Article
Gain-of-function human STAT1 mutations 
impair IL-17 immunity and underlie chronic 
mucocutaneous candidiasis
Luyan Liu,1 Satoshi Okada,2 Xiao-Fei Kong,2 Alexandra Y. Kreins,2  
Sophie Cypowyj,2 Avinash Abhyankar,2 Julie Toubiana,3 Yuval Itan,2  
Magali Audry,2 Patrick Nitschke,4 Cécile Masson,4 Beata Toth,9 Jérome Flatot,1 
Mélanie Migaud,1 Maya Chrabieh,1 Tatiana Kochetkov,2 Alexandre Bolze,1,2 
Alessandro Borghesi,1 Antoine Toulon,5 Julia Hiller,10 Stefanie Eyerich,10  
Kilian Eyerich,10,11 Vera Gulácsy,9 Ludmyla Chernyshova,12 Viktor Chernyshov,13 
Anastasia Bondarenko,12 Rosa María Cortés Grimaldo,14  
Lizbeth Blancas-Galicia,15 Ileana Maria Madrigal Beas,14 Joachim Roesler,16 
Klaus Magdorf,17 Dan Engelhard,18 Caroline Thumerelle,19  
Pierre-Régis Burgel,20 Miriam Hoernes,21 Barbara Drexel,21 Reinhard Seger,21 
Theresia Kusuma,22 Annette F. Jansson,22 Julie Sawalle-Belohradsky,22  
Bernd Belohradsky,22 Emmanuelle Jouanguy,1,2 Jacinta Bustamante,1  
Mélanie Bué,23 Nathan Karin,24 Gizi Wildbaum,24 Christine Bodemer,5  
Olivier Lortholary,6 Alain Fischer,7 Stéphane Blanche,7 Saleh Al-Muhsen,24 
Janine Reichenbach,21 Masao Kobayashi,26 Francisco Espinosa Rosales,15 
Carlos Torres Lozano,14 Sara Sebnem Kilic,27 Matias Oleastro,28 Amos 
Etzioni,24 Claudia Traidl-Hoffmann,10,11 Ellen D. Renner,22 Laurent Abel,1,2 
Capucine Picard,1,6,8 László Maródi,9 Stéphanie Boisson-Dupuis,1,2 Anne Puel,1 
and Jean-Laurent Casanova1,2,7,25
1Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Necker Medical School, Institut National de la Santé  
et de la Recherche Médicale U980 and University Paris Descartes, 75015 Paris, France
2St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065
3Department of Pediatrics, 4Bioinformatics Unit, 5Department of Dermatology, 6Department of Infectious Diseases, 7Pediatric Hematology-
Immunology Unit, and 8Center for Immunodeficiency, Necker Hospital, AP-HP, and University Paris Descartes, 75015 Paris, France
9Department of Infectious and Pediatric Immunology, Medical and Health Science Center, University of Debrecen, 4032 Debrecen, Hungary
10Center for Allergy and Environment, Helmholtz Center/TUM, 80802 Munich, Germany
11Department of Dermatology, Technische Universitat, 80802 Munich, Germany
12Department of Pediatric Infectious Diseases and Clinical Immunology, National Medical Academy for Post-Graduate Education, 01024 Kiev, Ukraine
13Laboratory of Immunology, Institute of Pediatrics, Obstetrics, and Gynecology, National Academy of Medical Sciences, 01024 Kiev, Ukraine
14Allergy and Immunology Department, UMAE-HE-CMNO-IMMS, 44500 Guadalajara, Mexico
15National Institute of Pediatrics, 04530 Mexico City, Mexico
16Department of Pediatrics, University Hospital Carl Gustav Carus, 01307 Dresden, Germany
17Department of Pediatric Pneumology and Immunology, Charité Medical School of Berlin, 11117 Berlin, Germany
18Department of Pediatrics, Hadassah University Hospital, 91120 Jerusalem, Israel
CORRESPONDENCE  19
Anne Puel:  Pneumology and Allergology Unit, Hospital Jeanne de Flandres, 59037 Lille, France
20
anne.puel@inserm.fr Pneumology and UPRES EA 2511, Hospital Cochin, AP-HP, 75014 Paris, France
21
OR Division of Immunology, Hematology, and BMT, Children’s Research Center, Children’s Hospital, University of Zurich, 8032 Zurich, Switzerland
Jean-Laurent Casanova:  22University Children’s Hospital at Dr. von Haunersches Kinderspital, Ludwig Maximilian University, 80337 Munich, Germany
jean-laurent.casanova@ 23University Hospital Center of Brest, 29609 Brest, France
rockefeller.edu 24Rappaport Faculty of Medicine, Technion, 31096 Haifa, Israel.
25Prince Naif Center for Immunology Research, Department of Pediatrics, College of Medicine, King Saud University, Riyadh, 11461Saudi Arabia
Abbreviations used: AD, auto- 26Department of Pediatrics, Hiroshima University Graduate School of Biomedical Sciences, 739-8511 Hiroshima, Japan
somal dominant; AR, autosomal 27Department of Pediatrics, Uludag University School of Medicine, 16059 Bursa, Turkey
recessive; CMC, chronic muco- 28National Children’s Hospital Prof. Dr. Juan P. Garrahan, 12049 Buenos Aires, Argentina
cutaneous candidiasis; CMCD, 
L. Liu, S. Okada, X.-F. Kong, A.Y. Kreins, and S. Cypowyj contributed equally to this paper.
CMC disease; EMSA, electro-
A. Abhyankar, J. Toubiana, Y. Itan, M. Audry, P. Nitschke, C. Masson, and B. Toth contributed equally to this paper.
phoretic mobility shift assay; S. Al-Muhsen, J. Reichenbach, M. Kobayashi, F. Espinoza Rosales, C. Torres Lozano, S. Sebnem Kilic, M. Oleastro, A. Etzioni,  
GAS, -activated sequence; C. Traidl-Hoffmann, E.D. Renner, L. Abel, and C. Picard contributed equally to this paper.
ISRE, IFN-stimulated response L. Maródi, S. Boisson-Dupuis, A. Puel, and J.-L. Casanova contributed equally to this paper.
element; MSMD, Mendelian 
© 2011 Liu et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the 
susceptibility to mycobacterial publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 
disease; WB, Western blotting. Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 208 No. 8 1635-1648 1635
www.jem.org/cgi/doi/10.1084/jem.20110958
The Journal of Experimental Medicine
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Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or 
autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germ-
line mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 
mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-
dependent cellular responses to IFN-. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular 
bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-/, IFN-, IFN-, and IL-27. In 
contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-
dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and 
IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated 
STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent  
IL-17 inhibitors IFN-/, IFN-, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 
inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function 
STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity.
