Biallelic TRAF3IP2 variants causing chronic mucocutaneous candidiasis in a child harboring a STAT1 variant

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is a accepted manuscript of a paper published in: Pediatric Allergy and Immunology: 21 July 2021 DOI: 10.1111/pai.13603 Copyright: ©2021 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. “This is the peer reviewed version of the following article: Blanco Lobo P, Lei WT, Pelham SJ, Guisado Hernández P, Villaoslada I, de Felipe B, Lucena JM, Casanova JL, Olbrich P, Puel A, Neth O. Biallelic TRAF3IP2 variants causing chronic mucocutaneous candidiasis in a child harboring a STAT1 variant. Pediatr Allergy Immunol. 2021 Nov;32(8):1804-1812. doi: 10.1111/pai.13603. Epub 2021 Aug 9. PMID: 34289170, which has been published in final form at https://doi.org/10.1111/pai.13603. 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For Peer Review Biallelic TRAF3IP2 variants causing chronic mucocutaneous candidiasis in a child harboring a STAT1 variant Journal: Pediatric Allergy and Immunology Manuscript ID PAI-21-O-0244.R1 Manuscript Type: Original Article Date Submitted by the Author: 09-Jul-2021 Complete List of Authors: Blanco Lobo, Pilar; Instituto de Biomedicina de Sevilla, Pediatric Infectious Diseases, Rheumatology and Immunology Unit, Hospital Universitario Virgen del Rocío. Universidad de Sevilla/CSIC, Red de Investigación Traslacional en Infectología Pediátrica RITIP Lei, Wei-Te; The Rockefeller University, St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch Pelham, Simon; The Rockefeller University, St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch Guisado Hernández, Paloma; Instituto de Biomedicina de Sevilla, Pediatric Infectious Diseases, Rheumatology and Immunology Unit, Hospital Universitario Virgen del Rocío.Universidad de Sevilla/CSIC, Red de Investigación Traslacional en Infectología Pediátrica RITIP Villaoslada, Isabel; Instituto de Biomedicina de Sevilla, Pediatric Infectious Diseases, Rheumatology and Immunology Unit, Hospital Universitario Virgen del Rocío. Universidad de Sevilla/CSIC, Red de Investigación Traslacional en Infectología Pediátrica RITIP, Seville, Spain de Felipe, Beatriz; Instituto de Biomedicina de Sevilla, Pediatric Infectious Diseases, Rheumatology and Immunology Unit, Hospital Universitario Virgen del Rocío. Universidad de Sevilla/CSIC, Red de Investigación Traslacional en Infectología Pediátrica RITIP, Seville, Spain Lucena, Jose Manuel; Hospital Universitario Virgen del Rocio, Immunology Casanova , Jean-Laurent; The Rockefeller University, St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch; INSERM UMR S1163, Laboratory of Human Genetics of Infectious Diseases, Necker Branch; University of Paris, Imagine Institute; Howard Hughes Medical Institute Olbrich, Peter; Hospital Infantil Universitario Virgen del Rocío, Infectious Diseases and Immunopathologies; Instituto de Biomedicina de Sevilla, Pediatric Infectious Diseases, Rheumatology and Immunology Unit, Hospital Universitario Virgen del Rocío.Universidad de Sevilla/CSIC, Red de Investigación Traslacional en Infectología Pediátrica RITIP Puel, Anne; The Rockefeller University, St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch; INSERM UMR S1163, Laboratory of Human Genetics of Infectious Diseases, Necker Branch; University of Paris, Imagine Institute Neth, Olaf; Hospital Infantil Universitario Virgen del Rocío, Infectious Diseases, Rheumatology and Immunology; Instituto de Biomedicina de Sevilla, Pediatric Infectious Diseases, Rheumatology and Immunology Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Unit, Hospital Universitario Virgen del Rocío. Universidad