Use of ruxolitinib to successfully treat chronic mucocutaneous candidiasis caused by gain-of-function signal transducer and activator of transcription 1 (STAT1) mutation
2015; Elsevier BV; Volume: 135; Issue: 2 Linguagem: Inglês
10.1016/j.jaci.2014.12.1867
ISSN1097-6825
AutoresEleanor Higgins, Tariq Al Shehri, Maeve A. McAleer, Niall Conlon, Conleth Feighery, Desa Lilić, Alan D. Irvine,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoRecently, Xing et al1Xing L. Dai Z. Jabbari A. Cerise J.E. Higgins C.A. Gong W. et al.Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.Nat Med. 2014; 20: 1043-1049Crossref PubMed Scopus (511) Google Scholar demonstrated the role of cytotoxic T lymphocytes in alopecia areata (AA) and provided important mechanistic information on the pathogenic T-cell inflammatory pathways in patients with this autoimmune condition. They described 3 patients with AA successfully treated with the oral Janus kinase (JAK) family protein tyrosine kinase inhibitor ruxolitinib. The inhibitory effectiveness of this small molecule in vitro was also reported in a novel interferonopathy coined stimulator of interferon genes (STING)–associated vasculopathy, which is caused by a gain-of-function (GOF) mutation in the TMEM173 gene encoding STING, resulting in hyperactivation of the signal transducer and activator of transcription (STAT) 1/STAT2 signaling pathways.2Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Montealegre Sanchez G.A. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (762) Google ScholarWe now demonstrate the potential utility of this drug for AA and other symptoms occurring in the wider context of a genetic immunodeficiency syndrome associated with autoimmunity. We recently treated AA associated with chronic mucocutaneous candidiasis (CMC; Online Mendelian Inheritance in Man #614162)3Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (82) Google Scholar, 4van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (486) Google Scholar in a 28-year-old woman also using ruxolitinib. Our patient had a sporadic autosomal dominant heterozygous GOF mutation, c.1159A>G p.Thr387Ala in exon 14 of STAT1 and had a 2-year history of AA. The AA had progressed despite previous intralesional corticosteroid injections, with greater than 40% scalp involvement (Fig 1, left panel). Our patient had recalcitrant oral candidiasis (CMC) since childhood, with frequent flares despite daily oral fluconazole, frequent throat and chest infections requiring antibiotics, a prior history of autoimmune hepatitis, and an episode in adolescence of severe laryngeal erythema multiforme with alopecia and full hair regrowth.STAT1 GOF caused by these mutations is mediated through enhanced phosphorylation caused by impaired nuclear dephosphorylation, resulting in increased expression of interferon-stimulated genes.5Liu 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 (569) Google Scholar Interestingly, patients with GOF STAT1 mutations have decreased expression of STAT3-stimulated genes (unpublished data), which might explain downstream reduction of IL-17 expression and consequent susceptibility to mucocutaneous Candida species infections.5Liu 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 (569) Google Scholar In canonical IFN-γ–JAK–STAT1 signaling, ligand engagement of the IFN-γ receptor leads to activation of receptor-associated JAK1 and JAK2. Therefore we hypothesized that JAK1/2 inhibition would target this pathway and could ameliorate both the CMC and the associated autoimmune AA phenotype in our patient. Ruxolitinib, a JAK1/2 inhibitor that is US Food and Drug Administration (FDA) approved for the treatment of myelofibrosis, targets JAK1/2 pathway signaling and activation of transcription (JAK-STAT) pathways.6Harrison C. Kiladjian J.J. Al-Ali H.K. Gisslinger H. Waltzman R. Stalbovskaya V. et al.JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis.N Engl J Med. 2012; 366: 787-798Crossref PubMed Scopus (1299) Google ScholarAfter 2 weeks of treatment with oral ruxolitinib (20 mg twice daily), our patient experienced dramatic hair regrowth in all affected areas of alopecia. The hair regrowth, which was uniform and thick, continued over the treatment course. Full hair regrowth was noted at 12 weeks and was sustained 6 months after completion of therapy (Fig 1, right panel). Unexpectedly, the patient also reported complete resolution of the oral candidiasis while taking ruxolitinib. The CMC returned within 2 weeks of discontinuing the treatment, despite oral fluconazole. The 3 patients with AA described by Xing et al1Xing L. Dai Z. Jabbari A. Cerise J.E. Higgins C.A. Gong W. et al.Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.Nat Med. 2014; 20: 1043-1049Crossref PubMed Scopus (511) Google Scholar demonstrated impressive clinical responses to ruxolitinib therapy. Our patient with AA demonstrated similar results with no adverse effects and also reported great relief of CMC symptoms.The heterozygous mutation reported here, c.1159A>G p.Thr387Ala in the DNA-binding domain of STAT1 in exon 14, has not been previously reported, although it has been previously found in patients with CMC (Anne Puel, personal communications). We confirmed GOF by demonstrating hyperphosphorylation of STAT1 (Tyr701) after stimulation with IFN-α (Fig 2), as previously demonstrated in other patients with CMC with GOF STAT1 mutations.5Liu 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 (569) Google Scholar GOF STAT1 mutations in the DNA-binding domain were first reported by Takezaki et al3Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (82) Google Scholar and subsequently repeatedly confirmed,7Soltesz B. Toth B. Shabashova N. Bondarenko A. Okada S. Cypowyj S. et al.New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.J Med Genet. 2013; 50: 567-578Crossref PubMed Scopus (79) Google Scholar although different STAT1 mutations in the DNA-binding domain have also been reported to cause loss of function.8Sharfe N. Nahum A. Newell A. Dadi H. Ngan B. Pereira S.L. et al.Fatal combined immunodeficiency associated with heterozygous mutation in STAT1.J Allergy Clin Immunol. 2014; 133: 807-817Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Patients with CMC caused by underlying GOF STAT1 mutations have chronic Candida species infection of the skin and mucous membranes and are thought to account for more than half of patients presenting with CMC.9Puel A. Cypowyj S. Marodi L. Abel L. Picard C. Casanova J.L. Inborn errors of human IL-17 immunity underlie chronic mucocutaneous candidiasis.Curr Opin Allergy Clin Immunol. 2012; 12: 616-622Crossref PubMed Scopus (220) Google Scholar The increased susceptibility to fungal infections is likely linked to reduced L17A and IL-22 production, as documented by us and others10Ng W.F. von Delwig A. Carmichael A.J. Arkwright P.D. Abinun M. Cant A.J. et al.Impaired T(H)17 responses in patients with chronic mucocutaneous candidiasis with and without autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.J Allergy Clin Immunol. 2010; 126 (e1-4): 1006-1015Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar and also demonstrated for our current patient (see Fig E1 in this article's Online Repository at www.jacionline.org). However, the mechanisms through which STAT1 GOF mutations lead to fungal susceptibility are still unclear, although inhibition of STAT3 activation and the consequent reduction in RORC gene transcription resulting in reduced levels of TH17 cytokines are likely to play a role. Additional inhibition of IL-17 production by CD4+ T cells might be mediated by IL-27 activation of STAT1, leading to overexpression of the inhibitory molecule programmed death ligand (PDL)-1 on naive T cells, preventing their commitment to the TH17 lineage.11Romberg N. Morbach H. Lawrence M.G. Kim S. Kang I. Holland S.M. et al.Gain-of-function STAT1 mutations are associated with PD-L1 overexpression and a defect in B-cell survival.J Allergy Clin Immunol. 2013; 131: 1691-1693Abstract Full Text Full Text PDF PubMed Scopus (63) Google ScholarFig 2Western blot of PBMC lysates from healthy control subjects (HC) and our patient with CMC (CMC pt). Cells were left unstimulated or stimulated with IFN-α (1 × 103 IU/mL) for 30 minutes in the presence or absence of ruxolitinib at 1 μmol/L. GAPDH, Glyceraldehgyde-3-phosphate dehydrogenase; pSTAT1, phosphorylated STAT1; pSTAT3, phosphorylated STAT3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We provide data demonstrating hyperphosphorylation of STAT1 consistent with a GOF STAT1 mutation in addition to the patient's clinical presentation of CMC. Western blotting (Fig 2) demonstrates hyperphosphorylation of the patient's STAT1 after stimulation with IFN-α, as well as complete inhibition of phosphorylation with the addition of ruxolitinib in doses that correspond to serum levels achieved in patients receiving 20 mg twice daily, as also shown by using flow cytometry (see Fig E2 in this article's Online Repository at www.jacionline.org). Importantly, in our and other patients with CMC, we do not see constitutive STAT1 phosphorylation, as has been reported in patients with STING-associated vasculopathy syndrome caused by an underlying GOF mutation in the gene encoding STING.2Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Montealegre Sanchez G.A. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (762) Google Scholar In addition, STAT3 phosphorylation is not reduced in this or other patients with CMC due to an underlying GOF STAT1 mutation (unpublished data), suggesting that the decreased IL-17A and IL-22 production repeatedly reported in these patients has a cause downstream of STAT3 activation. Although we did not demonstrate improvement of IL-17 production in vitro by addition of ruxolitinib, the clinical improvement in the patient's CMC was remarkable. Ruxolitinib might have differential effects on STAT1 and STAT3 in vivo, perhaps more so at the lower concentrations seen in tissues. Alternatively, a far more complex interaction between STAT1 and STAT3 must be envisaged, confirming that the underlying mechanisms remain unclear.Our case suggests the potential therapeutic benefit of ruxolitinib in the GOF STAT1 genetic immunodeficiency/autoimmunity syndrome, which can present with additional severe and often insufficiently appreciated problems for which there is no current treatment, such as debilitating superficial eye disease leading to blindness, esophageal dysmotility, and esophageal/oral cancer. Although further evaluation on additional patients is clearly needed, this therapy might represent an ideal personalized treatment for patients with GOF mutations in STAT1.For detailed methods, please see the Methods section in this article's Online Repository at www.jacionline.org. Recently, Xing et al1Xing L. Dai Z. Jabbari A. Cerise J.E. Higgins C.A. Gong W. et al.Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.Nat Med. 2014; 20: 1043-1049Crossref PubMed Scopus (511) Google Scholar demonstrated the role of cytotoxic T lymphocytes in alopecia areata (AA) and provided important mechanistic information on the pathogenic T-cell inflammatory pathways in patients with this autoimmune condition. They described 3 patients with AA successfully treated with the oral Janus kinase (JAK) family protein tyrosine kinase inhibitor ruxolitinib. The inhibitory effectiveness of this small molecule in vitro was also reported in a novel interferonopathy coined stimulator of interferon genes (STING)–associated vasculopathy, which is caused by a gain-of-function (GOF) mutation in the TMEM173 gene encoding STING, resulting in hyperactivation of the signal transducer and activator of transcription (STAT) 1/STAT2 signaling pathways.2Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Montealegre Sanchez G.A. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (762) Google Scholar We now demonstrate the potential utility of this drug for AA and other symptoms occurring in the wider context of a genetic immunodeficiency syndrome associated with autoimmunity. We recently treated AA associated with chronic mucocutaneous candidiasis (CMC; Online Mendelian Inheritance in Man #614162)3Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (82) Google Scholar, 4van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (486) Google Scholar in a 28-year-old woman also using ruxolitinib. Our patient had a sporadic autosomal dominant heterozygous GOF mutation, c.1159A>G p.Thr387Ala in exon 14 of STAT1 and had a 2-year history of AA. The AA had progressed despite previous intralesional corticosteroid injections, with greater than 40% scalp involvement (Fig 1, left panel). Our patient had recalcitrant oral candidiasis (CMC) since childhood, with frequent flares despite daily oral fluconazole, frequent throat and chest infections requiring antibiotics, a prior history of autoimmune hepatitis, and an episode in adolescence of severe laryngeal erythema multiforme with alopecia and full hair regrowth. STAT1 GOF caused by these mutations is mediated through enhanced phosphorylation caused by impaired nuclear dephosphorylation, resulting in increased expression of interferon-stimulated genes.5Liu 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 (569) Google Scholar Interestingly, patients with GOF STAT1 mutations have decreased expression of STAT3-stimulated genes (unpublished data), which might explain downstream reduction of IL-17 expression and consequent susceptibility to mucocutaneous Candida species infections.5Liu 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 (569) Google Scholar In canonical IFN-γ–JAK–STAT1 signaling, ligand engagement of the IFN-γ receptor leads to activation of receptor-associated JAK1 and JAK2. Therefore we hypothesized that JAK1/2 inhibition would target this pathway and could ameliorate both the CMC and the associated autoimmune AA phenotype in our patient. Ruxolitinib, a JAK1/2 inhibitor that is US Food and Drug Administration (FDA) approved for the treatment of myelofibrosis, targets JAK1/2 pathway signaling and activation of transcription (JAK-STAT) pathways.6Harrison C. Kiladjian J.J. Al-Ali H.K. Gisslinger H. Waltzman R. Stalbovskaya V. et al.JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis.N Engl J Med. 2012; 366: 787-798Crossref PubMed Scopus (1299) Google Scholar After 2 weeks of treatment with oral ruxolitinib (20 mg twice daily), our patient experienced dramatic hair regrowth in all affected areas of alopecia. The hair regrowth, which was uniform and thick, continued over the treatment course. Full hair regrowth was noted at 12 weeks and was sustained 6 months after completion of therapy (Fig 1, right panel). Unexpectedly, the patient also reported complete resolution of the oral candidiasis while taking ruxolitinib. The CMC returned within 2 weeks of discontinuing the treatment, despite oral fluconazole. The 3 patients with AA described by Xing et al1Xing L. Dai Z. Jabbari A. Cerise J.E. Higgins C.A. Gong W. et al.Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.Nat Med. 2014; 20: 1043-1049Crossref PubMed Scopus (511) Google Scholar demonstrated impressive clinical responses to ruxolitinib therapy. Our patient with AA demonstrated similar results with no adverse effects and also reported great relief of CMC symptoms. The heterozygous mutation reported here, c.1159A>G p.Thr387Ala in the DNA-binding domain of STAT1 in exon 14, has not been previously reported, although it has been previously found in patients with CMC (Anne Puel, personal communications). We confirmed GOF by demonstrating hyperphosphorylation of STAT1 (Tyr701) after stimulation with IFN-α (Fig 2), as previously demonstrated in other patients with CMC with GOF STAT1 mutations.5Liu 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 (569) Google Scholar GOF STAT1 mutations in the DNA-binding domain were first reported by Takezaki et al3Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (82) Google Scholar and subsequently repeatedly confirmed,7Soltesz B. Toth B. Shabashova N. Bondarenko A. Okada S. Cypowyj S. et al.New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.J Med Genet. 2013; 50: 567-578Crossref PubMed Scopus (79) Google Scholar although different STAT1 mutations in the DNA-binding domain have also been reported to cause loss of function.8Sharfe N. Nahum A. Newell A. Dadi H. Ngan B. Pereira S.L. et al.Fatal combined immunodeficiency associated with heterozygous mutation in STAT1.J Allergy Clin Immunol. 2014; 133: 807-817Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Patients with CMC caused by underlying GOF STAT1 mutations have chronic Candida species infection of the skin and mucous membranes and are thought to account for more than half of patients presenting with CMC.9Puel A. Cypowyj S. Marodi L. Abel L. Picard C. Casanova J.L. Inborn errors of human IL-17 immunity underlie chronic mucocutaneous candidiasis.Curr Opin Allergy Clin Immunol. 2012; 12: 616-622Crossref PubMed Scopus (220) Google Scholar The increased susceptibility to fungal infections is likely linked to reduced L17A and IL-22 production, as documented by us and others10Ng W.F. von Delwig A. Carmichael A.J. Arkwright P.D. Abinun M. Cant A.J. et al.Impaired T(H)17 responses in patients with chronic mucocutaneous candidiasis with and without autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.J Allergy Clin Immunol. 