Chronic mucocutaneous candidiasis (CMC) is characterized type 1 (Atkinson et al., 2001). It is unclear whether CMCD, 
by persistent or recurrent disease of the nails, skin, oral, or with these or other manifestations (Shama and Kirkpatrick, 
genital mucosae caused by Candida albicans (Puel et al., 2010b). 1980; Bentur et al., 1991; Germain et al., 1994), is immuno-
CMC may be caused by various inborn errors of immunity. logically and genetically related to pure CMCD. Low propor-
CMC is one of a multitude of infectious diseases observed in tions of IL-17A–producing T cells have been documented in 
patients with broad and profound T cell deficiencies. In con- five patients with CMCD (Eyerich et al., 2008). Moreover, a 
trast, patients with the autosomal dominant (AD) hyper IgE candidate gene approach centered on IL-17 immunity re-
syndrome, caused by dominant-negative mutations of STAT3, cently revealed the first genetic etiologies of pure CMCD. In 
are susceptible principally to CMC and staphylococcal dis- a consanguineous family from Morocco, a child with CMCD 
eases of the lungs and skin (Minegishi, 2009). These patients was found to display AR complete IL-17RA deficiency (Puel 
have very low proportions of circulating IL-17A– and IL-22– et al., 2011). His leukocytes and fibroblasts did not respond 
producing T cells, probably because of impaired responses to to IL-17A or IL-17F homodimers, or to IL-17A/F hetero-
IL-6, IL-21, and/or IL-23 (de Beaucoudrey et al., 2008; Ma dimers. Four patients from an Argentinean family were shown 
et al., 2008; Milner et al., 2008; Renner et al., 2008; Minegishi to harbor dominant-negative mutations in the IL17F gene 
et al., 2009). Patients with autosomal recessive (AR) IL-12p40 (Puel et al., 2011). Mutated IL-17F–containing homodimers 
or IL-12R1 deficiency suffer from Mendelian susceptibility and heterodimers were produced in normal amounts but 
to mycobacterial disease (MSMD) and occasionally develop were not biologically active, as they were unable to bind to 
mild CMC (Filipe-Santos et al., 2006; de Beaucoudrey et al., the IL-17 receptor. Morbid mutations in IL17RA and IL17F 
2010). Some have low proportions of IL-17A– and IL-22– demonstrated that CMCD could be caused by inborn errors 
producing T cells, presumably because of the abolition of of IL-17 immunity. However, no genetic etiology has yet 
IL-23 responses (de Beaucoudrey et al., 2008, 2010). The pro- been identified for most patients with CMCD. We set out to 
portion of IL-17A–producing T cells was also found to be low identify new genetic etiologies of CMCD through a recently 
in a family with AR CARD9 deficiency, dermatophytosis, developed genome-wide approach based on whole-exome 
invasive candidiasis, and CMC (Glocker et al., 2009). Finally, sequencing (Alcaïs et al., 2010; Bolze et al., 2010; Byun et al., 
CMC is the only infection in patients with autoimmune 2010; Ng et al., 2010).
polyendocrinopathy syndrome type 1, who have high titers of 
neutralizing autoantibodies against IL-17A, IL-17F, and IL-22 RESULTS
(Kisand et al., 2010; Puel et al., 2010a). Thus, regardless of the We investigated one sporadic case and the probands from five 
underlying illness, CMC pathogenesis apparently involves multiplex kindreds with AD CMCD, by whole-exome se-
the impairment of IL-17A, IL-17F, and IL-22 immunity (Puel quencing. The annotated data were analyzed with sequence 
et al., 2010b). analysis software that had been developed in-house and made 
The pathogenesis of CMC was eventually deciphered it possible to analyze and compare several exome sequences 
through investigations of patients with CMC disease (CMCD), simultaneously. A hierarchy of candidate variations was gener-
in which CMC is isolated, with no other infectious or auto- ated by filtering out known polymorphisms reported in dbSNP 
immune signs (Kirkpatrick, 2001; Puel et al., 2010b). The and 1,000-genome databases. We also used our own database 
definition of CMCD is not absolute, as illustrated in some of 250 exomes to filter out unreported polymorphisms 
patients by cutaneous staphylococcal disease, which is milder (Table S1). The only relevant gene displaying heterozygous 
than that in patients with AD hyper IgE syndrome (Herrod, variations in at least four of the six unrelated patients with AD 
1990), or by autoimmune features affecting the thyroid in CMCD was STAT1 (Fig. 1, A and B, Kindreds A, B, G, and L; 
particular, although fewer such features are observed than Table I; and Table S2).  Three different STAT1 mutations 
in patients with autoimmune polyendocrinopathy syndrome were found in four patients; they were confirmed by Sanger 
1636 Human STAT1 activating mutations impair IL-17 immunity | Liu et al.
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Article
sequencing and shown to be missense mutations. All these dephosphorylation (Fig. 1 C; Chen et al., 1998; Zhong et al., 
mutations affected the coiled-coil domain, which plays a key 2005; Mertens et al., 2006). In contrast, the other two morbid 
role in unphosphorylated STAT1 dimerization and STAT1 mutations (K201N and K211R) affect residues located on the 
nuclear dephosphorylation (Fig. 1, A and C; Chen et al., 1998; other side of the coiled-coil domain (Fig. 1 C). Moreover, 
Levy and Darnell, 2002; Braunstein et al., 2003; Zhong et al., these two hypomorphic alleles were shown to be pathogenic 
2005; Hoshino et al., 2006; Mertens et al., 2006). We therefore not because they were missense, but because they promoted 
sequenced the corresponding coding region of STAT1 (exons the splicing out of exon 8, resulting in AR partial STAT1 de-
6 to 10) in another 106 patients, including 57 with spo- ficiency, with the production of small amounts of intrinsically 
radic CMCD and 49 from 22 multiplex kindreds with AD functional STAT1 molecules (Kong et al., 2010; Kristensen 
CMCD. 29 patients from 16 kindreds were heterozygous for et al., 2011). These genetic data strongly suggest that hetero-
a STAT1 missense mutation (Fig. 1, A and B, Kindreds C-F, zygous missense mutations in the coiled-coil domain of STAT1 
H-K, and M-T; Fig. 1 C; and Table I; Table S3). In total, 36 may cause AD CMCD in a large fraction of patients. Never-
patients from 20 kindreds were heterozygous for 1 of the 12 theless, the occurrence of other germline mutations in STAT1 
missense mutations identified that affected the coiled-coil in patients without CMC and with an AD or AR predisposi-
domain of STAT1. 11 other CMCD patients in these kindreds tion to other infectious diseases raised questions about whether 
were not genotyped. The intrafamilial segregation of the mu- these mutations were really responsible for CMCD and the 
tations was consistent with an AD trait, as all patients with underlying mechanism of disease.
CMCD from the kindreds tested were heterozygous, whereas We functionally characterized the CMCD-causing STAT1 
none of these mutations was found in the heterozygous state allele R274Q, which was found in four kindreds (Fig. 1 B and 
in any of the healthy relatives tested (Fig. 1 B). Moreover, Table I). We compared it with a WT and an MSMD-causing 
the STAT1 haplotypes for common SNPs indicated that the loss-of-function STAT1 allele (L706S; Dupuis et al., 2001). 
five recurrent mutations were caused by mutation hotspots We transfected STAT1-deficient U3C fibrosarcoma cells with 
rather than founder effects (unpublished data). Finally, the WT, R274Q, or L706S STAT1 alleles. Upon stimulation with 
mutations were found to have occurred de novo in at least IFN-, IFN-, or IL-27, cells transfected with the R274Q 
four kindreds, which is consistent with a high clinical pene- allele responded two to three times more strongly than those 
trance of these alleles. The mutations were not found in the transfected with the WT allele, as shown by measurement of 
National Center for Biotechnology Information, Ensembl, the induction of -activated sequence (GAS)–dependent re-
and dbSNP databases. They were also absent from 1,052 con- porter gene transcription activity, with mock- and L706S-
trols from 52 ethnic groups in the Centre d’Etude du Poly- transfected cells serving as negative controls (Fig. 2 A and 
morphisme Humain and Human Genome Diversity panels, Fig. S1 A). All STAT1 alleles were expressed at an equal 
suggesting that they were rare, CMCD-inducing variants rather strength, as shown by Western blotting (WB; Fig. 2 B). Higher 
than irrelevant polymorphisms. levels of STAT1 phosphorylation were observed for the 
The 12 missense mutations were not conservative and R274Q allele than for the WT allele after stimulation with 
were therefore predicted to affect protein structure and func- IFN-, IFN-, and IL-27, whereas STAT3 phosphorylation 
tion. Moreover, most of the affected residues were found to levels were similar for the two alleles (Fig. 2 B). In contrast, 
have been conserved throughout evolution in the species in the induction of IFN-stimulated response element (ISRE)–
which STAT1 had been sequenced (Table S3). Accordingly, dependent transcription activity by IFN- was normal (Fig. S1, 
POLYphen II predicted that all but one of these mutations B and C). In the same experimental conditions, the other 10 
would be possibly or probably damaging (Adzhubei et al., 2010; CMCD-associated STAT1 alleles tested were also gain-of-
Table S3). None of the previously described nine patients function, unlike the K201N and K211R alleles (Fig. S1 D). 