de Sevilla/CSIC, Red de Investigación Traslacional en Infectología Pediátrica RITIP Topics of paper: immunodeficiency: primary immunodeficiencies, methods: cytokines, methods: lymphocytes, methods: monocytes/macrophages, methods: T cells Abstract: Background: Inherited chronic mucocutaneous candidiasis (CMC) is often caused by inborn errors of immunity, impairing the response to, or the production of IL-17A and IL-17F. About half of the cases carry STAT1 gain-of-function (GOF) mutations. Only few patients have been reported with mutations of TRAF3IP2, a gene encoding the adaptor ACT1 essential for IL-17-receptor(R) -signaling. We investigated a 10-year-old girl with CMC, carrying a heterozygous variant of STAT1 and compound heterozygous variants of TRAF3IP2. Methods: By flow cytometry STAT1 levels and phosphorylation (CD14+) as well as IL-17A-, IL-22, IFN-γ- and IL-4-production (memory CD4+ T cells) were determined. ACT1 expression and binding to IL-17RA were assessed by western blot and co-immunoprecipitation in HEK-293T cells transfected with plasmids encoding wild-type or mutant HA-tagged ACT1 and Flag-IL-17RA. We evaluated IL-17A responses by measuring luciferase induction under a NF-κB-driven reporter system in HEK-293T cells and Gro-a secretion in fibroblasts. Results: A STAT1 variant (c.1363G>A/p.V455I) was identified by next generation sequencing and classified as likely non-pathogenic as functional testing revealed normal STAT1 expression and phosphorylation upon IFN-γ. We also found compound heterozygous variants (c.1325A>G/p.D451G and c.1335delA/p.K454fs11*) of TRAF3IP2. By overexpression, despite normal protein expression, and impaired (K454fs11*) or normal (D451G) interaction with IL-17RA, both mutant alleles resulted in impaired NF-κB-activation. Patient’s fibroblasts displayed abolished GRO-α secretion upon IL-17A stimulation. Finally, ex vivo CD4+ T cells showed increased IL-17A, IL-22, and IL-4, and normal-low IFN-γ expression upon stimulation. Conclusion: We identify novel compound heterozygous variants of TRAFP3IP2 causing autosomal recessive ACT1 deficiency in a child with CMC, and provide a review of the current literature. Page 1 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 1Biallelic TRAF3IP2 variants causing chronic mucocutaneous candidiasis in a child harboring a 2STAT1 variant 3 Pilar Blanco Lobo1+, Wei-Te Lei2+, Simon J. Pelham2, Paloma Guisado Hernández1, Isabel 4 Villaoslada1, Beatriz de Felipe1, José Manuel Lucena3, Jean-Laurent Casanova2,4,5,6, Peter 5 Olbrich1#, Anne Puel2,4,5*, Olaf Neth1* 6 71Pediatric Infectious Diseases, Rheumatology and Immunology Unit, Hospital Universitario 8 Virgen del Rocío, Instituto de Biomedicina de Sevilla, IBiS/ Universidad de Sevilla/CSIC, Red de 9 Investigación Traslacional en Infectología Pediátrica RITIP, Seville, Spain 10 2St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The 11 Rockefeller University, New York, NY, USA 12 3Immunology Unit. University Hospital Virgen del Rocío, Seville, Spain 13 4Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM UMR 1163, Paris, 14 France, EU 15 5University of Paris, Imagine Institute, Paris, France, EU 16 6Howard Hughes Medical Institute, New York, NY, USA 17 18 +PB and WTL contributed equally 19 *AP and ON contributed equally 20 #Corresponding author: Peter Olbrich, MD, PhD 21 Pediatric Infectious Diseases, Rheumatology and Immunology Unit 22 Hospital Infantil Virgen del Rocío, 23 Instituto de Biomedicina de Sevilla, Research Group: “Inborn Errors of Immunity” 24 Av. Manuel Siurot, s/n. 41013, Sevilla, Spain. 