STIM1‐mediated calcium influx controls antifungal immunity and the metabolic function of non‐pathogenic Th17 cells
2020; Springer Nature; Volume: 12; Issue: 8 Linguagem: Inglês
10.15252/emmm.201911592
ISSN1757-4684
AutoresSascha Kahlfuß, Ulrike Kaufmann, Axel R. Concepcion, Lucile Noyer, Dimitrius Raphael, Martin Vaeth, Jun J. Yang, Priya Pancholi, Máté Maus, James Muller, Lina Kozhaya, Alireza Khodadadi‐Jamayran, Zhengxi Sun, Patrick J. Shaw, Derya Unutmaz, Peter B. Stathopulos, Cori Feist, Scott B. Cameron, Stuart E. Turvey, Stefan Feske,
Tópico(s)Pediatric health and respiratory diseases
ResumoArticle1 July 2020Open Access Source Data STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of non-pathogenic Th17 cells Sascha Kahlfuss orcid.org/0000-0001-8813-1061 Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Ulrike Kaufmann Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Axel R Concepcion Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Lucile Noyer Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Dimitrius Raphael Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Martin Vaeth Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Jun Yang Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Priya Pancholi Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Mate Maus Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author James Muller Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Lina Kozhaya The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA Search for more papers by this author Alireza Khodadadi-Jamayran Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Zhengxi Sun Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Patrick Shaw Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Derya Unutmaz The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA Search for more papers by this author Peter B Stathopulos orcid.org/0000-0002-0536-6656 Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada Search for more papers by this author Cori Feist Department of Obstetrics & Gynecology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Scott B Cameron Division of Allergy and Clinical Immunology, Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Stuart E Turvey Division of Allergy and Clinical Immunology, Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Stefan Feske Corresponding Author [email protected] orcid.org/0000-0001-5431-8178 Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Sascha Kahlfuss orcid.org/0000-0001-8813-1061 Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Ulrike Kaufmann Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Axel R Concepcion Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Lucile Noyer Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Dimitrius Raphael Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Martin Vaeth Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Jun Yang Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Priya Pancholi Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Mate Maus Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author James Muller Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Lina Kozhaya The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA Search for more papers by this author Alireza Khodadadi-Jamayran Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Zhengxi Sun Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Patrick Shaw Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Derya Unutmaz The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA Search for more papers by this author Peter B Stathopulos orcid.org/0000-0002-0536-6656 Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada Search for more papers by this author Cori Feist Department of Obstetrics & Gynecology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Scott B Cameron Division of Allergy and Clinical Immunology, Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Stuart E Turvey Division of Allergy and Clinical Immunology, Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Stefan Feske Corresponding Author [email protected] orcid.org/0000-0001-5431-8178 Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Search for more papers by this author Author Information Sascha Kahlfuss1,†, Ulrike Kaufmann1,†, Axel R Concepcion1, Lucile Noyer1, Dimitrius Raphael1, Martin Vaeth1,†, Jun Yang1, Priya Pancholi1, Mate Maus1,†, James Muller1, Lina Kozhaya2, Alireza Khodadadi-Jamayran1, Zhengxi Sun1, Patrick Shaw1,†, Derya Unutmaz2, Peter