · 2 citations