with AD STAT1 deficiency and MSMD was heterozygous Upon stimulation with IFN-, IFN-, or IL-27, an increase 
for mutations affecting the coiled-coil domain (Fig. 1, A and C; in GAS-binding activity was detected in cells transfected with 
Dupuis et al., 2001; Chapgier et al., 2006a; Averbuch et al., the R274Q allele (Fig. S1 E). Accordingly, the transcription of 
2011; unpublished data). However, three of the eight patients the CXCL9 and CXCL10 target genes was enhanced (Fig. 2, 
with AR STAT1 deficiency and susceptibility to intracellular C and D). Overall, these data indicate that at least 11 of the 
bacterial and viral diseases, who, like their heterozygous rela- 12 CMCD-linked STAT1 missense alleles are intrinsically 
tives, did not display CMC, carried mutations affecting the gain-of-function.
coiled-coil domain (Fig. 1, A and C; Chapgier et al., 2009; The mechanism involved an increase in STAT1 tyrosine 
Chapgier et al., 2006b; Dupuis et al., 2003; Kong et al., 2010; 701 residue phosphorylation, as shown for R274Q by WB 
Kristensen et al., 2011; Averbuch et al., 2011). These three pa- after stimulation with IFN-, IFN-, and IL-27 (Fig. 2 B). 
tients from two kindreds carried the K201N or K211R mu- STAT1 was not constitutively activated, and STAT3 was nor-
tation (Kong et al., 2010; Kristensen et al., 2011). Nevertheless, mally activated in R274Q-transfected cells (Fig. 2 B and not 
the three-dimensional structure of phosphorylated STAT1 depicted). Almost all the mutant STAT1 molecules, which 
molecules revealed that the 12 CMCD-linked missense mu- were phosphorylated in response to IFN-, translocated to 
tations affected a cluster of residues located in a specific pocket and accumulated in the nucleus, as shown by immunofluores-
of the coiled-coil domain, near residues essential for STAT1 cence (Fig. S1 F). WB showed R274Q STAT1 to be more 
JEM Vol. 208, No. 8 1637
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Figure 1. Heterozygous missense mutations affecting the STAT1 coiled-coil domain in kindreds with AD CMCD. (A) The human STAT1  iso-
form is shown, with its known pathogenic mutations. Coding exons are numbered with roman numerals and delimited by a vertical bar. Regions corre-
sponding to the coiled-coil domain (CC), DNA-binding domain (DNA-B), linker domain (L), SH2 domain (SH2), tail segment domain (TS), and transactivator 
domain (TA) are indicated, together with their amino-acid boundaries, and are delimited by bold lines. Tyr701 (pY) and Ser727 (pS) are indicated. Muta-
tions in green are dominant and associated with partial STAT1 deficiency and MSMD. Mutations in brown are recessive and associated with complete 
STAT1 deficiency and intracellular bacterial and viral disease. Mutations in blue are recessive and associated with partial STAT1 deficiency and intracellular 
bacterial and/or viral disease. Mutations in red are dominant and associated with a gain-of-function of STAT1 and CMCD. (B) Pedigrees of 20 families 
with AD “gain-of-function” STAT1 mutations. Each kindred is designated by a letter (A to T), each generation is designated by a roman numeral (I-II-III-IV), 
and each individual is designated by an Arabic numeral (each individual studied is identified by a code of this type, organized from left to right). Black 
indicates CMCD patients. The probands are indicated by arrows. When tested, the genotype for STAT1 is indicated below each individual. (C) Three- 
dimensional structure of phosphorylated STAT1 in complex with DNA. Connolly surface representation, with the following amino acids highlighted: red, 
amino acids mutated in patients with CMCD; blue, amino acids located in the coiled-coil domain and mutated in patients with MSMD and viral diseases; 
yellow, amino acids identified in vitro as affecting the dephosphorylation process.
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Table I. Summary of the clinical and genetic data for the patients
Patient Age at Origin Clinical features of CMC Cause of death (age/yr) Autoimmunity Genotype
presentation 
A-I-1 - France Nails Not related to the disease (old None -
age)
A-II-1 - France Nails Not related to the disease (old None -
age)
A-III-1 1 mo France Nails, oral cavity, oropharynx, None WT/R274Q
genital mucosa
A-III-3 - France Nails, oral cavity Not related to the disease (40) None -
A-III-4 - France Nails, oral cavity None -
A-IV-1 1 mo France Nails, oral cavity, oropharynx None WT/R274Q
B-II-1 - France - None -
B-III-2 3 yr France Skin, nails, oral cavity, oropharynx, None WT/K286I
genital mucosa
B-IV-1 5 yr France & Skin, nails, oral cavity, oropharynx None WT/K286I
Congo
B-IV-2 5 mo France & Skin, nails, oral cavity, oropharynx Cerebral aneurysm (8) None -
Congo
C-III-1 - Turkey Nails, oral cavity, genital mucosa Cerebral aneurysm (34) Thyroid WT/R274Q
autoimmunity
C-IV-1 - Turkey Nails, oral cavity None WT/R274Q
D-II-1 - France Nails, oral cavity, genital mucosa - -
D-III-2 7 yr France Skin, oral cavity, oropharynx None WT/M202V
D-IV-2 1 mo France Skin, nails, oropharynx Thyroid WT/M202V
autoimmunity
E-II-1 1 yr Germany Skin, oral cavity, oropharynx Squamous cell carcinoma (54) - -
E-III-2 1 yr Germany Nails, oral cavity, oropharynx, Thyroid WT/C174R
genital mucosa autoimmunity
E-III-3 9 mo Germany Skin, nails, oral cavity, oropharynx, Thyroid WT/C174R
genital mucosa autoimmunity
E-IV-1 18 mo Germany Skin, oral cavity, oropharynx, genital None WT/C174R
mucosa
E-IV-2 2 yr Germany Skin, oral cavity, oropharynx Thyroid WT/C174R
autoimmunity
E-IV-4 2 yr Germany Skin, oral cavity, oropharynx, genital None WT/C174R
mucosa
E-IV-5 1 yr Germany Skin, nails, oral cavity, oropharynx None WT/C174R
F-III-2 1 mo Argentina Nails, oral cavity, oropharynx, - WT/R274W
genital mucosa
F-IV-2 1 mo Argentina Skin, nails, oral cavity, oropharynx - WT/R274W
F-IV-3 6 mo Argentina Nails, oral cavity, genital mucosa - WT/R274W
G-II-1 3 mo Ukrainian Nails, skin, oral cavity, oropharynx, None WT/D165G
esophagus
H-I-2 1 yr Japan Skin, oropharynx, esophagus - WT/R274Q
H-II-2 5 yr Japan Oral cavity, oropharynx - WT/R274Q
I-II-3 9 mo Mexico Skin, nails, oral cavity, genital None WT/T288A
mucosa
J-I-2 - Switzerland Oral cavity, oropharynx None WT/T288A
J-II-2 3 mo Switzerland Oral cavity, oropharynx - WT/T288A
K-II-2 11 mo Switzerland Nails, oral cavity, oropharynx Thyroid WT/Y170N
autoimmunity
L-I-2 7 yr France Skin, nails, oropharynx, esophagus Thyroid WT/R274Q
autoimmunity
L-II-1 1 mo France Skin, nails, oropharynx, esophagus None WT/R274Q
M-II-2 6 mo Germany Skin, nails, oropharynx, genital Thyroid WT/D165H
mucosa autoimmunity
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Table I. Summary of the clinical and genetic data for the patients (Continued)
Patient Age at Origin Clinical features of CMC Cause of death (age/yr) Autoimmunity Genotype
presentation 
N-II-2 1 yr Germany Skin, nails, oropharynx Squamous cell carcinoma (54) None WT/R274W
O-II-1 18 mo Germany Oral cavity, oropharynx None WT/M202I
P-I-1 1 yr Israel Oropharynx, genital mucosa Not related to the disease (46) None -
P-II-1 <2 yr Israel Skin, nails, oropharynx None WT/A267V
P-II-2 <2 yr Israel Skin, nails, oropharynx None WT/A267V
Q-II-1 1 mo France Skin, oral cavity, oropharynx, genital None WT/R274W
mucosa
R-I-1 4 yr France Skin, nails, oropharynx Squamous cell carcinoma (55) None -
R-II-1 18 mo France Lips, oropharynx None WT/M202V
S-I-2 6 mo France Skin, oral cavity, oropharynx Systemic lupus WT/M202I
erythematosus
S-II-2 1 yr France Nails None -
S-II-3 1 mo France Skin, oropharynx None WT/M202I
T-II-3 1 yr Germany Skin, nails, oropharynx Squamous cell carcinoma (41) None WT/Q271P
None of the patients displays autoantibodies against IL-17A, IL-17F, and IL-22. -, unknown.