B Stathopulos3, Cori Feist4, Scott B Cameron5, Stuart E Turvey5 and Stefan Feske *,1 1Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA 2The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA 3Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada 4Department of Obstetrics & Gynecology, Oregon Health & Science University, Portland, OR, USA 5Division of Allergy and Clinical Immunology, Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada †Present address: Institute of Molecular and Clinical Immunology, Health Campus Immunology, Infectiology and Inflammation, Otto-von-Guericke University Magdeburg, Magdeburg, Germany †Present address: Genentech, South San Francisco, CA, USA †Present address: Institute for Systems Immunology, Julius Maximilians University of Wuerzburg, Wuerzburg, Germany †Present address: Institute for Research in Biomedicine (IRB), Barcelona, Spain †Present address: Bristol-Myers Squibb, Princeton, NJ, USA *Corresponding author. Tel: +1 212 263 9066; E-mail: [email protected] EMBO Mol Med (2020)12:e11592https://doi.org/10.15252/emmm.201911592 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Immunity to fungal infections is mediated by cells of the innate and adaptive immune system including Th17 cells. Ca2+ influx in immune cells is regulated by stromal interaction molecule 1 (STIM1) and its activation of the Ca2+ channel ORAI1. We here identify patients with a novel mutation in STIM1 (p.L374P) that abolished Ca2+ influx and resulted in increased susceptibility to fungal and other infections. In mice, deletion of STIM1 in all immune cells enhanced susceptibility to mucosal C. albicans infection, whereas T cell-specific deletion of STIM1 impaired immunity to systemic C. albicans infection. STIM1 deletion impaired the production of Th17 cytokines essential for antifungal immunity and compromised the expression of genes in several metabolic pathways including Foxo and HIF1α signaling that regulate glycolysis and oxidative phosphorylation (OXPHOS). Our study further revealed distinct roles of STIM1 in regulating transcription and metabolic programs in non-pathogenic Th17 cells compared to pathogenic, proinflammatory Th17 cells, a finding that may potentially be exploited for the treatment of Th17 cell-mediated inflammatory diseases. Synopsis Pathogenic Th17 cells have been implicated in autoimmune diseases, while non-pathogenic Th17 cells provide immunity to fungal pathogens. Patients with mutations in ORAI1 or STIM1 have impaired Ca2+ signaling in immune cells and are more susceptible to infections with fungal pathogens. A novel missense mutation in STIM1 (p.L374P) abolishes Ca2+ signals in immune cells by interfering with the activation of ORAI1, the pore-forming subunit of the calcium release-activated calcium (CRAC) channel. T cells of patients with STIM1 p.L374P mutation fail to produce cytokines when challenged with C. albicans and have severe defects in metabolic functions including glycolysis and oxidative phosphorylation (OXPHOS). Deletion of STIM1 and its homologue STIM2 in all immune cells results in enhanced severity of mucosal C. albicans infection, which is associated with defective T cell and neutrophil function. T cell-specific deletion of STIM1 reduces resistance to systemic C. albicans infection and is associated with impaired effector functions of Th1 and non-pathogenic Th17 cells. STIM1 is required for the transcriptional regulation of aerobic glycolysis and OXPHOS in non-pathogenic Th17 cells, whereas glycolysis in pathogenic Th17 cells is independent of STIM1 and CRAC channel function. The paper explained Problem Calcium signals are critical for the function of cells of the innate and adaptive immune system and their ability to mediate protective immune responses to infection. Calcium signals in immune cells are mediated by CRAC channels that are formed by ORAI1 and STIM1 proteins. We had previously reported that defects in this pathway render patients and mice susceptible to viral infections. The role of CRAC channels for immunity to infection with fungal pathogens has not been studied and the mechanisms by which calcium signals regulate antifungal immunity are largely unexplored. Results We here describe patients with an inherited novel loss-of-function mutation in the STIM1 gene that abolishes calcium influx through CRAC channels and therefore the function of immune cells. These patients, like others with mutations in the same pathway described before, are more susceptible to