Flow cytometric measurement of STAT1 and STAT3 phosphorylation in CD4 + and CD8 + T cells—clinical applications in primary immunodeficiency diagnostics

Bitar, Michael; Boldt, Andreas; Binder, Stefanie Petrou; Borte, Michael; Kentouche, Karim; Borte, Stephan; Sack, Ulrich

doi:10.1016/j.jaci.2017.05.017

Cited by 10 publications

(11 citation statements)

References 11 publications

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“…STAT1 (pSTAT1) in addition to genetic testing and western blotting [5,6]. Here, we report a case of GOF STAT1 mutation in a patient diagnosed with AD-CMC by whole-exome sequencing and present the results of FCM, which are concordant with the sequencing results.…”

supporting

confidence: 53%

“…Compared with the normal control group, the patient showed a significant expression of pSTAT1 (greater than two-fold SI) and a decreased reduction rate in response to the inhibitor. Although it is dif cult to determine the cut-off value for MFI or SI for the diagnosis of AD-CMC because of the limited number of pSTAT1 measurements performed using FCM worldwide, we suggest that FCM may be used as a simple and fast supplementary diagnostic tool for the GOF STAT1 mutations [5]. In addition to the potential of FCM as a diagnostic tool, FCM-based phosphorylation assays may be useful for monitoring treatment response in patients harboring a GOF or loss-of-function STAT1 mutations [5,18].…”

Section: Discussionmentioning

confidence: 99%

“…Although genetic testing is crucial to diagnose PIDs, fast and anticipative diagnosis of PIDs is also important to manage and reduce complications associated with the infection. In this respect, FCM has recently been suggested as a diagnostic tool for PIDs [5,6,16]. STAT1 is a key transcription factor that mediates the IFN-α/β signaling and regulates the immune response via factors such as IFN-γ and interleukin-17 [3], and both loss-and gain-of-function STAT1 mutations have been described in PIDs [9].…”

Section: Discussionmentioning

confidence: 99%

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The Usefulness of Flow Cytometry for Measuring Phosphorylated Signal Transducer and Activator of Transcription 1 to Diagnose and Manage Chronic Mucocutaneous Candidiasis: A Case Report

et al. 2020

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ized by a chronic and recurrent mucocutaneous fungal infection [1]. Genetic mutation of the signal transducer and activator of transcription 1 (STAT1) protein has been known to cause AD-CMC [2]. The STAT1 protein is a member of the STAT family regulated by the Janus kinase (JAK), which is translocated to the nucleus and regulates the expression of genes related to STAT signaling and immune system regulation [3]. In particular, gain-of-function (GOF) STAT1 mutation disrupts interferon-γ, interleukin-17, and interleukin-22 signaling, causing defective Th1 and Th17 responses (Fig. 1A) [2, 4]. Although GOF STAT1 mutation can be identi ed by genetic testing or western blotting, both methods are laborious, time-consuming, and expensive [5]. Recently, ow cytometry (FCM) has been increasingly used to measure intracellular phosphorylatedprotein levels. Some studies have suggested that FCM could be used as a diagnostic tool to monitor the phosphorylation of INTRODUCTION Autosomal dominant chronic mucocutaneous candidiasis (AD-CMC) is a primary immunode ciency disorder (PID) character

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confidence: 53%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The Usefulness of Flow Cytometry for Measuring Phosphorylated Signal Transducer and Activator of Transcription 1 to Diagnose and Manage Chronic Mucocutaneous Candidiasis: A Case Report

et al. 2020

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“…To investigate for phosphorylation defects in STAT1 and STAT3, PBMCs from SLT patients ( n = 13) were isolated and cells were stimulated for 15 min with IL-6 (100 ng/ml), IL-21 (100 ng/ml), and IFNα (11,500 U/ml; R&D Systems, cat#11101-1) according to previously described diagnostic protocols 71 , 72 . Cells were fixed immediately (Cytofix Fixation Buffer; BD Biosciences, cat#554655), permeabilised (Phosflow Perm Buffer III; BD Biosciences, cat#558050), and stained for CD4 (CD4-FITC), pSTAT1 (STAT1 pY701—Alexa647; BD Biosciences, cat#612597) and pSTAT3 (STAT3 pY705 – PE; BD Biosciences, cat#612569).…”