25 Phone: +34 695600725; Fax: +34-955012991 26 e-mail: [email protected] 27 28 Key words: autosomal recessive ACT1 deficiency, CMC, IL-17, Inborn error of immunity 29 30 31 32 33 Page 2 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 34 Abstract 35 Background: Inherited chronic mucocutaneous candidiasis (CMC) is often caused by inborn 36 errors of immunity, impairing the response to, or the production of IL-17A and IL-17F. About half 37 of the cases carry STAT1 gain-of-function (GOF) mutations. Only few patients have been 38 reported with mutations of TRAF3IP2, a gene encoding the adaptor ACT1 essential for IL-17- 39 receptor(R) -signaling. We investigated a 10-year-old girl with CMC, carrying a heterozygous 40 variant of STAT1 and compound heterozygous variants of TRAF3IP2. 41 Methods: By flow cytometry STAT1 levels and phosphorylation (CD14+) as well as IL-17A-, IL-22, 42 IFN-γ- and IL-4-production (memory CD4+ T cells) were determined. ACT1 expression and binding 43 to IL-17RA were assessed by western blot and co-immunoprecipitation in HEK-293T cells 44 transfected with plasmids encoding wild-type or mutant HA-tagged ACT1 and Flag-IL-17RA. We 45 evaluated IL-17A responses by measuring luciferase induction under a NF-κB-driven reporter 46 system in HEK-293T cells and Gro- secretion in fibroblasts. 47 Results: A STAT1 variant (c.1363G>A/p.V455I) was identified by next generation sequencing and 48 classified as likely non-pathogenic as functional testing revealed normal STAT1 expression and 49 phosphorylation upon IFN-. We also found compound heterozygous variants 50 (c.1325A>G/p.D451G and c.1335delA/p.K454fs11*) of TRAF3IP2. By overexpression, despite 51 normal protein expression, and impaired (K454fs11*) or normal (D451G) interaction with IL- 52 17RA, both mutant alleles resulted in impaired NF-κB-activation. Patient’s fibroblasts displayed 53 abolished GRO-α secretion upon IL-17A stimulation. Finally, ex vivo CD4+ T cells showed 54 increased IL-17A, IL-22, and IL-4, and normal-low IFN-γ expression upon stimulation. 55 Conclusion: We identify novel compound heterozygous variants of TRAFP3IP2 causing 56 autosomal recessive ACT1 deficiency in a child with CMC, and provide a review of the current 57 literature. 58 Page 3 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 59 Key message 60 This article describes the diagnostic approach to a child with chronic mucocutaneous candidiasis 61 (CMC) identifying variants in two genes (STAT1 and TRAF3IP2). Whilst basic routine immunology 62 results were normal for age, detailed immunological work-up identified the compound 63 heterozygous (D451G and K454fs11*) variants of TRAF3IP2 to be responsible of her clinical 64 symptoms. In contrast, the STAT1 variant (p.V455I) was shown to be non-pathogenic. 65 These findings highlight the importance of performing detailed functional studies in order to 66 better understand the clinical impact of identified genetic variants, thereby potentially 67 contributing in the clinical management of patients suffering from immune dysregulation 68 syndromes. In addition, we provide an update of clinical and laboratory features and discuss the 69 current available information of all published ACT1 deficiency cases so far. 70 71 Introduction 72 Chronic mucocutaneous candidiasis (CMC) is a disorder characterized by recurrent or 73 persistent symptomatic infections caused by Candida species affecting the nails, skin, oral cavity, 74 and genital mucosa [1]. CMC has been identified as an important infectious phenotype in 75 patients suffering from inborn errors of immunity in particular those with (severe) T cell 76 deficiencies, or altering the interleukin (IL)-17 pathway [2, 3]. For about half of CMC cases, signal 77 transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations were found 78 as the disease-causing etiology, and it has been suggested that the identification of a STAT1 79 variant in the setting of CMC should be followed by a thorough functional variant work-up [2, 80 4]. Patients with STAT1 GOF mutations suffer from a broad clinical phenotype including (myco-) 81 bacterial, viral, and fungal, infections, autoimmunity, malignancies, and the development of 82 aneurysms [4]. In contrast, patients with defects of the IL-17-response pathway such as 83 autosomal dominant (AD) IL-17F or JNK1 deficiency, or autosomal recessive (AR) IL-17RA, IL- Page 4 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 84 17RC, or ACT1 deficiencies are characterized by a narrow infectious spectrum mostly restricted 85 to superficial C. albicans and Staphylococcus aureus diseases [2, 5-7]. 86 The IL-17A- and IL-17F- (IL-17A/F)-mediated response is critical for the host defense against 87 fungal pathogens affecting epithelium and mucosa [6, 8]. IL-17A/F-signaling requires the binding 88 to a heterodimeric receptor(R) (IL-17RA/IL-17RC) and subsequent recruitment of the ubiquitin 89 ligase adaptor protein ACT1, encoded by TRAF3IP2, ubiquitously expressed in human tissues. 90 ACT1 further recruits tumor necrosis factor receptor-associated factor 6 (TRAF6) to trigger 91 downstream activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase 92 (MAPK) pathways (Figure 1). This results in the upregulation of pro-inflammatory cytokines and 93 chemokines with critical roles in protective mucosal immunity [2, 9-11] . 94 Mutations of ACT1 were first reported in two siblings harboring a bi-allelic missense mutation 95 (c.1607C>T, p.T536I) in the SEFIR (similar expression to fibroblast growth factor genes/IL-17) 96 domain [12]. Both patients had early onset CMC and seborrheic dermatitis, and one patient had 97 in addition bilateral persistent blepharitis due to S. aureus. The mutation impaired the 98 homotypic interaction of ACT1 with IL-17RA and IL-17RC, abrogating IL17A/F-dependent 99 signaling in fibroblasts, explaining the CMC phenotype. It also impaired ACT1 interaction with IL- 100 17RB, abolishing IL-17E-dependent signaling in leukocytes, through IL-17RA/IL-17RB. In contrast, 101 patients displayed enhanced proportions of Th17 cells, as measured ex vivo upon 102 PMA/ionomycin stimulation [12]. More recently, eight additional patients with mutations of 103 TRAF3IP2 have been described [13-16]. We present a 10-year-old girl with CMC due to AR ACT1 104 deficiency and review important clinical and laboratory characteristics of the so far published 105 cases. 106 107 1. Methods 108 Study participants Page 5 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 109 The study was conducted in accordance to the protocol approved by the Ethics Committee of 110 the Hospital Universitario Virgen del Rocío and Virgen del Macarena (Seville, Spain) (0641-N-20). 111 Genetic analysis 112 DNA was isolated from peripheral blood samples using the MagNa Pure Compact Nucleic Acid 113 Isolation Kit I (Roche) according to the manufacturer’s instructions. A next generation 114 sequencing (NGS) panel including genes related to CMC (STAT1, TRAF3IP2, CARD9, IL17F, IL17RA, 115 and IL17RC), was performed using an AmpliSeq strategy on the Ion Torrent PGM platform. 116 Primary sequence data analysis was performed with Torrent server 5.1 software. Single- 117 nucleotide polymorphisms were denoted as proposed by the Torrent Variant Caller v5.12.0.40 118 and analyzed by Ion Reporter Sofware v5.12. Identified specific mutations were validated by 119 Sanger sequencing using specific primers amplifying exon 6 of TRAF3IP2 gene and exon 17 of 120 STAT1. 