fungal infections with C. albicans, A. fumigatus, P. jirovecii, and potentially other fungal pathogens. In this study, we describe the molecular mechanisms by which the mutation abolishes the ability of STIM1 to activate CRAC channels and show that lack of calcium influx in the patients' T cells suppresses several metabolic pathways that are required for normal T-cell function. To understand the mechanisms by which CRAC channels control immunity to fungal infections, we used mice with genetic deletion of STIM1 and its homologue STIM2 to abolish calcium influx in all immune cells or more selectively only in T cells. Mice lacking STIM1 or both STIM1 and STIM2 in all immune cells showed increased susceptibility to oral C. albicans infection, which was associated with defective neutrophil function. Deletion of STIM1 only in T cells, by contrast, had little effect on immunity to oral C. albicans infection but rendered mice susceptible to systemic fungal infection. A subset of CD4+ T cells, T helper (Th) 17 cells, are important mediators of antifungal immunity. Deletion of STIM1 in Th17 cells impaired not only the expression of several Th17 cytokines but also that of many genes which regulate the metabolic function of Th17 cells. This included genes controlling the utilization of glucose by aerobic glycolysis and the generation of ATP in mitochondria by oxidative phosphorylation (OXPHOS). In contrast to Th17 cells that mediate antifungal immunity, a related subset of Th17 cells that cause inflammation in the context of many autoimmune diseases required CRAC channel function only to regulate OXPHOS but not glycolysis. Impact Our study offers new insights into the role of calcium influx through CRAC channels in cells of the innate and adaptive immune system and how this signaling pathway provides immunity to fungal pathogens. Furthermore, we describe distinct roles of CRAC channels in regulating the metabolic function of Th17 cell subsets that contribute to antifungal immunity and those that mediate inflammation in autoimmune diseases like multiple sclerosis, Crohn's disease, and rheumatoid arthritis. We propose that the latter finding may potentially be exploited for the treatment of Th17 cell-mediated autoimmune diseases. Introduction Over 150 million people worldwide are estimated to suffer from fungal diseases, with the severity ranging from asymptomatic-mild to life-threatening systemic infections resulting in ~1.6 million deaths associated with fungal disease each year (Bongomin et al, 2017). Aspergillus, Candida, Cryptococcus species, and Pneumocystis jirovecii are the main fungal pathogens responsible for the majority of serious fungal disease cases. Candida species are part of the normal human microflora of the gastrointestinal and reproductive tracts in 50–80% of healthy individuals, but can become pathogenic in immune compromised hosts (Brown et al, 2012). Common causes of increased susceptibility to Candida infections include HIV/AIDS, immunosuppressive therapies, antibiotic use, and inherited immunodeficiencies (Lanternier et al, 2013; Bongomin et al, 2017; Mengesha & Conti, 2017). Infections with Candida (C.) albicans manifest as mucosal or mucocutaneous candidiasis, onychomycosis or systemic fungal infection. Systemic C. albicans infection can occur after dissemination of local fungal infections or as nosocomial, often catheter-associated, infections in patients receiving critical care (Villar & Dongari-Bagtzoglou, 2008; Lanternier et al, 2013). Immunity to C. albicans infections involves innate and adaptive immune responses (Hernandez-Santos & Gaffen, 2012; Conti & Gaffen, 2015; Netea et al, 2015; Sparber & LeibundGut-Landmann, 2015). C. albicans is initially recognized by cells of the innate immune system including dendritic cells, macrophages, and neutrophils. At skin and mucosal surfaces, C. albicans hyphae may enter epithelial cells resulting in their activation and production of IL-1β, TNF-α, and IL-6, which activate neutrophils and other innate immune cells. The recruitment and activation of neutrophils also depend on TNF-α, IFN-γ, and IL-17A produced by Th1, Th17 cells, type 3 innate lymphoid cells (ILC3) as well as NK cells and γδ T cells (Bar et al, 2014; Conti & Gaffen, 2015; Netea et al, 2015). Neutrophils are required for clearing fungal pathogens, and C. albicans is among the most frequently isolated pathogens in neutropenic patients with nosocomial systemic candidiasis (Delaloye & Calandra, 2014). On the adaptive