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

2017; Elsevier BV; Volume: 140; Issue: 5 Linguagem: Inglês

10.1016/j.jaci.2017.05.017

1097-6825

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

Tuberculosis Research and Epidemiology

It is well known that cytokines achieve their biologic function by triggering Janus kinases, which are a family of proteins that regulate signal transducer and activator of transcription (STAT) proteins pathways, in order to control cellular activation processes.1Ivashkiv L.B. Hu X. Signaling by STATs.Arthritis Res Ther. 2004; 6: 159-168Crossref PubMed Google Scholar Loss- or gain-of-function (GOF) gene mutations in STAT1 and STAT3 were described in primary immunodeficiencies (PIDs).2Casanova J.-L. Holland S.M. Notarangelo L.D. Inborn errors of human JAKs and STATs.Immunity. 2012; 36: 515-528Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar Patients with autosomal-dominant (AD) loss-of-function STAT1 mutation are predisposed to mycobacterial infections, whereas the autosomal-recessive (AR) form is accompanied by mycobacterial and viral infections.2Casanova J.-L. Holland S.M. Notarangelo L.D. Inborn errors of human JAKs and STATs.Immunity. 2012; 36: 515-528Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 3Chapgier A. Kong X.-F. Boisson-Dupuis S. Jouanguy E. Averbuch D. Feinberg J. et al.A partial form of recessive STAT1 deficiency in humans.J Clin Invest. 2009; 119: 1502-1514Crossref PubMed Scopus (146) Google Scholar In contrast, GOF STAT1 mutations may cause chronic mucocutaneous candidiasis (CMC).4Liu L. Okada S. Kong X.-F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (588) Google Scholar, 5Zheng J. van de Veerdonk F.L. Crossland K.L. Smeekens S.P. Chan C.M. Al Shehri T. et al.Gain-of-function STAT1 mutations impair STAT3 activity in patients with chronic mucocutaneous candidiasis (CMC).Eur J Immunol. 2015; 45: 2834-2846Crossref PubMed Scopus (81) Google Scholar Dominant-negative mutations in STAT3 were found in the AD form of hyper-IgE syndrome (HIES), also called Job syndrome.6Yong P.F.K. Freeman A.F. Engelhardt K.R. Holland S. Puck J.M. Grimbacher B. An update on the hyper-IgE syndromes.Arthritis Res Ther. 2012; 14: 228Crossref PubMed Scopus (108) Google Scholar STAT1 and STAT3 proteins have been studied by different methods, that is, genome sequencing and Western blot (WB), which is limited due to its time-consuming nature and the requirement of extensive cell numbers.7Lafarge S. Hamzeh-Cognasse H. Chavarin P. Genin C. Garraud O. Cognasse F. A flow cytometry technique to study intracellular signals NF-kappaB and STAT3 in peripheral blood mononuclear cells.BMC Mol Biol. 2007; 8: 64Crossref PubMed Scopus (21) Google Scholar Therefore, we established a rapid flow cytometric whole blood assay to evaluate STAT1 and STAT3 protein phosphorylation and to provide reference values based on 21 healthy adult controls (for characteristics, see Table E1 in this article's Online Repository at www.jacionline.org). In addition, we evaluated the test in 4 patients with PID selected by their clinical representation: recurrent opportunistic infections, candidiasis, or an HIES phenotype. As a result, we conclude that our approach provides a diagnostics benefit by avoiding labor and time consumption needed for WB or other classical protein analyses, thereby achieving earlier diagnoses, potentially leading to improved treatment options and patient outcome. Written informed consent was obtained from all included individuals. To evaluate phosphorylation of STAT 1 (pSTAT1) expression, peripheral EDTA