121 Whole blood stimulation and flow cytometry 122 Levels of STAT1 and phosphorylated (p)STAT1 were evaluated by flow cytometry as previously 123 described [17]. Briefly, heparinized whole blood samples were stimulated with IFN-γ; 400 UI/mL; 124 Imukin, Horizon Pharma) for 15 min at 37°C. Cells were permeabilized by 1-hour incubation with 125 ice-cold methanol and stained with antibodies against CD14, STAT1, and pSTAT1 (supplementary 126 Table 1), for 1h at 4°C in the dark. Stained cells were fixed (1% paraformaldehyde, Sigma), and 127 acquired (BD LSRII FORTESSA™). Data was analyzed with the FlowJo (Tristar Treestar, Ashland, 128 OR, USA) software package. 129 Protein expression 130 HEK-293T cells (4 x 105) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) 131 supplemented with 10% fetal calf serum (FCS, GIBCO). Cells were transfected with plasmids 132 encoding wild type (WT) or mutant ACT1 (p.D451G, p.K454fs11*, or p.T536I already reported Page 6 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 133 [12] serving as positive control), using Lipofectamine 2000 according to the manufacturer’s 134 protocol. After 24 hours, cells were lysed with NP40 lysis buffer (1M Tris-HCl, 5M NaCl, 1% 135 NP40). Samples were separated by SDS-PAGE on a 4-20% gradient gel (BioRad, 456-1093), and 136 proteins were transferred to a PVDF membrane (Millipore). The membrane was blocked using 137 5% bovine serum albumin with 0.1% Tween 20 in PBS, followed by incubation with the primary 138 antibodies against ACT1 and GAPDH. Visualization of the proteins was assessed by enhanced 139 chemiluminescence. 140 Co-Immunoprecipitation 141 HEK-293T cells (1 x 106) were co-transfected with an N-terminal HA-tagged plasmid encoding 142 WT or mutant (p.D451G, p.K454fs11* or p.T536I) ACT1 along with a C-terminal Flag-tagged 143 plasmid encoding IL-17RA. After 24 hours, cells were lysed as above. An aliquot of the lysate was 144 reserved for the input control and the co-immunoprecipitation was performed on the remaining 145 lysate using agarose beads with anti-Flag antibodies conjugated (Sigma-Aldrich, F2426) followed 146 by a 3-hour incubation and washing with NP40 lysis buffer. Western blots were performed as 147 above using primary antibodies against HA (R&D systems, HAM0601) or Flag (Sigma-Aldrich, 148 A8592). 149 Luciferase assay 150 HEK-293T cells (3 x 104) were co-transfected, using Lipofectamine 2000, with WT or mutant 151 (D451G, K454fs11* or T536I) ACT1 plasmids along with NF-κB–dependent firefly luciferase 152 plasmids pGL4.32 (Promega) and a Renilla luciferase reporter pRLTK plasmid as control of 153 transfection efficiency. After 24 hours, cells were stimulated with 100 ng of IL-17A (R&D) for 24 154 hours or left unstimulated. Luciferase was then detected using the Dual-Glo Stop & Glo system 155 (Promega). 156 ELISA Page 7 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 305 (red) display impaired NF-κB activation upon IL-17. IL: interleukin; TNF: tumor necrosis 306 factor; TRAF: tumor necrosis factor receptor-associated factor; NF-κB: nuclear factor κB; 307 MAPK: mitogen-activated protein kinase; AP-1: activator protein-1. 308 Figure 2. Autosomal recessive ACT1 deficiency. A) Clinical features (from left to right) of the 309 patient with oral thrush, angular cheilitis, Type 1 hypersensitivity allergic reaction post insect 310 bites, fungal infection of the toe, and impetigo of the trunk. B) Pedigree of the kindred showing 311 the familial segregation of STAT1 and ACT1 alleles. C) Schematic representation of the ACT1 312 protein including the previously reported amino acid changes found in previously reported ACT1 313 deficiency cases (black) and in our patient (red). 