side of the immune system, non-pathogenic Th17 cells are critical for antifungal immunity as shown by studies in mice and human patients with inherited defects in Th17 cell differentiation and/or function (Mengesha & Conti, 2017). Individuals with mutations in IL-17A, IL-17 receptor A (IL-17RA), or IL-17RC (Puel et al, 2011; Ling et al, 2015; Levy et al, 2016) are susceptible to chronic mucocutaneous candidiasis (CMC) as are patients with dominant-negative mutations in the transcription factor signal transducer and activator of T cells (STAT) 3 (Milner et al, 2008) and gain-of-function (GOF) mutations in STAT1 (Toubiana et al, 2016), which result in defects of Th17 cell differentiation. Furthermore, neutralizing autoantibodies to IL-17A, IL-17F, and IL-22 are associated with an increased susceptibility to C. albicans infections in patients with autoimmune polyglandular syndrome type 1 (APS1) due to mutations in autoimmune regulator (AIRE) (Kisand et al, 2010). In mice, deletion of IL-23p19, which is required for the differentiation of Th17 cells, or the IL-17A receptor (IL-17RA) causes severe oropharyngeal candidiasis (OPC; Conti et al, 2009). By contrast, Il-12−/− mice lacking Th1 cells do not develop OPC but fail to prevent Candida dissemination to the kidney. Besides immunity to local candidiasis, non-pathogenic Th17 cells are also crucial for immunity to systemic Candida infection (Huang et al, 2004). Mice lacking IL-17RA had a C. albicans dose-dependent survival defect after systemic infection (Huang et al, 2004). Intestinal colonization of mice with C. albicans was recently shown to mediate Th17 cell differentiation and expansion, which provides immunity to systemic Candida infection (Shao et al, 2019). Collectively, these studies show that IL-17 signaling is critical for immunity to local Candida infection and identify Th17 cells as critical during systemic candidiasis. The function of T cells depends on calcium (Ca2+) influx and signaling (Feske, 2007). The main mode of Ca2+ influx in T cells is store-operated Ca2+ entry (SOCE) that is mediated by Ca2+ release-activated Ca2+ (CRAC) channels. CRAC channels are hexamers of ORAI1 proteins located in the plasma membrane that are activated by STIM1 and its homologue STIM2. STIM1 and STIM2 are single-pass membrane proteins located in the endoplasmic reticulum (ER) membrane. T-cell receptor stimulation results in the production of inositol (1,4,5) trisphosphate (IP3) and opening of IP3 receptors in the ER membrane, followed by Ca2+ release from the ER. This triggers a conformational change of STIM proteins and their binding to ORAI1 in the plasma membrane, resulting in CRAC channel opening and SOCE (Deng et al, 2009; Maus et al, 2015). Loss-of-function (LOF) mutations in ORAI1 or STIM1 genes (OMIM 610277 and 605921) abolish SOCE and cause CRAC channelopathy, which is characterized by combined immunodeficiency (CID), humoral autoimmunity, and ectodermal dysplasia (Lacruz & Feske, 2015; Concepcion et al, 2016). CID typically presents in early infancy and can be severe resulting in death of patients from viral and bacterial infections (Feske et al, 2006, 2005; McCarl et al, 2009; Lacruz & Feske, 2015). A common feature of CRAC channelopathy are fungal infections with a variety of pathogens including, most commonly, C. albicans, Aspergillus fumigatus, and Pneumocystis jirovecii (Table 1). Table 1. Synopsis of patients with CRAC channelopathy due to loss-of-function mutations in ORAI1 or STIM1 and associated fungal infections Gene Mutation Fungal infections References Local Systemic ORAI1 p.V181SfsX8 Pneumocystis jirovecii pneumonia n.r. Lian et al (2018) p.L194P n.r. C. albicans sepsis Lian et al (2018) p.G98R Aspergillus fumigatus, Candida albicans n.r. Lian et al (2018) p.R91W Oral candidiasis, gastrointestinal candidiasis n.r. Feske et al (1996), Feske et al (2000), McCarl et al (2009) p.A88SfsX25 n.r. n.r. Partiseti et al (1994), McCarl et al (2009) p.A103E/p.L194P n.r. n.r. Le Deist et al (1995), McCarl et al (2009) p.H165PfsX1 n.r. n.r. Chou et al (2015) p.R270X Pneumocystis jirovecii pneumonia Badran et al (2016) p.I148S Pneumocystis jirovecii pneumonia, oral thrush, diaper rash Klemann et al (2017) STIM1 p.E128RfsX9 n.r. n.r. Picard et al (2009) C1538-1 G>A n.r. n.r. Sahin et al (2010), Byun et al (2010) p.R429C n.r. n.r. Fuchs et al (2012), Maus et al (2015) p.R426C n.r. n.r. Wang et al (2014) p.P165Q n.r. n.r. Schaballie et al (2015) p.L74P n.r. n.r. Parry et al (2016) p.L374P