blood was treated with IFN-α (PBL science, Piscataway, NJ, 40,000 U/mL), whereas IL-6 (BD Biosciences, Heidelberg, Germany, 100 ng/mL) was applied to stimulate the STAT3 signal cascade. Live and dead cells were discriminated on the basis of BD Horizon Fixable viability stain 450 (FVS450). The phosphorylation status of STAT1 and STAT3 in CD4+ and CD8+ T cells was analyzed both by flow cytometry (FCM) and by WB (see Table E2 and Fig E1 in this article's Online Repository at www.jacionline.org). All measurements were adapted according to DIN EN ISO 15189 requirements. The validation of our data set included the definition of intraassay and interassay precision values.8Sack U. Barnett D. Demirel G.Y. Fossat C. Fricke S. Kafassi N. et al.Accreditation of flow cytometry in Europe.Cytometry B Clin Cytom. 2013; 84: 135-142Crossref PubMed Scopus (25) Google Scholar Response to stimulation (phosphorylation) was calculated as percentage (%) and increase in mean fluorescence intensity (MFI) of pSTAT1-and pSTAT3-expressing cells within CD4+ or CD8+ subsets. The adjusted P values were considered significant based on Wilcoxon test when less than .05 (Graph Pad Prism, San Diego, Calif). The summary of FCM analysis showed that IFN-α and IL-6 induce a significant increase in pSTAT1 and pSTAT3, respectively, magnitude (vs unstimulated cells), as depicted in Fig 1 (A-D; see Tables E3 and E4 in this article's Online Repository at www.jacionline.org). However, pSTAT3 responsiveness of CD8+ cells was moderate and produced a bimodal peak (see Fig E2, A, in this article's Online Repository at www.jacionline.org). More specifically, because of this bimodal peak within CD8+ subsets, we investigated which phenotype-naive or memory cells were involved in phosphorylation of STAT3. Thus, these cells were stained for CD45RA expression to discriminate between naive (CD45RA+) and memory T cells (CD45RA−). Following stimulation with IL-6, the expression of pSTAT3 in naive CD8+ subsets was more pronounced compared with memory subsets, in contrast to a very weak phosphorylation of STAT3 in unstimulated controls (see Fig E3 in this article's Online Repository at www.jacionline.org). Oberg et al9Oberg H.-H. Wesch D. Grussel S. Rose-John S. Kabelitz D. Differential expression of CD126 and CD130 mediates different STAT-3 phosphorylation in CD4+CD25− and CD25high regulatory T cells.Int Immunol. 2006; 18: 555-563Crossref PubMed Scopus (83) Google Scholar described that IL-6 induces intracellular signals by ligand-binding to IL-6R-α chain (CD126) and gp130 (CD130), while only about 50% of human CD8+ lymphocytes express the surface markers CD126 and CD130, in contrast to about 80% on CD4+ T cells. Moreover, a study by Betz and Muller10Betz U.A. Muller W. Regulated expression of gp130 and IL-6 receptor alpha chain in T cell maturation and activation.Int Immunol. 1998; 10: 1175-1184Crossref PubMed Scopus (62) Google Scholar described that peripheral T cells with a cell surface marker profile of memory cells exhibited a gp130− and IL-6R-α− immune phenotype due to downregulation upon activation. This suggests that naive subsets are responsible for most pSTAT3 signals in CD8+ cells (see Fig E3 in this article's Online Repository at www.jacionline.org). To examine whether our results were specific, the samples were also analyzed by WB in parallel. As displayed in Fig E4, B, in this article's Online Repository at www.jacionline.org, IFN-α–stimulated cells generated specific 92-kDa protein bands (the upper line) representing pSTAT1, in contrast to untreated cells, which depicted very weak or absent bands. However, stimulated CD4+ cells exhibited a strong increase in phosphorylation as indicated by specific band intensities, according to FCM analysis. In contrast, the CD8+ subsets presented a slight increase in phosphorylation as indicated by the band intensities, compared with FCM (see Fig E4, A). For pSTAT3 (see Fig E2, B), the bands in the upper line illustrated a strong expansion of pSTAT3 within CD4+ and a moderate pSTAT3 increase in CD8+ T cells corresponding to a 92-kDa fragment. β-Actin blotting was performed as loading control. The band intensities correlated with a strong increase in CD4+ and a moderate increase in CD8+ T cells, respectively, detected by FCM analyses (see Fig E2, A). Subsequently, we investigated 4 patients with PID with defects in the STAT1 or STAT3 signaling pathway. Patient 1 (recurrent opportunistic infections, candidiasis) exhibited almost completely deficient STAT1 phosphorylation in both CD4+ and CD8+, in contrast to normal pSTAT3 expression (Fig 2). This result resembles AR STAT1 deficiency, which is characterized by the occurrence of recurrent infections with opportunistic bacteria.3Chapgier A. Kong X.-F. Boisson-Dupuis S. Jouanguy E. Averbuch D. Feinberg J. et al.A partial form of recessive STAT1 deficiency in humans.J Clin Invest. 2009; 119: 1502-1514Crossref PubMed Scopus (146) Google Scholar Patient 2 (patient with CMC), who was diagnosed with a GOF-STAT1 mutation, exhibited a strong increase in MFI of pSTAT1 in response to IFN-α stimulation (Fig 2). In healthy controls, additional treatment with staurosporine (tyrosine kinas inhibitor, Sigma-Aldrich, Carlsbad, Calif) inhibited a pSTAT1 overexpression, in contrast to patient 2 with GOF-STAT1 mutation. The MFI of pSTAT1 remained at a high level (see Fig E5 in this article's Online Repository at www.jacionline.org). As illustrated in Fig 2, we were also able to distinguish between different HIES traits: in patient 3 (AD with STAT3 mutation) pSTAT3 was defective, in contrast to patient 4 (AR without STAT3 mutation) with normal pSTAT3 expression. Similar results were also described by Yong et al.6Yong P.F.K. Freeman A.F. Engelhardt K.R. Holland S. Puck J.M. Grimbacher B. An update on the hyper-IgE syndromes.Arthritis Res Ther. 2012; 14: 228Crossref PubMed Scopus (108) Google Scholar In conclusion, disorders in the Janus kinases/STAT signal pathway in CD4+ and CD8+ T cells may result in insufficient response to pathogens. Therefore, FCM-based pSTAT1 and pSTAT3 profiling is an effective tool for clinical laboratory diagnostics to (1) understand the susceptibility to recurrent opportunistic infections, (2) identify patients with functional CD4+ and CD8+ T-cell defects, and (3) identify and characterize well-known PIDs such as CMC, AD-HIES, or AR-HIES. We are thankful to Heike Knaack, Katrin Bauer, Bettina Glatte, Prof. Dr Sunna Hauschildt, Dr Anja Grahnert, Dr Ronald Weiß, Dr Erik Schilling, Dr Danny Issa, and Dr Tewodros Debebe for support and excellent laboratory work. Fig E2Detection of pSTAT3 in CD4+, CD8+ subsets by FCM and WB. A, Phosphorylation analysis of STAT3 by FCM; human peripheral blood was either left without stimulation (gray histogram) or stimulated with IL-6 (black histogram). STAT3 phosphorylation is presented as both percentage and the MFI of cells positive for pSTAT3. B, WB analysis, pSTAT3 bands (upper line at 92 KDa), β-actin blotting (bottom). stim, Stimulated; w/o, without stimulation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Evaluation of pSTAT3 in CD8+ CD45RA+ as naive cells and CD8+ CD45RA− as memory cells. After IL-6 stimulation, an efficient expression of pSTAT3 within CD8+ CD45RA+ naive cells was increased distinctly rather than memory cells CD45RA−. Percentages of naive and memory cells that were involved in phosphorylation of STAT3 are given within the histogram boxes. stim, Stimulated; w/o, without stimulation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Detection of pSTAT1 in CD4+, CD8+ subsets by FCM and WB. A, Phosphorylation analysis of STAT1 by FCM; human peripheral blood was either left without stimulation (gray histogram) or stimulated with IFN-α (black histogram). STAT1 phosphorylation is presented as both percentage and the MFI of cells positive for pSTAT1. B, WB analysis, pSTAT1 bands (upper line at 92 KDa), β-actin blotting (bottom). stim, Stimulated; w/o, without stimulation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Time-dependent STAT1 phosphorylation and its inhibition in a patient with GOF-STAT1 mutation compared with healthy control. PBMCs were isolated and stimulated with IFN-α for 15, 30, 60, and 90 minutes. After reaching the maximum of pSTAT1-MFI at 15 minutes, the phosphorylation decreased to a normal level in healthy control and in the patient, in both CD4+ (A) and CD8+ (C) T cells. Interestingly, the maximum pSTAT1-MFI was multiple higher in the GOF-STAT1 patient compared with healthy control (Fig E5, A and C). Furthermore, PBMCs were stimulated with IFN-α and after 15 minutes treated with staurosporine (B and D). Staurosporine treatment inhibited strongly the STAT1 phosphorylation in healthy control in contrast to the patient with GOF-STAT1 mutation (Fig E5, B and D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Clinical characteristics of 21 adult healthy controlsCharacteristicControls (n = 21)Age (y), mean ± SD35.1 ± 9.8SexMale: 8/Female: 13Immune diseases/medical treatmentNo Open table in a new tab Table E2The antibodies used for staining and measurement of the cells by FCMAntibodyCloneVolume (μL)BD phosflow PerCP-Cy 5.5 mouse anti-human CD3UCHT12.5BD phosflow Alexafluor 488 mouse anti-human CD8RPA-T82.5BD phosflow PE mouse anti-human CD4RPA-T45Alexa Fluor 647 mouse anti-human STAT1 (PY701)Clone 4a10Alexa Fluor 647 mouse anti-human STAT3 (PY705)4/P-STAT310BD Horizon V450 mouse anti-human CD45RAHI1002.5 Open table in a new tab Table E3Expression of pSTAT1 (% of positive cells and MFI) in adult healthy control whole blood samplesControl(n = 21)CD4+ pSTAT1w/oCD4+ pSTAT1 IFN-α–stimulatedCD8+ pSTAT1w/oCD8+ pSTAT1IFN-α–stimulatedPercentage∗Percentage of cells positive for pSTAT1; the value was calculated as mean ± SD.1.4 ± 0.3684.65 ± 8.341.47 ± 0.4562.3 ± 13.8MFI†MFI for entire CD4+, CD8+ populations, calculated as mean ± SD.115 ± 17778 ± 154109 ± 14641 ± 131w/o, Without stimulation.∗ Percentage of cells positive for pSTAT1; the value was calculated as mean ± SD.† MFI for entire CD4+, CD8+ populations, calculated as mean ± SD. Open table in a new tab Table E4Expression of pSTAT3 (% of positive cells and MFI) in adult healthy control whole blood samplesControl group(n = 11)CD4+ pSTAT3w/oCD4+ pSTAT3IL-6–stimulatedCD8+ pSTAT3w/oCD8+ pSTAT3IL-6–stimulatedPercentage∗Percentage of cells positive for pSTAT3; the value calculated as mean ± SD.1.42 ± 0.3884.3 ± 8.431.32 ± 0.2744.9 ± 14MFI†MFI for entire CD4+, CD8+ populations, calculated as mean ± SD.112 ± 15707 ± 104101 ± 10660 ± 92w/o, Without stimulation.∗ Percentage of cells positive for pSTAT3; the value calculated as mean ± SD.† MFI for entire CD4+, CD8+ populations, calculated as mean ± SD. Open table in a new tab w/o, Without stimulation. w/o, Without stimulation.