314 Figure 3. IFN-γ-dependent STAT1 activation in patient’s CD14+ monocytes: Geometric mean 315 fluorescence intensity (gMFI) of total STAT1 (A), and pSTAT1 (B), evaluated by flow cytometry in 316 non-stimulated (black), or IFN-γ-stimulated (gray) CD14+ monocytes from healthy controls, 317 patient’s relatives (father, mother and sister), and the patient. 318 Figure 4. Patient’s ACT1 allele molecular characterization and impact on IL-17A responses. A) 319 HEK-293T cells were transfected with plasmids encoding WT or mutant (D451G, K454fs11*, or 320 T536I) ACT1. Protein expression was analyzed western blot using antibodies against ACT1 and 321 GAPDH, as a loading control. B) HEK-293T cells were co-transfected with plasmids encoding Flag- 322 tagged IL-17RA, together with HA-tagged WT or mutant D451G, K454fs11*, or T536I ACT1. Cell 323 lysates were immunoprecipitated with anti-Flag antibodies. Left panel shows the input, and right 324 panel the immunoprecipitation. Immunoblotting analysis was performed with anti-HA or anti- 325 Flag specific antibodies. Empty plasmid (EV) was used as negative control. C) HEK-293T cells 326 were co-transfected with WT or mutant (D451G, K454fs11*, or T536I) ACT1-encoding plasmids 327 along with NF-κB–dependent firefly luciferase plasmids and a Renilla luciferase reporter. An 328 empty plasmid (EV) was used as negative control. Luciferase activity was evaluated after 24 329 hours of stimulation with IL-17A and normalized to Renilla signal using the Dual-Glo Stop & Glo Page 14 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 330 system (Promega). D) GRO-α production, as measured by ELISA, after stimulation (IL-17A, TNF- 331 α, IL-17A+TNF-α, or IL-1β) of SV40-fibroblasts from healthy individuals, patients with IL-17RA, IL- 332 17RC, or ACT1 (ACT1T536I/T536I) deficiency, and the patient under study (D451G, K454fs11*). 333 Figure 5. Proportions of IL-17A-producing memory CD4+ T cells in the patient. Controls and 334 patient’s PBMCs were stimulated with PMA/ionomycin for 5 hours. Levels of memory CD4+ T 335 cells expressing IL-17, IL-22, IFN-γ, and IL-4 were evaluated by flow cytometry. White symbols 336 are the healthy controls, with the square representing a travel control. The black symbols are 337 the patient’s relatives, with the circle representing the father, the square the mother, and the 338 sister by the diamond, and red symbols represent the patient. 339 Figure 6: Clinical (blue) and laboratory phenotype (red) and therapeutic management (green) 340 of previously reported patients with ACT1 deficiency (n=11). 341 342 References 343 1. Eyerich, K., et al., Chronic mucocutaneous candidiasis, from bench to bedside. Eur J 344 Dermatol, 2010. 20(3): p. 260-5. 345 2. Puel, A., Human inborn errors of immunity underlying superficial or invasive candidiasis. 346 Hum Genet, 2020. 139(6-7): p. 1011-1022. 347 3. Puel, A., et al., Chronic mucocutaneous candidiasis in humans with inborn errors of 348 interleukin-17 immunity. Science, 2011. 332(6025): p. 65-8. 349 4. Toubiana, J., et al., Heterozygous STAT1 gain-of-function mutations underlie an 350 unexpectedly broad clinical phenotype. Blood, 2016. 127(25): p. 3154-64. 351 5. Levy, R., et al., Genetic, immunological, and clinical features of patients with bacterial 352 and fungal infections due to inherited IL-17RA deficiency. Proc Natl Acad Sci U S A, 2016. 353 113(51): p. E8277-E8285. 354 6. Hernandez-Santos, N. and S.L. Gaffen, Th17 cells in immunity to Candida albicans. Cell 355 Host Microbe, 2012. 11(5): p. 425-35. 356 7. Ling, Y., et al., Inherited IL-17RC deficiency in patients with chronic mucocutaneous 357 candidiasis. J Exp Med, 2015. 