Diaper rash, Onychomycosisaa Present prior to, but aggravated by, treatment with infliximab and inhalative corticosteroids. n.r. This study n.r., not reported. a Present prior to, but aggravated by, treatment with infliximab and inhalative corticosteroids. We previously reported that the function of pathogenic Th17 cells, which orchestrate inflammation in many autoimmune diseases, depends on CRAC channels. Mice with T cell-specific deletion of ORAI1, STIM1, or both STIM1 and STIM2 had profound defects in Th17 cell function resulting in partial or complete protection from experimental autoimmune encephalomyelitis (EAE), a Th17 cell-dependent murine model of multiple sclerosis (MS; Ma et al, 2010; Schuhmann et al, 2010; Kaufmann et al, 2016). Furthermore, pathogenic Th17 cells that differentiate under the influence of a hyperactive form of STAT3 require STIM1 for their function and ability to cause multiorgan inflammation (Kaufmann et al, 2019). By contrast, the role of CRAC channels in non-pathogenic Th17 cells and their ability to mediate immunity to infection with bacterial and fungal pathogens is unknown. In addition, the cause of impaired antifungal immunity in CRAC channel-deficient patients remains elusive. In this study, we report patients with a novel LOF mutation in STIM1 (p.L374P) that abolishes STIM1 function and SOCE. Like many other SOCE-deficient patients, they suffer from chronic fungal infections. Using mice with conditional deletion of STIM1, we demonstrate that SOCE in non-pathogenic Th17 cells is essential for antifungal immunity to systemic infection with C. albicans. Mechanistically, the lack of functional STIM1 and SOCE in non-pathogenic Th17 cells resulted in impaired production of IL-17A and other cytokines, but did not impair the expression of Th17 signature molecules including RORγt. An unbiased transcriptome analysis of non-pathogenic Th17 cells revealed that STIM1 regulates two key metabolic pathways, aerobic glycolysis, and mitochondrial oxidative phosphorylation (OXPHOS), whose function was impaired in STIM1-deficient non-pathogenic Th17 cells. In pathogenic Th17 cells, by contrast, STIM1 only controlled OXPHOS but not glycolysis. The greater reliance of pathogenic Th17 cells on STIM1 and OXPHOS may be exploitable for the treatment of autoimmune diseases in which pathogenic Th17 cells play an important role without affecting the antimicrobial function of non-pathogenic Th17 cells. Results Mutation of p.L374 in the C terminus of STIM1 abolishes SOCE and causes CRAC channelopathy We here report two patients (P1, P2) born to parents who are first-cousins-once-removed. The patients presented with combined immune deficiency (CID) and recurrent infections since childhood with varicella zoster virus (VZV), pneumonias caused by atypical mycobacteria, onychomycosis, and skin infections with C. albicans species (Fig 1A, Table EV1). Patients also had non-immunological symptoms including congenital muscular hypotonia and anhidrotic ectodermal dysplasia, which are typical of CRAC channelopathy due to LOF mutations in STIM1 and ORAI1 (Lacruz & Feske, 2015). Detailed case reports are provided in the Appendix. Laboratory analyses demonstrated overall normal immune cell populations in both patients. P1 had a profound defect in T-cell proliferation after T-cell receptor (TCR) stimulation with several viruses and C. albicans whereas proliferative responses to mitogens (PHA, PWA) were normal (Appendix Table S1). Sequencing of genomic DNA (gDNA) of P1, P2, and their asymptomatic mother revealed a homozygous missense mutation (c.1121T>C) within exon 8 of STIM1 in both patients, whereas the mother was heterozygous for the same mutation (Fig 1B). gDNA of the father and a sister of P1 and P2, who are both asymptomatic, was not available for analysis. The STIM1 c.1121T>C mutation is predicted to cause a single amino acid substitution (p.L374P) in the second coiled-coil domain (CC2, aa 345–391) within the C terminus of STIM1. Analysis of 71,702 genomes from unrelated individuals using the gnomAD v3 database (GRCh38) (https://gnomad.broadinstitute.org) demonstrated that the p.L374P mutation of STIM1 is extremely rare with an allele frequency of 6.98e-6 (1 allele out of 143,330). As CRAC channelopathy due to mutations in ORAI1 or STIM1 genes follows an autosomal recessive inheritance, the homozygous STIM1 c.1121T>C mutation detected in both patients is compatible with their disease. The scaled CADD score of the STIM1 c.1121T>C, p.L374P