strongly phosphorylated than the WT protein in both cyto- shift assay (EMSA; Fig. 3, A and C). In contrast, the DNA-
plasmic and nuclear extracts (Fig. S1 G). The mechanism binding activity of ISGF-3 seemed to be normal in cells from 
underlying the gain of R274Q phosphorylation was explored the patient stimulated with IFN-/ (Fig. S3 A). These data 
with the tyrosine kinase inhibitor staurosporine and the strongly suggest that the heterozygous R274Q allele is domi-
phosphatase inhibitor pervanadate. The dephosphorylation of nant for STAT1-dependent responses and gain-of-function for 
IFN-–activated R274Q STAT1 was impaired by stauro- GAF-dependent cellular responses to key STAT1-activating 
sporine, but less than that of the known dephosphorylation cytokines, such as IFN-/, IFN-, and IL-27. The mutation 
mutant F77A (Fig. 2 E; Zhong et al., 2005). In contrast, per- may also affect IFN- responses.
vanadate normalized the phosphorylation of R274Q to We then tested cytokines that predominantly activate 
WT levels (Fig. 2 F). Another CMCD-linked mutation, STAT3, rather than STAT1, such as IL-6, IL-21, IL-22, and 
D165G (Fig. 1, A–C), also resulted in impaired dephosphory- IL-23 (Hunter, 2005; Kishimoto, 2005; Kastelein et al., 2007; 
lation that could be normalized by adding pervanadate (Fig. 2 F Spolski and Leonard, 2008; Donnelly et al., 2010; Sabat, 2010; 
and Fig. S1 H). Thus, at least two CMCD-linked STAT1 mis- Ouyang et al., 2011). Peripheral T cell blasts from a patient 
sense alleles (R274Q and D165G) are gain-of-function displayed normal STAT3 activation in response to IL-23, as 
caused by the impairment of nuclear dephosphorylation. shown by WB (Fig. S3 B). No increase in STAT1 phosphory-
These alleles may therefore enhance cellular responses to lation was detected in cells from a patient or controls upon 
cytokines activating STAT1 predominantly and STAT3 to a IL-23 stimulation. Furthermore, fibroblasts from a patient 
lesser extent, such as IFN-/, IFN-, IFN-, and IL-27, and displayed normal activation of STAT3 in response to IL-22 
possibly also responses to cytokines activating STAT3 pre- (Fig. S3 C). In the same conditions, no STAT1 phosphorylation 
dominantly and STAT1 to a lesser extent, such as IL-6, IL-21, was detected in cells from the patient or controls (unpublished 
IL-22, and IL-23 (Fig. S2). data). In contrast, the levels of STAT1 phosphorylation in re-
We investigated the dominance of the STAT1 alleles at the sponse to IL-6 and IL-21 were higher in the patient’s EBV-B 
cellular level by testing EBV-B–transformed (EBV-B) cells and cells than in cells from healthy controls and from a patient 
SV-40–transformed dermal fibroblasts from a CMCD patient with MSMD heterozygous for the L706S allele, whereas 
heterozygous for the STAT1 R274Q allele. We observed en- STAT3 activation was normal as shown by WB (Fig. 3, F 
hanced IFN-/–, IFN-–, and IL-27–dependent STAT1 and H). Consistent with these findings, stronger GAS activity 
phosphorylation in EBV-B cells from a patient heterozygous was observed in cells from the patient in response to IL-6 and 
for the STAT1 R274Q allele, as shown by WB (Fig. 3, B IL-21 stimulation (Fig. 3, E and G). These data suggest that 
and D). Phospho-STAT1 accumulated in the nucleus of heterozygous missense mutations in the coiled-coil domain 
R274Q heterozygous SV-40 fibroblasts upon IFN- stimulation, of STAT1 are dominant and gain-of-function for GAF- 
as well as in EBV-B cells (Fig. 3 I and Fig. S3 D). Moreover, the dependent cellular responses for cytokines that predominantly 
IFN-/–, IFN-–, and IL-27–induced DNA-binding activity activate STAT3, such as IL-6 and IL-21. Overall, these data 
of GAF was stronger in cells from the CMCD patient than in suggest that the STAT1 alleles are truly responsible for CMCD 
those from a healthy control or from a MSMD patient carrying in these kindreds and raise questions about the immuno-
the L706S mutant allele, as shown by electrophoretic mobility logical basis of CMCD.
1640 Human STAT1 activating mutations impair IL-17 immunity | Liu et al.
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Figure 2. The mutant R274Q STAT1 allele 
is gain-of-phosphorylation and gain-of-
function for GAF-dependent cellular  
responses. U3C cells were transfected with a 
mock vector, a WT, or two mutant alleles of 
STAT1 (R274Q and L706S). The response to  
IFN-, IL-27, and IFN- was then evaluated by 
determining luciferase activity of a reporter 
gene under the control of the GAS promoter 
(A), and by determining STAT1 and STAT3 phos-
phorylation by Western blot (B). Experiments 
were performed at least three times indepen-
dently. (C and D) Quantitative RT-PCR was used 
to measure the induction of CXCL9 (C) and 
CXCL10 (D) 2–8 h after stimulation with IFN-. 
Experiments were performed two times inde-
pendently. (E) The nuclear dephosphorylation 
of STAT1 was tested by WB in U3C cells trans-
fected with a mock vector, WT STAT1, the 
R274Q, or the F77A (a known loss-of-dephos-
phorylation mutant) STAT1 mutant alleles, and 
treated with IFN- with or without the tyrosine 
kinase inhibitor staurosporine for the indicated 
periods of time (in minutes). Three independent 
experiments were performed. (F) Western blot 
of U3C cells transfected with mock, WT, R274Q, 
D165G, and F77A alleles of STAT1, nontreated 
or treated with IFN- in the absence or pres-
ence of the phosphatase inhibitor pervanadate. 
Two independent experiments were performed. 
Error bars represent SD of one experiment done 
in triplicate (Fig. S1 D).
Villarino et al., 2010). Moreover, mouse 
IFN- (Feng et al., 2008; Tanaka 
et al., 2008; Villarino et al., 2010) 
and human IFN-/ (Chen et al., 
2009; Ramgolam et al., 2009) have 
been shown to antagonize the devel-
opment of IL-17–producing T cells 
via STAT1. In addition, IL-6, IL-21, 
and IL-23 are prominent inducers of 
IL-17–producing T cells, via a mecha-
nism dependent on STAT3 and antag-
onized by STAT1 (Hirahara et al., 2010). 
Finally, we recently showed that in-
born errors of IL-17F or IL-17RA 
were genetic etiologies of CMCD 
(Puel et al., 2010b, 2011). We thus 
determined the proportion of IL-17A– 
and IL-22–producing T cells by flow 
IL-27 is a potent inhibitor of the development of IL-17– cytometry in patients with heterozygous STAT1 mutations 
producing T cells in mice (Batten et al., 2006; Stumhofer and AD CMCD. The 18 CMCD patients carrying gain-of-
et al., 2006; Yoshimura et al., 2006; Amadi-Obi et al., 2007; function mutations in STAT1 that were tested had lower 
Diveu et al., 2009; El-behi et al., 2009; Villarino et al., proportions of circulating IL-17A– and IL-22–producing 
2010) and humans (Diveu et al., 2009; Liu and Rohowsky- T cells ex vivo than 28 healthy controls (P < 104) and six 
Kochan, 2011), through a mechanism dependent on STAT1 patients bearing loss-of-function STAT1 alleles (P < 2.103; 
(Amadi-Obi et al., 2007; Batten et al., 2006; Diveu et al., 2009; Fig. 4, A and B; and Fig. S4 G). In contrast, they displayed 
Liu and Rohowsky-Kochan, 2011; Stumhofer et al., 2006; normal proportions of IFN-–producing T cells (Fig. S4 F). 
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Figure 3. The mutant R274Q STAT1  
allele is dominant for GAF-dependent 
cellular responses at the cellular level. The 
responses of the patient’s EBV-B cells (R274Q/
WT) were evaluated independently at least 
twice, by EMSA, with a GAS probe (A, C, E,  
and G), and by Western blot (B, D, F, and H). 
This response was compared with that of one 
or two healthy controls (WT/WT1 and WT/
WT2), heterozygous cells with a WT and a 
loss-of-function STAT1 allele (STAT1+/), cells 
heterozygous for a dominant loss-of-function 
mutation of STAT1 (L706S/WT), cells with 
complete STAT1 deficiency (STAT1/), and 
cells from two patients heterozygous for 
dominant loss-of-function mutations of 
STAT3 (STAT3+/1 and STAT3+/2). Cells were 
left nonstimulated (NS) or stimulated, as indi-
cated, with IFN-, IFN-, IL-27, IL-6, and  
IL-21. pSTAT is an antibody specific for STAT 
with a phosphorylated tyrosine residue. (I) The 
nuclear and cytoplasmic fractions of EBV-B 
cells from a control (WT/WT), a CMCD patient 
(R274Q/WT), a heterozygous patient with a 
dominant loss-of-function mutation of STAT1 
(L706S/WT) and a patient with complete 
STAT1 deficiency (/) stimulated with IFN- 
and IFN- were tested for the presence of 
phosphorylated STAT1 and STAT1 by WB. Anti-
bodies directed against GAPDH and Lamin B1 
were used to normalize the amount of cyto-
plasmic and nuclear proteins, respectively. The 
experiment was performed twice.
T cells and the amounts of IL-17A, 
IL-17F, and IL-22 secreted were small-
est for the four patients with the most 
apparently severe clinical phenotype 
(Fig. 4, A–E and not depicted).
After the culture of PBMCs in 
vitro in the presence of various cyto-
kines, including IL-6, TGF-, IL-1, 
and IL-23, the proportion of IL-17A– 
and IL-22–producing T cell blasts re-
mained significantly lower (P < 104) 
in CMCD patients carrying STAT1 
mutations than in controls (Fig. S4, A 
and B; and not depicted). In contrast, 
the proportions of IL-17A– and IL-22–
producing T cell blasts were normal in 
patients with loss-of-function STAT1 
Moreover, only very small amounts of IL-17A, IL-17F, and mutations (Fig. S4, A and B; and not depicted). The amounts 
IL-22 were secreted by freshly prepared leukocytes after of IL-17A, IL-17F, and IL-22 in the supernatant of T cell 
ex vivo stimulation with PMA and ionomycin (P < 8.103), blasts stimulated with PMA and ionomycin after culture in 
as shown by ELISA (Fig. 4, C–E). In contrast, the amounts of vitro were also significantly lower in patients with STAT1 
secreted IL-17A, IL-17F, and IL-22 were normal in patients mutations and CMCD (P < 4.104; Fig. S4, C–E; and not 
heterozygous or homozygous for loss-of-function or hypo- depicted). In contrast, patients with loss-of-function mutant 
morphic STAT1 mutations (Fig. 4, C–E). Interestingly, in all STAT1 alleles displayed normal levels of cytokine secretion 
assays, the proportions of IL-17A– and IL-22–producing (Fig. S4, C–E; and not depicted). Finally, levels of IL-12p70 and 
1642 Human STAT1 activating mutations impair IL-17 immunity | Liu et al.
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IL-12p40 production by whole blood stimulated with IFN- the gain-of-function, which manifests itself in terms of DNA-
were higher in CMCD patients bearing gain-of-function binding activity, reporter gene induction, and target gene in-
STAT1 alleles than in patients bearing loss-of-function duction, may not necessarily increase the transcription of all 
STAT1 alleles and healthy controls (Fig. 4 F and not depicted). target genes, possibly even resulting in the repression of some 
Thus, patients with familial or sporadic AD CMCD hetero- genes. In addition, the various STAT1 mutations, although 
zygous for mutations affecting the coiled-coil domain of they all affect the coiled-coil domain and are probably all loss-
STAT1, including the dominant gain-of-function R274Q of-dephosphorylation and gain-of-function, may somewhat 
mutant allele, displayed lower levels of IL-17 cytokine pro- differ from each other in terms of their functional impact. 
duction by peripheral T cells, providing a molecular mecha- The genome-wide impact of these mutations on the tran-
nism for the disease. scriptome remains to be assessed in various cell types stimulated 
with a range of cytokines. In any case, the gain-of-function 
DISCUSSION mutant STAT1 alleles were dominant for GAF activation in 
We have shown that several germline missense mutations all cell types tested. They affected cellular responses to various 
affecting the coiled-coil domain of STAT1 may cause spo- cytokines, including IFN-/, IFN-, and IL-27, which pre-
radic and familial AD CMCD. The underlying mechanism dominantly activate STAT1 over STAT3, and IL-6 and IL-21, 
involves a gain of STAT1 phosphorylation caused by the loss which predominantly activate STAT3 over STAT1. These 
of nuclear dephosphorylation, resulting in a gain-of-function mutations probably also strengthen cellular responses to 
of GAF in response to various cytokines. Impaired dephos- IFN-. However, they do not seem to affect STAT1-containing 
phorylation may not be the only mechanism influencing the ISGF-3 activation by IFN-/, at least in the conditions 
impact of these mutations on the transcription of STAT1 target tested. Moreover, STAT3 activation by IL-6, IL-21, IL-22, and 
genes, as these mutations may also affect other processes, such IL-23 is maintained, suggesting that STAT3 activation by 
as the dimerization of unphosphorylated STAT1. Moreover, IL-26 is also intact.
Figure 4. Impaired development and function of IL-17– and IL-22–producing T cells ex vivo in patients with AD CMCD and STAT1 muta-
tions. Each symbol represents a value from a healthy control individual (black circles), a patient bearing a STAT1 gain-of-function (GOF) allele (red upright 
triangles), or a patient bearing one or two STAT1 loss-of-function (LOF) alleles (black upside-down triangles). (A and B) Percentage of CD3+/IL-17A+ (A) 
and CD3+/IL-22+ (B) cells, as determined by flow cytometry, in nonadherent PBMCs activated by incubation for 12 h with PMA and ionomycin. (C–E) Secre-
tion of IL-17F (C), IL-17A (D) and IL-22 (E) by whole blood cells, as determined by ELISA, in the absence of stimulation (open symbols) and after stimu-
lation with PMA and ionomycin for 48 h (closed symbols). Horizontal bars represent medians. The p-values for the nonparametric Wilcoxon test, between 
patients with STAT1 GOF mutations (n = 18) and controls (n = 28) and patients with STAT1 LOF mutations (n = 6) are indicated. All differences between 
healthy controls and patients with STAT1 LOF alleles were not significant. (F) Secretion of IL-12p70 by whole blood cells, as determined by ELISA, in the 
absence of stimulation (open symbols), after stimulation with BCG (lightly colored symbols), or BCG + IFN- for 48 h (closed symbols). Horizontal bars 
represent medians. The p-values for differences between patients with STAT1 GOF mutations (n = 15) and controls (n = 23) and patients with STAT1 LOF 
mutations (n = 6) are indicated and were calculated in nonparametric Wilcoxon tests. All experiments were performed at least two times independently.
JEM Vol. 208, No. 8 1643
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The mutant STAT1 alleles described herein enhance suffer from mycobacterial disease caused by the impairment 
cellular responses to cytokines such as IFN-/, IFN-, and of IFN- immunity (Chapgier et al., 2006a; Dupuis et al., 
IL-27, which potently inhibit the development of IL-17– 2001). Overall, mutations impairing STAT1 function confer AD 
producing T cells via STAT1 (Batten et al., 2006; Yoshimura or AR susceptibility to intracellular agents, through the im-
et al., 2006; Stumhofer et al., 2006; Amadi-Obi et al., 2007; pairment of IFN-/ (viral diseases) and/or IFN- immu-
Feng et al., 2008; Kimura et al., 2008; Tanaka et al., 2008; nity (mycobacterial diseases). In contrast, the gain-of-function 
Chen et al., 2009; Ramgolam et al., 2009; Crabé et al., 2009; STAT1 mutations reported here confer AD CMCD because 
Diveu et al., 2009; El-behi et al., 2009; Guzzo et al., 2010; of the enhancement of STAT1-mediated cellular responses 
Villarino et al., 2010; Liu and Rohowsky-Kochan, 2011). to STAT1-dependent repressors and STAT3-dependent induc-
These mutant alleles also increase cellular responses to IL-6 ers of IL-17–producing T cells. These studies neatly demonstrate 
and IL-21, which normally induce IL-17–producing T cells that severe infectious diseases in otherwise healthy patients 
via STAT3 rather than STAT1 (Hirahara et al., 2010). En- may be subject to genetic determinism (Casanova and Abel, 
hanced STAT1-dependent cellular responses to these two 2005, 2007; Alcaïs et al., 2009, 2010). They also highlight the 
groups of cytokines probably impair the development of profoundly different effects that germline mutations in the same 
IL-17–producing T cells. It remains unclear whether this human gene may have, resulting in different infectious dis-
mechanism predominantly involves IL-17–inhibiting cytokines eases through different molecular and cellular mechanisms.
(IFN-/, IFN-, and IL-27), either individually or in combi-
nation. The available data from the mouse model suggest that MATERIALS AND METHODS
IL-27 is the most potent of the three inhibitors. There is also Massively parallel sequencing
evidence that these cytokines inhibit IL-17–producing T cell DNA (3 µg) extracted from EBV-B cells from the patient was sheared with a 
S2 Ultrasonicator (Covaris). An adapter-ligated library was prepared with the 
development in humans (Ramgolam et al., 2009; Liu and Paired-End Genomic DNA Sample Prep kit (Illumina). The SureSelect 
Rohowsky-Kochan, 2011). Enhanced STAT1 and GAF acti- Human All Exon kit (Agilent Technologies) was then used for exome capture. 
vation in response to the IL-17 inducers IL-6 and IL-21, and Single-end sequencing was performed on a Genome Analyzer IIx (Illumina), 
perhaps IL-23, may also play a key role in disease, by antago- generating 72-base reads.
nizing STAT3 responses. The effect of the aryl hydrocarbon Sequence alignment, variant calling, and annotation
receptor on IL-17 T cell development might also be enhanced BWA aligner (Li and Durbin, 2009) was used to align the sequences obtained 
by gain-of-function STAT1 alleles (Kimura et al., 2008). with the human genome reference sequence (hg18 build). Downstream pro-
Moreover, enhanced STAT1 activity downstream from IL-22 cessing was performed with the Genome analysis toolkit (GATK; McKenna 
and IL-26 in cells, not detected in our study, might also contrib- et al., 2010), SAMtools (Li et al., 2009), and Picard Tools (http://picard 
ute to the CMCD phenotype. Finally, thyroid autoimmunity .sourceforge.net). Substitution calls were made with a GATK UnifiedGeno-
typer, whereas indel calls were made with a GATK IndelGenotyperV2. All calls 
in eight patients and systemic lupus erythematosus in another with a read coverage ≤2x and a Phred-scaled SNP quality of ≤20 were filtered 
patient in our series probably resulted from the enhancement out. All the variants were annotated with annotation software that was developed 
of IFN-/ responses, as such autoimmunity is a frequent in-house. The data were further analyzed with sequence analysis software that 
adverse effect of treatment with recombinant IFN- or IFN- had been developed in-house (SQL database query–driven system).
(Oppenheim et al., 2004; Selmi et al., 2006). Importantly, Molecular genetics
no autoantibodies against IL-17A, IL-17F, or IL-22 were de- EBV-B cells and the STAT1-deficient cell line U3C were cultured as previ-
tected in the patients’ serum (Table I and unpublished data). ously described (Chapgier et al., 2006a). Primary fibroblasts were cultured in 
Remarkably, germline mutations in human STAT1 un- DME supplemented with 10% fetal calf serum. Cells were stimulated with 
derlie susceptibility to three different types of infectious dis- the indicated doses (in IU/ml or ng/ml) of IFN- (Imukin; Boehringer 
ease: mycobacterial diseases, viral diseases, and CMC. Patients Ingelheim), IFN-2b (IntronA; Schering-Plough), IL-27 (R&D Systems), 
bearing STAT1 mutations and displaying mycobacterial IL-21 (R&D Systems), IL-22 (R&D Systems), IL-23 (R&D Systems), and 
IL-6 (R&D Systems). Genomic DNA and total RNA were extracted from 
and/or viral disease do not suffer from CMC, and the patients cell lines and fresh blood cells, as previously described (Chapgier et al., 
with CMCD caused by other STAT1 alleles described here 2006a). Genomic DNA was amplified with specific primers encompassing 
present no mycobacterial or viral disease. The pathogenic exons 6–10 of STAT1 (available upon request), sequenced with the Big Dye 
mechanisms involved are clearly different, with loss-of-function Terminator cycle sequencing kit (Applied Biosystems), and analyzed on an 
mutations in STAT1 underlying mycobacterial and viral dis- ABI Prism 3730 (Applied Biosystems). We used the various alleles of STAT1 
eases (Dupuis et al., 2001, 2003; Chapgier et al., 2006b, 2009; in the pcDNA3 STAT1-V5 vector (Chapgier et al., 2006a; Kong et al., 2010). 
We generated the various STAT1 mutations by site-directed mutagenesis 
Kong et al., 2010; Averbuch et al., 2011; Kristensen et al., (QuikChange Site-Directed Mutagenesis kit; Stratagene) with the mis-
2011). Human AR STAT1 deficiency impairs cellular re- matched primers listed in Table S4. U3C cells were harvested by trypsin 
sponses to IFN-/, IFN-, IFN-, and IL-27 (Dupuis treatment 24 h before transfection and replated at a density of 2.5 × 105 
et al., 2003; Chapgier et al., 2006b, 2009; Kong et al., 2010; cells/ml in 6-well plates. Plasmid DNA (5 µg per plate) carrying the WT or 
Kristensen et al., 2011). Viral diseases probably result from all the various mutant STAT1 alleles was used for cell transfection with the 
impaired IFN-/ and, perhaps, IFN- immunity, although Calcium Phosphate Transfection kit (Invitrogen).
impaired IFN- and IL-27 immunity may also contribute to Luciferase reporter assay
the phenotype. Patients with AD MSMD, heterozygous for U3C cells were dispensed into 96-well plates (1 × 104/well) and trans-
loss-of-function dominant-negative mutations of STAT1, fected with reporter plasmids (Cignal GAS and ISRE Reporter Assay kit; 
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SABiosciences) and plasmids carrying the various alleles of STAT1 or a incubated for 30 min with 100 ng/ml IL-23. Activation was stopped by add-
mock vector, in the presence of Lipofectamine LTX (Invitrogen). 6 h after ing 1X cold PBS, and cells were processed for immunoblot analysis.
transfection, the cells were transferred back into medium containing 10% 
FBS and cultured for 24 h. The transfectants were then stimulated with Modeling
IFN- (500 and 1,000 IU/ml), IL-27 (20 and 100 ng/ml), and IFN- Images of the three-dimensional structure of STAT1 (Chen et al., 1998) were 
(500, 1,000, and 5,000 IU/ml) for 16 h and subjected to luciferase assays generated with the 2002 PyMOL Molecular Graphics System (DeLano Sci-
with the Dual-Glo luciferase assay system (Promega). Experiments were per- entific), using PDB accession no. 1BF5.
formed in triplicate and firefly luciferase activity was normalized with respect 
to Renilla luciferase activity. The data are expressed as fold induction with re- Whole-blood assay of the IL-12–IFN- circuit
spect to nonstimulated cells. Whole-blood assays were performed as previously described (Feinberg et al., 
2004). Heparin-treated blood samples from healthy controls and patients 
Immunoblot analysis and electrophoretic mobility shift assays were stimulated in vitro with live Mycobacterium bovis BCG (Pasteur) alone or 
The following optimal stimulation conditions were used. EBV-B or U3C with IFN- (5,000 IU/ml; Boehringer Ingelheim). Supernatants were col-
cells were stimulated by incubation for 20 min with 100 µg/ml IL-21 or lected after 48 h of stimulation, and ELISA were performed with specific 
25 ng of IL-22; 30 min with 103 or 105 IU/ml IFN- and IFN-; 15 min antibodies directed against IL-12p40 or IL-12p70, using kits from R&D Sys-
with 50 ng/ml IL-6; or 30 min with 50 or 100 ng/ml IL-27. WB was per- tems according to the manufacturer’s instructions.
formed as previously described (Dupuis et al., 2003). In brief, cell activation 
was blocked with cold 1X PBS, cells were lysed in 1% NP-40 lysis buffer, and Production of IL-17A, IL-17F, and IL-22 by leukocytes
the proteins were recovered and subjected to SDS-PAGE. We used antibodies Cell activation. IL-17A– and IL-22–producing T cells were evaluated by 
directed against phosphorylated STAT1 (pY701; BD), STAT1 (C-24; Santa intracellular staining or by ELISA, as previously described (de Beaucoudrey 
Cruz Biotechnology), V5 (Invitrogen), -tubulin (Santa Cruz Biotechnol- et al., 2008). In brief, PBMCs were purified by centrifugation on a gradient 
ogy), phosphorylated STAT3 (Cell Signaling Technology), lamin B1 (Santa (Ficoll-Paque PLUS; GE Healthcare) and resuspended in RPMI supple-
Cruz Biotechnology), GAPDH (Santa Cruz Biotechnology), and STAT3 mented with 10% FBS (RPMI/10% FBS; Invitrogen). Adherent monocytes 
(Santa Cruz Biotechnology). EMSA was performed as previously described were removed from the PBMC preparation by incubation for 2 h at 37°C, 
(Chapgier et al., 2006a). In brief, cell activation was blocked by incubation under an atmosphere containing 5% CO2.
with cold 1X PBS, and the cells were gently lysed to remove cytoplasmic For ex vivo evaluation of IL-17– and IL-22–producing T cells by flow 
proteins while keeping the nucleus intact. We then added nuclear lysis cytometry, we resuspended 5 × 106 nonadherent cells in 5 ml RPMI/10% 
buffer and recovered the nuclear proteins, which were subjected to nonde- FBS in 25 cm2 flasks and stimulated them by incubation with 40 ng/ml PMA 
naturing electrophoresis with radiolabeled GAS (from the FCR1 promoter: (Sigma-Aldrich) and 105 M ionomycin (Sigma-Aldrich) in the presence of 
5-ATGTATTTCCCAGAAA-3) and ISRE (from the ISG15 promoter: a secretion inhibitor (1 µl/ml GolgiPlug; BD) for 12 h.
5-GATCGGGAAAGGGAAACCGAAACTGAA-3) probes. For evaluation of the IL-17– and IL-22–producing T cell blasts after in 
vitro differentiation, the nonadherent PBMCs were dispensed into 24-well 
Staurosporine and pervanadate treatment of cells plates at a density of 2.5 × 106 cells/ml in RPMI/10% FBS and activated 
We assessed dephosphorylation by stimulating U3C transfectants with 105 IU/ml with 2 µg/ml of an antibody directed against CD3 (Orthoclone OKT3; 
IFN-. The cells were then washed and incubated with 1 µM staurosporine Janssen-Cilag) alone, or together with 5 ng/ml TGF-1 (240-B; R&D Sys-
in DME for 15, 30, or 60 min. The cells were then lysed with 1% NP-40 lysis tems), 20 ng/ml IL-23 (1290-IL; R&D Systems), 50 ng/ml IL-6 (206-IL; 
buffer, and the proteins recovered were subjected to immunoblot analysis. R&D Systems), 10 ng/ml IL-1 (201-LB; R&D Systems), or combinations 
Pervanadate was prepared by mixing orthovanadate with H2O2 for 15 min of these four cytokines. After 3 d, the cells were restimulated in the same acti-
at 22°C. U3C transfectants were treated with pervanadate (0.8 mM orthovana- vation conditions, except that the anti-CD3 antibody was replaced with 
date and 0.2 mM H2O2) 5 min before stimulation. They were then stimulated 40 IU/ml IL-2 (Proleukin i.v.; Chiron). We added 1 ml of the appropriate 
with IFN- for 20 min. The stimulation was stopped by adding cold 1X PBS. medium, resuspended the cells by gentle pipetting, and then split the cell sus-
The proteins were recovered and subjected to immunoblot analysis. pension from each well into two. Flow cytometry was performed on one of 
the duplicated wells 2 d later, after stimulation by incubation for 12 h with 
Extraction of nuclear and cytoplasmic proteins 40 ng/ml PMA and 105 M ionomycin in the presence of 1 µl/ml GolgiPlug. 
U3C transfectants or EBV-B cells were stimulated with IFN- or IFN- for FACS analysis was performed as described in the following section. The 
20 min and subjected to nuclear and cytoplasmic protein extraction with other duplicated well was split into two, with one half left unstimulated and 
NE-PER Nuclear and Cytoplasmic Extraction Regents (Thermo Fisher the other stimulated by incubation with 40 ng/ml PMA and 105 M iono-
Scientific) according to the manufacturer’s protocol. mycin for another 2 d. Supernatants were collected after 48 h of incubation, 
for ELISA.
Immunofluorescence staining
Immunofluorescence experiments were performed as previously described Flow cytometry. Cells were washed in cold PBS, and surface labeling was 
(Chapgier et al., 2006a). In brief, cells (transfected U3C or SV-40 fibroblasts) achieved by incubating the cells with PECy5-conjugated anti–human CD3 
were stimulated for the times indicated with 10,000 IU/ml of IFN-. Cells were antibody (BD) in PBS/2% FBS for 20 min on ice. Cells were then washed 
then washed with cold PBS and fixed with 4% PFA. The cells were washed and twice with 2% FBS in cold PBS, fixed by incubation with 100 µl of BD 
incubated with an antibody against STAT1, which was then detected by incuba- Cytofix for 30 min on ice, and washed twice with BD Cytoperm (Cytofix/
tion with an Alexa Fluor 488–conjugated anti–mouse antibody. Cytoperm Plus, fixation/permeabilization kit; BD). Cells were then incu-
bated for 1 h on ice with Alexa Fluor 488–conjugated anti–human IL-17A 
T cell blast differentiation and stimulation (53–7179-42; eBioscience), PE-conjugated anti–human IL-22 (IC7821P; 
PBMCs were recovered by centrifuging blood samples on Ficoll gradients, as R&D Systems), or PE-conjugated anti–human IFN- (IC285P; R&D Sys-
previously described (Chapgier et al., 2006a). They were then cultivated, at a tems) antibodies, washed twice with Cytoperm, and analyzed with a FACS-
density of 1 million cells per ml in RPMI supplemented with 10% fetal calf Canto II system (BD).
serum and stimulated with phytohemagglutinin (1 µg/ml) for 3 d. Cells were 
then recovered, centrifuged on a Ficoll gradient, cultivated (at a density of ELISA. IL-17A, IL-17F, and IL-22 levels were determined by ELISA on the 
0.2 million cells/ml) to Panserin 401 supplemented with 10% FCS and supernatants harvested after 48 h of whole-blood stimulation with 40 ng/ml 
glutamine 1X, and stimulated with 40 IU/ml IL-2 (Roche). Cells were then PMA and 105 M ionomycin, or after in vitro PHA blast differentiation and 
JEM Vol. 208, No. 8 1645
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48 h of stimulation with 40 ng/ml PMA and 105 M ionomycin. We used uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/
anti–human IL-17A and anti–human IL-22 Duoset kits (R&D Systems) STAT1. Nat. Med. 13:711–718. doi:10.1038/nm1585
and the anti–human IL-17F ELISA Ready-SET-GO! set (eBioscience). Atkinson, T.P., A.A. Schäffer, B. Grimbacher, H.W. Schroeder Jr., C. 
Woellner, C.S. Zerbe, and J.M. Puck. 2001. An immune defect causing 
Statistical analysis. We assessed differences between controls, MSMD pa- dominant chronic mucocutaneous candidiasis and thyroid disease maps 
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gain-of-function STAT1 alleles in terms of the percentages of IL-17A– and doi:10.1086/323611
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Fig. S1 shows that STAT1-CMCD mutants are gain-of-function alleles by producing T cells. Nat. Immunol. 7:929–936. doi:10.1038/ni1375
loss of nuclear dephosphorylation. Fig. S2 is a schematic representation of Bentur, L., E. Nisbet-Brown, H. Levison, and C.M. Roifman. 1991. Lung 
disease associated with IgG subclass deficiency in chronic muco-
the cytokines and transcription factors directing the development of naive 
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CD4 cells into IL-17–producing T cells. Fig. S3 shows the normal response 3476(05)81852-9
of CMCD patient cells to IFN- in terms of ISGF3 activation, to IFN- Bolze, A., M. Byun, D. McDonald, N.V. Morgan, A. Abhyankar, L. 
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genes shared by more than one patient. Table S3 lists conservation and pre- Byun, M., A. Abhyankar, V. Lelarge, S. Plancoulaine, A. Palanduz, L. Telhan, B. 
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We thank the members of the laboratory for helpful discussions; Yelena jem.20050854
Nemiroskaya, Eric Anderson, Martine Courat, and Michele N’Guyen for secretarial Casanova, J.L., and L. Abel. 2007. Primary immunodeficiencies: a field in its 
assistance; and Tony Leclerc and Tiffany Nivare for technical assistance. We also infancy. Science. 317:617–619. doi:10.1126/science.1142963
thank Alekszandra Barsony, Dmitriy Samarin, Fedir Lapiy, Maxim Vodyanik, Marcela Chapgier, A., S. Boisson-Dupuis, E. Jouanguy, G. Vogt, J. Feinberg, A. 
Moncada Velez, Bertrand Boisson, and Astrid Research, Inc. Prochnicka-Chalufour, A. Casrouge, K. Yang, C. Soudais, C. Fieschi, et al. 
This work was supported by grants from Institut National de la Santé et de la 2006a. Novel STAT1 alleles in otherwise healthy patients with mycobac-
Recherche Médicale, University Paris Descartes, the Rockefeller University, the terial disease. PLoS Genet. 2:e131. doi:10.1371/journal.pgen.0020131
Rockefeller University CTSA grant number 5UL1RR024143-04, the St. Giles Chapgier, A., R.F. Wynn, E. Jouanguy, O. Filipe-Santos, S. Zhang, J. 
Foundation, and the Candidoser Association awarded to Jean-Laurent Casanova. Feinberg, K. Hawkins, J.L. Casanova, and P.D. Arkwright. 2006b. 
Janine Reichenbach was supported by the Gebert Rüf Stiftung, program “Rare Human complete Stat-1 deficiency is associated with defective type 
Diseases – New Approaches”; Ellen Renner by the DFG RE2799/3-1 and a Fritz- I and II IFN responses in vitro but immunity to some low virulence 
Thyssen research foundation grant (Az. 10.07.1.159). Support was also provided by viruses in vivo. J. Immunol. 176:5078–5083.
TÁMOP 4.2.1./B-09/1/KONV-2010-0007 and TÁMOP 4.2.2-08/1-2008-0015 grants to Chapgier, A., X.F. Kong, S. Boisson-Dupuis, E. Jouanguy, D. Averbuch, 
László Maródi and LMU Munich FöFoLe grant #680/658. Sophie Cypowyj was J. Feinberg, S.Y. Zhang, J. Bustamante, G. Vogt, J. Lejeune, et al. 2009. 
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Chen, M., G. Chen, H. Nie, X. Zhang, X. Niu, Y.C. Zang, S.M. Skinner, 
Submitted: 11 May 2011 J.Z. Zhang, J.M. Killian, and J. Hong. 2009. Regulatory effects of IFN-
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