212(5): p. 619-31. 358 8. McGeachy, M.J., D.J. Cua, and S.L. Gaffen, The IL-17 Family of Cytokines in Health and 359 Disease. Immunity, 2019. 50(4): p. 892-906. 360 9. Dainichi, T., et al., Immune Control by TRAF6-Mediated Pathways of Epithelial Cells in 361 the EIME (Epithelial Immune Microenvironment). Front Immunol, 2019. 10: p. 1107. 362 10. Sonder, S.U., et al., IL-17-induced NF-kappaB activation via CIKS/Act1: physiologic 363 significance and signaling mechanisms. J Biol Chem, 2011. 286(15): p. 12881-90. Page 15 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 364 11. Qian, Y., et al., The adaptor Act1 is required for interleukin 17-dependent signaling 365 associated with autoimmune and inflammatory disease. Nat Immunol, 2007. 8(3): p. 366 247-56. 367 12. Boisson, B., et al., An ACT1 mutation selectively abolishes interleukin-17 responses in 368 humans with chronic mucocutaneous candidiasis. Immunity, 2013. 39(4): p. 676-86. 369 13. Bhattad, S., et al., Chronic Mucocutaneous Candidiasis in an Adolescent Boy Due to a 370 Novel Mutation in TRAF3IP2. J Clin Immunol, 2019. 39(6): p. 596-599. 371 14. Shafer, S., et al., Two patients with chronic mucocutaneous candidiasis caused by 372 TRAF3IP2 deficiency. J Allergy Clin Immunol, 2021. 373 15. Nemer, G., et al., A novel TRAF3IP2 variant causing familial scarring alopecia with mixed 374 features of discoid lupus erythematosus and folliculitis decalvans. Clin Genet, 2020. 375 98(2): p. 116-125. 376 16. Marujo, F., et al., A Novel TRAF3IP2 Mutation Causing Chronic Mucocutaneous 377 Candidiasis. J Clin Immunol, 2021. 378 17. Zimmerman, O., et al., STAT1 Gain-of-Function Mutations Cause High Total STAT1 Levels 379 With Normal Dephosphorylation. Front Immunol, 2019. 10: p. 1433. 380 18. van de Veerdonk, F.L., et al., STAT1 mutations in autosomal dominant chronic 381 mucocutaneous candidiasis. N Engl J Med, 2011. 365(1): p. 54-61. 382 19. Bai, W., et al., TRAF1 suppresses antifungal immunity through CXCL1-mediated 383 neutrophil recruitment during Candida albicans intradermal infection. Cell Commun 384 Signal, 2020. 18(1): p. 30. 385 Page 16 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Schematic representation of the cytokine pathways tested in the patient. Pathways evaluated in this manuscript include: IFN-γ-JAK-STAT pathway (purple): binding of IFN-γ to its receptor leads to activation of Janus-kinase and phosphorylation (p) of STAT1. Dimers of pSTAT1 translocate to the nucleus to activate interferon stimulated genes (ISG). Patients with gain-of-function mutation in STAT1 are characterized by high levels of STAT1 and pSTAT1 resulting in enhanced ISG expression. IFN: interferon; JAK: Janus Kinase; STAT: signal transducer and activator of transcription; NF-κB pathway: this pathway can be triggered by cytokines such as IL-17 (red), IL-1β (green), and TNF-α (pink), leading to the recruitment of TRAF proteins to trigger downstream activation of NF-κB and MAPK/AP-1 leading to the expression of pro-inflammatory molecules and chemokines (eg. CXCL1). Patients with defects of the adaptor molecule ACT1 (red) display impaired NF-κB activation upon IL-17. IL: interleukin; TNF: tumor necrosis factor; TRAF: tumor necrosis factor receptor-associated factor; NF-κB: nuclear factor κB; MAPK: mitogen-activated protein kinase; AP-1: activator protein-1. 52x34mm (1200 x 1200 DPI) Page 17 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Autosomal recessive ACT1 deficiency. A) Clinical features (from left to right) of the patient with oral thrush, angular cheilitis, Type 1 hypersensitivity allergic reaction post insect bites, fungal infection of the toe, and impetigo of the trunk. B) Pedigree of the kindred showing the familial segregation of STAT1 and ACT1 alleles. C) Schematic representation of the ACT1 protein including the previously reported amino acid changes found in previously reported ACT1 deficiency cases (black) and in our patient (red). 