mutation is 28.5, indicating that it is within the top 0.1% of the most deleterious variants in the human genome. Figure 1. p.L374P mutation in the C terminus of STIM1 abolishes SOCE and causes CRAC channelopathy A. Pedigree of the patients P1 (II-1) and P2 (II-2) presenting with CRAC channelopathy. Filled circles (females) and squares (males) indicate homozygous patients; dotted symbols indicate heterozygous asymptomatic carriers; symbols with "?" indicate asymptomatic family members not available for DNA sequencing. B. Sanger sequencing of genomic DNA isolated from PBMC of the mother (I-2), P1, and P2. C. STIM1 and STIM2 mRNA expression in PBMC of P1, P2, mother, and a healthy donor (HD) that were left unstimulated or stimulated with anti-CD3/CD28 for 24 h. mRNA levels of STIM1/2 normalized to 18S rRNA. Graph shows the mean ± SEM of duplicates from one experiment. D. STIM1 protein expression in CD4+ and CD8+ T cells from P1, P2, the mother, and a HD analyzed by flow cytometry. Shaded histograms: polyclonal rabbit IgG control antibody; open histograms: polyclonal anti-STIM1 antibody. Bar graphs show the delta MFI calculated as MFISTIM1 – MFIIgG control that was normalized to HD T cells. Bar graphs are mean ± SEM of two independent repeat experiments. E. STIM1 protein expression in expanded human T cells analyzed by immunoblotting. One representative Western blot of 3 is shown. Bar graphs are the mean ± SEM of STIM1 expression normalized to actin from three independent experiments. F. Ca2+ influx in Fura-2 loaded PBMC of P1, P2, the mother, and a HD. T cells were stimulated with 1 μM thapsigargin (TG) in the absence of extracellular Ca2+ followed by addition of 1 mM extracellular Ca2+. Bar graphs show the integrated Ca2+ influx response (AUC, area under the curve) from 400 to 800 s and the peak Ca2+ influx normalized to baseline Ca2+ levels (F340/380) at 400 s. Data represent the mean ± SEM from 2 experiments. Statistical analysis by unpaired Student's t-test. **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 1 [emmm201911592-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint The c.1121T>C mutation does not interfere with STIM1 mRNA expression as transcript levels were comparable in PBMC from both patients, their mother, and a healthy donor (HD) control either before and after stimulation with anti-CD3/CD28 (Fig 1C). No compensatory upregulation of STIM2 was observed in P1 and P2. Intracellular staining of CD4+ and CD8+ T cells from P1, P2, their mother and a HD also showed comparable STIM1 protein expression (Fig 1D). The specificity of STIM1 staining was verified using fibroblasts of a patient homozygous for a STIM1 p.E128RfsX9 frameshift mutation that abolishes STIM1 protein expression (Appendix Fig S1A) (Picard et al, 2009). Similar STIM1 protein expression in T cells of P1, P2, their mother, and two HD controls was confirmed by Western blot analysis (Fig 1E). Despite normal STIM1 mRNA and protein expression, SOCE in PBMC isolated from P1 and P2 was severely impaired after stimulation with the sarcoplasmic/endoplasmic Ca2+ ATPase inhibitor thapsigargin (TG) compared to PBMC from the patients' mother and a HD control (Fig 1F). Abolished SOCE was also observed in T cells from P1 and P2 cultured in vitro compared to those of a HD (Appendix Fig S1B). SOCE in the mother's T cells was reduced, likely because she is a heterozygous carrier of the STIM1 p.L374P mutation. STIM1 p.L374P mutation abolishes STIM1 puncta formation and co-localization with ORAI1 Because SOCE is strongly impaired but STIM1 protein expression is normal, we hypothesized that the p.L374P mutation affects STIM1 protein function. Leucine 374 is located in the second coiled-coil (CC2) domain of the cytoplasmic C-terminal segment of STIM1. CC2 is part of a functional domain that is necessary and sufficient for STIM1 binding to ORAI1 and that is alternatively referred to as CRAC activation domain (CAD, aa 342–448) (Park et al, 2009), STIM-ORAI activation region (SOAR, 344–442) (Yuan et al, 2009), or CC fragment b9 (CCb9, 339–444) (Kawasaki et al, 2009; Fig 2A). We modeled the impact of the p.L374P mutation on the structure of the STIM1 CC2 and CC3 domains using available crystal structures of CAD/SOAR/CCb9 and NMR structures of C-terminal STIM1 fragments (Yang et al, 2012; Stathopulos et al, 2013). Based on the L374M/V419A/C437T triple mutant crystal structure (Yang et al, 2012), the human CAD/SOAR/CCb9
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