2010; 126 (e1-4): 1006-1015Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar and also demonstrated for our current patient (see Fig E1 in this article's Online Repository at www.jacionline.org). However, the mechanisms through which STAT1 GOF mutations lead to fungal susceptibility are still unclear, although inhibition of STAT3 activation and the consequent reduction in RORC gene transcription resulting in reduced levels of TH17 cytokines are likely to play a role. Additional inhibition of IL-17 production by CD4+ T cells might be mediated by IL-27 activation of STAT1, leading to overexpression of the inhibitory molecule programmed death ligand (PDL)-1 on naive T cells, preventing their commitment to the TH17 lineage.11Romberg N. Morbach H. Lawrence M.G. Kim S. Kang I. Holland S.M. et al.Gain-of-function STAT1 mutations are associated with PD-L1 overexpression and a defect in B-cell survival.J Allergy Clin Immunol. 2013; 131: 1691-1693Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar We provide data demonstrating hyperphosphorylation of STAT1 consistent with a GOF STAT1 mutation in addition to the patient's clinical presentation of CMC. Western blotting (Fig 2) demonstrates hyperphosphorylation of the patient's STAT1 after stimulation with IFN-α, as well as complete inhibition of phosphorylation with the addition of ruxolitinib in doses that correspond to serum levels achieved in patients receiving 20 mg twice daily, as also shown by using flow cytometry (see Fig E2 in this article's Online Repository at www.jacionline.org). Importantly, in our and other patients with CMC, we do not see constitutive STAT1 phosphorylation, as has been reported in patients with STING-associated vasculopathy syndrome caused by an underlying GOF mutation in the gene encoding STING.2Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Montealegre Sanchez G.A. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (762) Google Scholar In addition, STAT3 phosphorylation is not reduced in this or other patients with CMC due to an underlying GOF STAT1 mutation (unpublished data), suggesting that the decreased IL-17A and IL-22 production repeatedly reported in these patients has a cause downstream of STAT3 activation. Although we did not demonstrate improvement of IL-17 production in vitro by addition of ruxolitinib, the clinical improvement in the patient's CMC was remarkable. Ruxolitinib might have differential effects on STAT1 and STAT3 in vivo, perhaps more so at the lower concentrations seen in tissues. Alternatively, a far more complex interaction between STAT1 and STAT3 must be envisaged, confirming that the underlying mechanisms remain unclear. Our case suggests the potential therapeutic benefit of ruxolitinib in the GOF STAT1 genetic immunodeficiency/autoimmunity syndrome, which can present with additional severe and often insufficiently appreciated problems for which there is no current treatment, such as debilitating superficial eye disease leading to blindness, esophageal dysmotility, and esophageal/oral cancer. Although further evaluation on additional patients is clearly needed, this therapy might represent an ideal personalized treatment for patients with GOF mutations in STAT1. For detailed methods, please see the Methods section in this article's Online Repository at www.jacionline.org. We thank Lisa Turnbull, Clinical Scientist in our Northern Molecular Genetics Service, Newcastle, United Kingdom, for assistance with genetic analysis of our patient. MethodsWestern blottingEqual amounts of protein were separated on an SDS-PAGE gel, transferred to membranes, and probed with antibodies against STAT1 (9172), phosphorylated STAT1 (Tyr701/9171), STAT3 (4904), phosphorylated STAT3 (Tyr705/9145), and glyceraldehgyde-3-phosphate dehydrogenase (GAPDH 4134; all from Cell Signaling, Danvers, Mass) overnight at 4°C, followed by horseradish peroxidase–conjugated goat anti-rabbit antibody (P0448; DAKO, Glostrup, Denmark). Proteins were visualized with the Immobilon Western detection system (Millipore, Temecula, Calif).Flow cytometryPBMCs were fixed (Lyse/Fix buffer; BD Biosciences, San Jose, Calif) and permeablized (Phosflow Perm buffer III, BD Biosciences), according to the manufacturer's instructions. Cells were stained with antibodies to phosphorylated STAT1 (pSTAT1-Alexa Fluor 647, pY701) phosphorylated STAT3 (pSTAT3-Alexa Fluor 488, pY705), CD3– Pacific Blue, and CD4-Bv510 (all from BD Biosciences) in PBS containing 0.2% BSA for 1 hour. Data were collected with a FACSCaliber (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, Ore).Cytokine productionPBMCs from healthy control subjects and our patient with CMC were stimulated with 1:15,000 live Candida albicans (18804; ATCC, Manassas, Va) or PHA (2 mg/mL) or left unstimulated in the presence or absence of 1 μmol/L ruxolitinib (Selleckchem, Houston, Tex) for 5 days in RPMI 10% FCS and amphotericin (2.5 μg/mL; Sigma, St Louis, Mo) added on days 0 and 3. Supernatants were collected, and cytokine levels were measured as per the manufacturer's instructions with Ready-Set-Go kits (eBioscience, San Diego, Calif).Ethical approvalFig E2Flow cytometric trace of phosphorylated STAT1 (pSTAT1) gated on CD3+ lymphocytes. Whole blood from a patient with CMC was stimulated with IFN-γ (1 × 103 IU/mL) for 15 minutes with and without ruxolitinib (Rx) or left unstimulated and then stained with antibodies to CD3–Pacific Blue and pSTAT1–Alexa Fluor 647 antibody by using the BD Biosciences Phosphlow kit.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Western blottingEqual amounts of protein were separated on an SDS-PAGE gel, transferred to membranes, and probed with antibodies against STAT1 (9172), phosphorylated STAT1 (Tyr701/9171), STAT3 (4904), phosphorylated STAT3 (Tyr705/9145), and glyceraldehgyde-3-phosphate dehydrogenase (GAPDH 4134; all from Cell Signaling, Danvers, Mass) overnight at 4°C, followed by horseradish peroxidase–conjugated goat anti-rabbit antibody (P0448; DAKO, Glostrup, Denmark). Proteins were visualized with the Immobilon Western detection system (Millipore, Temecula, Calif). Equal amounts of protein were separated on an SDS-PAGE gel, transferred to membranes, and probed with antibodies against STAT1 (9172), phosphorylated STAT1 (Tyr701/9171), STAT3 (4904), phosphorylated STAT3 (Tyr705/9145), and glyceraldehgyde-3-phosphate dehydrogenase (GAPDH 4134; all from Cell Signaling, Danvers, Mass) overnight at 4°C, followed by horseradish peroxidase–conjugated goat anti-rabbit antibody (P0448; DAKO, Glostrup, Denmark). Proteins were visualized with the Immobilon Western detection system (Millipore, Temecula, Calif). Flow cytometryPBMCs were fixed (Lyse/Fix buffer; BD Biosciences, San Jose, Calif) and permeablized (Phosflow Perm buffer III, BD Biosciences), according to the manufacturer's instructions. Cells were stained with antibodies to phosphorylated STAT1 (pSTAT1-Alexa Fluor 647, pY701) phosphorylated STAT3 (pSTAT3-Alexa Fluor 488, pY705), CD3– Pacific Blue, and CD4-Bv510 (all from BD Biosciences) in PBS containing 0.2% BSA for 1 hour. Data were collected with a FACSCaliber (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, Ore). PBMCs were fixed (Lyse/Fix buffer; BD Biosciences, San Jose, Calif) and permeablized (Phosflow Perm buffer III, BD Biosciences), according to the manufacturer's instructions. Cells were stained with antibodies to phosphorylated STAT1 (pSTAT1-Alexa Fluor 647, pY701) phosphorylated STAT3 (pSTAT3-Alexa Fluor 488, pY705), CD3– Pacific Blue, and CD4-Bv510 (all from BD Biosciences) in PBS containing 0.2% BSA for 1 hour. Data were collected with a FACSCaliber (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, Ore). Cytokine productionPBMCs from healthy control subjects and our patient with CMC were stimulated with 1:15,000 live Candida albicans (18804; ATCC, Manassas, Va) or PHA (2 mg/mL) or left unstimulated in the presence or absence of 1 μmol/L ruxolitinib (Selleckchem, Houston, Tex) for 5 days in RPMI 10% FCS and amphotericin (2.5 μg/mL; Sigma, St Louis, Mo) added on days 0 and 3. Supernatants were collected, and cytokine levels were measured as per the manufacturer's instructions with Ready-Set-Go kits (eBioscience, San Diego, Calif). PBMCs from healthy control subjects and our patient with CMC were stimulated with 1:15,000 live Candida albicans (18804; ATCC, Manassas, Va) or PHA (2 mg/mL) or left unstimulated in the presence or absence of 1 μmol/L ruxolitinib (Selleckchem, Houston, Tex) for 5 days in RPMI 10% FCS and amphotericin (2.5 μg/mL; Sigma, St Louis, Mo) added on days 0 and 3. Supernatants were collected, and cytokine levels were measured as per the manufacturer's instructions with Ready-Set-Go kits (eBioscience, San Diego, Calif). Ethical approval
Referência(s)