Section: Methodsmentioning

confidence: 99%

Inherited salt-losing tubulopathies are associated with immunodeficiency due to impaired IL-17 responses

et al. 2020

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Increased extracellular sodium activates Th17 cells, which provide protection from bacterial and fungal infections. Whilst high salt diets have been shown to worsen autoimmune disease, the immunological consequences of clinical salt depletion are unknown. Here, we investigate immunity in patients with inherited salt-losing tubulopathies (SLT). Forty-seven genotyped SLT patients (with Bartter, Gitelman or EAST Syndromes) are recruited. Clinical features of dysregulated immunity are recorded with a standardised questionnaire and immunological investigations of IL-17 responsiveness undertaken. The effects of altering extracellular ionic concentrations on immune responses are then assessed. Patients are hypokalaemic and hypomagnesaemic, with reduced interstitial sodium stores determined by 23 Na-magnetic resonance imaging. SLT patients report increased mucosal infections and allergic disease compared to age-matched controls. Aligned with their clinical phenotype, SLT patients have an increased ratio of Th2:Th17 cells. SLT Th17 and Tc17 polarisation is reduced in vitro, yet STAT1 and STAT3 phosphorylation and calcium flux following T cell activation are unaffected. In control cells, the addition of extracellular sodium (+40 mM), potassium (+2 mM), or magnesium (+1 mM) reduces Th2:Th17 ratio and augments Th17 polarisation. Our results thus show that the ionic environment typical in SLT impairs IL-17 immunity, but the intracellular pathways that mediate salt-driven Th17 polarisation are intact and in vitro IL-17 responses can be reinvigorated by increasing extracellular sodium concentration. Whether better correction of extracellular ions can rescue the immunophenotype in vivo in SLT patients remains unknown.

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“…In the study of PID patients, it is a limitation since it is usual that not all cell lineages are present; (B) choosing the correct recombinant cytokine is crucial as cytokine receptors are expressed differently in distinct lymphocyte lineages; (C) and selecting the appropriate antibodies against the STAT phosphorylation sites [30]. These studies can also be performed by WB, which is a very sensitive but more time-consuming technique and delays results [38].…”

Section: Functional Analysis Studying Stat Phosphorylationmentioning

confidence: 99%

Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies

Martínez-Gallo¹,

García-Prat²

2020

Innovations in Cell Research and Therapy

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Primary immunodeficiencies are the result of biological defects associated with functional immune abnormalities. It consists of a group of disorders showing a higher incidence and severity of infections, expression of immunological dysregulation such as inflammation and lymphoproliferation. The immunophenotyping and in vitro functional characterization of immunodeficient patients contribute, together with the clinical aspects, to define the underlying immune defect particularities. Flow cytometry applications in primary immunodeficiency assessment are multiple and include the study of a wide range of specific cell lymphocyte subpopulations. This chapter describes the main techniques used in the diagnosis of a wide variety of primary immunodeficiencies, in which intracellular proteins or activation markers involved in immunity are evaluated, as well as functional proliferation, cytokine production, phosphorylation of transcription factors, cytotoxic and degranulation capacity. Flow cytometry is a tool that allows rapid and accurate evaluation of multiple lymphocyte populations and immunological function, and this information is essential for the diagnosis and evaluation of patients with primary immunodeficiencies.

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Flow cytometric measurement of STAT1 and STAT3 phosphorylation in CD4 + and CD8 + T cells—clinical applications in primary immunodeficiency diagnostics

Cited by 10 publications

References 11 publications

The Usefulness of Flow Cytometry for Measuring Phosphorylated Signal Transducer and Activator of Transcription 1 to Diagnose and Manage Chronic Mucocutaneous Candidiasis: A Case Report

The Usefulness of Flow Cytometry for Measuring Phosphorylated Signal Transducer and Activator of Transcription 1 to Diagnose and Manage Chronic Mucocutaneous Candidiasis: A Case Report

Inherited salt-losing tubulopathies are associated with immunodeficiency due to impaired IL-17 responses

Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies

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