46x33mm (1200 x 1200 DPI) Page 18 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review IFN-γ-dependent STAT1 activation in patient’s CD14+ monocytes: Geometric mean fluorescence intensity (gMFI) of total STAT1 (A), and pSTAT1 (B), evaluated by flow cytometry in non-stimulated (black), or IFN-γ- stimulated (gray) CD14+ monocytes from healthy controls, patient’s relatives (father, mother and sister), and the patient. 47x18mm (1200 x 1200 DPI) Page 19 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Patient’s ACT1 allele molecular characterization and impact on IL-17A responses. A) HEK-293T cells were transfected with plasmids encoding WT or mutant (D451G, K454fs11*, or T536I) ACT1. Protein expression was analyzed western blot using antibodies against ACT1 and GAPDH, as a loading control. B) HEK-293T cells were co-transfected with plasmids encoding Flag-tagged IL-17RA, together with HA-tagged WT or mutant D451G, K454fs11*, or T536I ACT1. Cell lysates were immunoprecipitated with anti-Flag antibodies. Left panel shows the input, and right panel the immunoprecipitation. Immunoblotting analysis was performed with anti-HA or anti-Flag specific antibodies. Empty plasmid (EV) was used as negative control. C) HEK-293T cells were co-transfected with WT or mutant (D451G, K454fs11*, or T536I) ACT1-encoding plasmids along with NF-κB–dependent firefly luciferase plasmids and a Renilla luciferase reporter. An empty plasmid (EV) was used as negative control. Luciferase activity was evaluated after 24 hours of stimulation with IL-17A and normalized to Renilla signal using the Dual-Glo Stop & Glo system (Promega). D) GRO-α production, as measured by ELISA, after stimulation (IL-17A, TNF-α, IL-17A+TNF-α, or IL-1β) of SV40- fibroblasts from healthy individuals, patients with IL-17RA, IL-17RC, or ACT1 (ACT1T536I/T536I) deficiency, and the patient under study (D451G, K454fs11*). 46x28mm (1200 x 1200 DPI) Page 20 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Proportions of IL-17A-producing memory CD4+ T cells in the patient. Controls and patient’s PBMCs were stimulated with PMA/ionomycin for 5 hours. Levels of memory CD4+ T cells expressing IL-17, IL-22, IFN-γ, and IL-4 were evaluated by flow cytometry. White symbols are the healthy controls, with the square representing a travel control. The black symbols are the patient’s relatives, with the circle representing the father, the square the mother, and the sister by the diamond, and red symbols represent the patient. 46x33mm (1200 x 1200 DPI) Page 21 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Clinical (blue) and laboratory phenotype (red) and therapeutic management (green) of previously reported patients with ACT1 deficiency (n=11). 46x34mm (1200 x 1200 DPI) Page 22 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Supplementary Table 1. Monoclonal antibodies used in flow cytometry Evaluation of JAK/STAT1 pathway Target Fluorophore Clone Company CD14 FITC M5E2 BD STAT1 Alexa Fluor 647 1/STAT1 BD pY701 -STAT1 PerCP Cy5.5 4A BD Cytokine expression in memory CD4+ T cells Target Fluorophore Clone Company CD8 BUV805 SK1 BD CCR7 BUV737 3D12 BD CD45RA BUV395 HI100 BD CD3 Spark Blue 550 SK7 BD CD4 BV480 SK3 BD IL-17A BV785 BL168 BioLegend IL-4 BV605 MP4-25D2 BioLegend IL-22 APC 2G12A41 BioLegend IFN- eFluor405 4S.B3 ThermoFisher Page 23 of 25 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology