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\begin{document}

\title{TRAF3IP2 variants in a child with chronic mucocutaneous candidiasis-
keeping an open mind!}



\author[1]{Pilar Blanco Lobo}%
\author[2]{Wei-Te Lei}%
\author[2]{Simon Pelham}%
\author[1]{Paloma Guisado Hernández}%
\author[1]{Isabel Villaoslada}%
\author[1]{Beatriz de Felipe}%
\author[3]{Jose Lucenas}%
\author[2]{Jean-Laurent Casanova }%
\author[4]{Peter Olbrich}%
\author[2]{Anne Puel}%
\author[4]{Olaf Neth}%
\affil[1]{Instituto de Biomedicina de Sevilla}%
\affil[2]{The Rockefeller University}%
\affil[3]{Virgen del Rocio University Hospital}%
\affil[4]{Hospital Infantil Universitario Virgen del Rocío}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\selectlanguage{english}
\begin{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-\selectlanguage{greek}γ-
\selectlanguage{english}and IL-4-production (memory CD4+ T cells) were determined. ACT1
expression and binding to IL-17RA 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 response using an NF-\selectlanguage{greek}κ\selectlanguage{english}B-driven luciferase reporter
system in HEK-293T cells, and by measuring GRO-\selectlanguage{greek}α \selectlanguage{english}secretion by
fibroblasts. Results: A likely non-pathogenic STAT1 variant
(c.1363G\textgreater{}A/p.V455I) was identified by next generation
sequencing., STAT1 expression and phosphorylation upon IFN-? were
normal. We also found compound heterozygous variants
(c.1325A\textgreater{}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 an impaired NF-\selectlanguage{greek}κ\selectlanguage{english}B-activation. Patient's fibroblasts
displayed abolished GRO-\selectlanguage{greek}α \selectlanguage{english}secretion upon IL-17A. Finally, ex vivo CD4+ T
cells showed increased IL-17A, IL-22, and IL-4, and normal-low IFN-\selectlanguage{greek}γ
\selectlanguage{english}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.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Chronic mucocutaneous candidiasis (CMC) is a disorder characterized by
recurrent or persistent symptomatic infections caused by
\emph{Candida}species affecting the nails, skin, oral cavity, and
genital mucosa {[}1{]}. CMC has been identified as an important
infectious phenotype in patients suffering from inborn errors of
immunity in particular those with (severe) T cell deficiencies, or
altering the interleukin (IL)-17 pathway {[}2, 3{]}. For about half of
CMC cases, signal transducer and activator of transcription 1 (STAT1)
gain-of-function (GOF) mutations were found as the disease-causing
etiology, and it has been suggested that the identification of a STAT1
variant in the setting of CMC should be followed by a thorough
functional variant work-up {[}2, 4{]}. Patients with STAT1 GOF mutations
suffer from a broad clinical phenotype including (myco-) bacterial,
viral, and fungal, infections, autoimmunity, malignancies, and the
development of aneurysms {[}4{]}. In contrast, patients with defects of
the IL-17-response pathway such as autosomal dominant (AD) IL-17F or
JNK1 deficiency, or autosomal recessive (AR) IL-17RA, IL-17RC, or ACT1
deficiencies are characterized by a narrow infectious spectrum mostly
restricted to superficial\emph{C.} \emph{albicans} and
\emph{Staphylococcus aureus} diseases {[}2, 5-7{]}.

The IL-17A- and IL-17F- (IL-17A/F)-mediated response is critical for the
host defense against fungal pathogens affecting epithelium and mucosa
{[}6, 8{]}. IL-17A/F-signaling requires the binding to a heterodimeric
receptor(R) (IL-17RA/IL-17RC) and subsequent recruitment of the
ubiquitin ligase adaptor protein ACT1, encoded by \emph{TRAF3IP2} ,
ubiquitously expressed in human tissues. ACT1 further recruits tumor
necrosis factor receptor-associated factor 6 (TRAF6) to trigger
downstream activation of nuclear factor-\selectlanguage{greek}κ\selectlanguage{english}B (NF-\selectlanguage{greek}κ\selectlanguage{english}B) and mitogen-activated
protein kinase (MAPK) pathways \textbf{(Figure 1).} This results in the
upregulation of pro-inflammatory cytokines and chemokines with critical
roles in protective mucosal immunity {[}2, 9-11{]} .

Mutations of ACT1 were first reported in two siblings harboring a
bi-allelic missense mutation (c.1607C\textgreater{}T, p.T536I) in the
SEFIR (similar expression to fibroblast growth factor genes/IL-17)
domain {[}12{]}. Both patients had early onset CMC and seborrheic
dermatitis, and one patient had in addition bilateral persistent
blepharitis due to \emph{S. aureus} . The mutation impaired the
homotypic interaction of ACT1 with IL-17RA and IL-17RC, abrogating
IL17A/F-dependent signaling in fibroblasts, explaining the CMC
phenotype. It also impaired ACT1 interaction with IL-17RB, abolishing
IL-17E-dependent signaling in leukocytes, through IL-17RA/IL-17RB. In
contrast, patients displayed enhanced proportions of Th17 cells, as
measured ex vivo upon PMA/ionomycin stimulation {[}12{]}. More recently,
eight additional patients with mutations of \emph{TRAF3IP2} have been
described {[}13-16{]}. We present a 10-year-old girl with CMC due to AR
ACT1 deficiency and review important clinical and laboratory
characteristics of the so far published cases.

\textbf{Methods}

\textbf{Study participants}

The study was conducted in accordance to the protocol approved by the
Ethics Committee of the Hospital Universitario Virgen del Roc\selectlanguage{ngerman}ío and
Virgen del Macarena (Seville, Spain) (0641-N-20).

\textbf{Genetic analysis}

DNA was isolated from peripheral blood samples using the MagNa Pure
Compact Nucleic Acid Isolation Kit I (Roche) according to the
manufacturer's instructions. A next generation sequencing (NGS) panel
including genes related to CMC (\emph{STAT1, TRAF3IP2, CARD9, IL17F,
IL17RA,} and \emph{IL17RC} ), was performed using an AmpliSeq strategy
on the Ion Torrent PGM platform. Primary sequence data analysis was
performed with Torrent server 5.1 software. Single-nucleotide
polymorphisms were denoted as proposed by the Torrent Variant Caller
v5.12.0.40 and analyzed by Ion Reporter Sofware v5.12. Identified
specific mutations were validated by Sanger sequencing using specific
primers amplifying exon 6 of \emph{TRAF3IP2} gene and exon 17
of\emph{STAT1} .

\textbf{Whole blood stimulation and flow cytometry}

Levels of STAT1 and phosphorylated (p)STAT1 were evaluated by flow
cytometry as previously described {[}17{]}. Briefly, heparinized whole
blood samples were stimulated with IFN-\selectlanguage{greek}γ; \selectlanguage{english}400 UI/mL; Inmukin, Horizon
Pharma) for 15 min at 37\selectlanguage{ngerman}°C. Cells were permeabilized by 1-hour
incubation with ice-cold methanol and stained with antibodies against
CD14, STAT1, and pSTAT1 (supplementary Table 1), for 1h at 4°C in the
dark. Stained cells were fixed (1\% paraformaldehyde, Sigma), and
acquired (BD LSRII FORTESSA\selectlanguage{english}). Data was analyzed with the FlowJo
(Tristar Treestar, Ashland, OR, USA) software package.

\textbf{Protein expression}

HEK-293T cells (4 x 10\textsuperscript{5}) were cultured in Dulbecco's
Modified Eagle Medium (DMEM, GIBCO) supplemented with 10\% fetal calf
serum (FCS, GIBCO). Cells were transfected with plasmids encoding wild
type (WT) or mutant ACT1 (p.D451G, p.K454fs11*, or p.T536I already
reported {[}12{]} serving as positive control), using Lipofectamine 2000
according to the manufacturer's protocol. After 24 hours, cells were
lysed with NP40 lysis buffer (1M Tris-HCl, 5M NaCl, 1\% NP40). Samples
were separated by SDS-PAGE on a 4-20\% gradient gel (BioRad, 456-1093),
and proteins were transferred to a PVDF membrane (Millipore). The
membrane was blocked using 5\% bovine serum albumin with 0.1\% Tween 20
in PBS, followed by incubation with the primary antibodies against ACT1
and GAPDH. Visualization of the proteins was assessed by enhanced
chemiluminescence.

\textbf{Co-Immunoprecipitation}

HEK-293T cells (1 x 10\textsuperscript{6}) were co-transfected with an
N-terminal HA-tagged plasmid encoding WT or mutant (p.D451G, p.K454fs11*
or p.T536I) ACT1 along with a C-terminal Flag-tagged plasmid encoding
IL-17RA. After 24 hours, cells were lysed as above. An aliquot of the
lysate was reserved for the input control and the co-immunoprecipitation
was performed on the remaining lysate using agarose beads with anti-Flag
antibodies conjugated (Sigma-Aldrich, F2426) followed by a 3-hour
incubation and washing with NP40 lysis buffer. Western blots were
performed as above using primary antibodies against HA (R\&D systems,
HAM0601) or Flag (Sigma-Aldrich, A8592).

\textbf{Luciferase assay}

HEK-293T cells (3 x 10\textsuperscript{4}) were co-transfected, using
Lipofectamine 2000, with WT or mutant (D451G, K454fs11* or T536I) ACT1
plasmids along with NF-\selectlanguage{greek}κ\selectlanguage{english}B--dependent firefly luciferase plasmids pGL4.32
(Promega) and a Renilla luciferase reporter pRLTK plasmid as control of
transfection efficiency. After 24 hours, cells were stimulated with 100
ng of IL-17A (R\&D) for 24 hours or left unstimulated. Luciferase was
then detected using the Dual-Glo Stop \& Glo system (Promega).

\textbf{ELISA}

Fibroblasts were immortalized by transfection using a simian virus 40
large (SV40) T antigen gene-containing plasmid and then cultured in DMEM
(GIBCO) supplemented with 10\% FCS. Fibroblasts (1 x
10\textsuperscript{5}) from healthy control, and from patients with
IL17RA- {[}3{]} or IL17RC- {[}7{]} deficiency, or
ACT1\textsuperscript{T536I/T536I} {[}12{]}, or our patient were
stimulated with IL-17A (100 ng/ml), TNF-\selectlanguage{greek}α \selectlanguage{english}(20 ng/ml), alone or in
combination, or IL-1\selectlanguage{greek}β \selectlanguage{english}(10ng/ml) (R\&D, 317-ILB, 210-TA, 201-LB), or left
unstimulated. After 24 hours, levels of GRO-\selectlanguage{greek}α \selectlanguage{english}in the supernatants were
measured by ELISA (R\&D, DY275) following manufacturer's instructions.

\textbf{Cytokine expression in memory CD4\textsuperscript{+} T cells}

Cytokine expression in memory CD4\textsuperscript{+} T cells were
evaluated ex vivo as previously described {[}16{]}. Briefly, peripheral
blood mononuclear cells (PBMCs) were activated with PMA (50 ng/mL) and
ionomycin (1 \selectlanguage{greek}µ\selectlanguage{english}M) for 5 hours in presence of GolgiStop and GolgiPlug
(BD). Cells were stained with LIVE/DEAD Fixable Blue (ThermoFisher
Scientific) with the addition of FcR blocking reagent (Miltenyi
Biotech). Cells were then stained with (CD8, CCR7, CD45RA, supplementary
Table 1), fixed, permeabilized with FOXP3/Transcription factor staining
buffer kit (eBioscience), followed by staining with antibodies against
CD3, CD4, IL-17A, IL-4, IL-22, and IFN-\selectlanguage{greek}γ-\selectlanguage{english}(supplementary Table 1). Data
was acquired on a Cytek Aurora 5 laser flow cytometer and data was
analyzed with FlowJo (Tree Star).

\textbf{Results}

\textbf{Clinical, immunological, and genetic findings}

The patient is a 10-year-old girl born to healthy non-consanguineous
Spanish parents. She presented with oral thrush at the age of 12 months.
Whilst topical treatment with miconazole failed, she responded to
systemic oral fluconazole therapy. Thrush recurrence (every three to
four months) due to \emph{C. albicans} prompted referral to our
immunodeficiency clinic when she was 6 years old. Other clinical
manifestations included persistent angular cheilitis with scaring,
recurrent wheezing, intermittent skin alterations such as eczema and
marked reactions to insect bites \textbf{(Figure 2A)} . In addition, she
suffered from two episodes of extensive impetigo secondary to local
fungal infections \textbf{(Figure 2A)} . So far, she has not experienced
invasive infections, scalp lesions, folliculitis, or blepharitis.
Physical examination including growth and weight was normal. There were
no facial, dental, or skeletal abnormalities. A full blood count, basic
immunology work-up including extended lymphocyte immunophenotyping and
immunoglobulin levels as well as serum INF-\selectlanguage{greek}γ \selectlanguage{english}levels over the last 4
years were normal for age apart from mildly elevated IgE
levels\textbf{(supplementary Table 2)} . Targeted NGS sequencing
revealed a heterozygous variant (c.1363C\textgreater{}A/p.D451G) of
\emph{STAT1}inherited from her father; both mother and sister were WT.
In addition, the patient carries private compound heterozygous variants
of\emph{TRAF3IP2} : c.1325A\textgreater{}G (p.D451G), inherited from her
mother, and c.1335delA (resulting in a frameshift and a premature stop
codon p.K454fs11*) inherited from her father, as did her sister
(\textbf{Figure 2B-2C)} .

\textbf{Testing of V455I STAT1 variant}

Although extremely rare in the general population (MAF: 0,00006), the
variant (c.1363G\textgreater{}A, p.V455I) was classified as being likely
benign (Sift 0.19, Polyphen 0.115, CADD 28.3). As the performance of
those algorithms in the setting of GOF mutations is limited, we decided
to evaluate the functional impact of this novel variant. Total STAT1
levels in monocytes were determined by flow cytometry in the patient and
her family and were similar to that of healthy controls \textbf{(Figure
3A)} . Similarly, we did not observe any differences in terms of STAT1
phosphorylation after 15min of INF-\selectlanguage{greek}γ \selectlanguage{english}stimulation \textbf{(Figure 3B)} .
In addition, family testing (Sanger sequencing) revealed that this
variant is inherited from the asymptomatic father \textbf{(Figure 2B)} ,
suggesting this variant not to be disease causing.

\textbf{Testing of D451G and K454fs11* ACT1 variants}

Neither c.1325A\textgreater{}G or c.1335delA \emph{TRAF3IP2} variants
were found in any public databases (GnomAd or ExAc). Moreover,
c.1325A\textgreater{}G (p.D451G) was classified as pathogenic (Sift
0.01, Polyphen 0.998, CADD 24.8). The c.1335delA variant causes a frame
shift resulting in a premature stop codon (p.K454fs11*). Familial
segregation showed that the c.1325A\textgreater{}G mutant allele was
inherited from the mother and the c.1335delA allele from the
father\textbf{(Figure 2B)} . Both mutations are located within the SEFIR
domain (\textbf{Figure 2C)} .

We then assessed WT and mutant (D451G or K454fs11*) ACT1 protein
expression by overexpression in HEK-293T cells, together with the
previously characterized T536I ACT1 variant {[}12{]}. We found similar
levels of protein expression for all variants, comparable to WT,
although the frameshift (K454fs11*) variant resulted in a truncated ACT1
protein \textbf{(Figure 4A)} . We next tested the interaction of WT and
mutant ACT1 with IL-17RA by co-immunoprecipitation assay in HEK-293T
cells \textbf{(Figure 4B)} . As compared to input
fraction\textbf{(Figure 4B, left)} , we observed a reduced interaction
of IL-17RA with K454fs11* ACT1, whereas, D451G did not apparently affect
the binding of ACT1 to IL-17RA \textbf{(Figure 4B, right).}

We then tested the functional activity of the variants by using an NF-\selectlanguage{greek}κ\selectlanguage{english}B
reporter assay. ACT1 over-expression is known to constitutively activate
NF-\selectlanguage{greek}κ\selectlanguage{english}B, a feature that is dependent on the SEFIR domain of ACT1 {[}10{]}.
We found a strong induction of NF-\selectlanguage{greek}κ\selectlanguage{english}B transcriptional activity when we
over-expressed wild type ACT1, as compared to the empty vector (EV),
which was further increased after IL-17A stimulation \textbf{(Figure
4C)} . In contrast, all three variants showed impaired constitutive
NF-\selectlanguage{greek}κ\selectlanguage{english}B activity, with barely any further induction after IL-17A
stimulation, suggesting that these variants are loss-of-function in this
overexpression system.

We then tested the response of the patient's SV40-fibroblasts upon
stimulation with IL-17A, TNF-\selectlanguage{greek}α, \selectlanguage{english}alone or combination, or IL-1\selectlanguage{greek}β, \selectlanguage{english}as
compared with fibroblasts from a healthy control, patients with AR
IL-17RA or IL-17RC deficiency, or from the previously reported ACT1
deficient patient \textbf{(Figure 4D)} . We observed an induction of
GRO-\selectlanguage{greek}α \selectlanguage{english}(also known as CXCL1), after 24 hours of stimulation of control's
fibroblasts upon IL-17A, further increased by TNF-\selectlanguage{greek}α. \selectlanguage{english}In contrast,
patient's fibroblasts, similarly those of patients with AR IL-17RA or
IL-17RC deficiency, or ACT1\textsuperscript{T536I/T536I} fibroblasts,
showed no GRO-\selectlanguage{greek}α \selectlanguage{english}induction upon IL-17A stimulation, and no further
induction as compared to TNF-\selectlanguage{greek}α \selectlanguage{english}alone, when stimulated with a combination
of IL17A and TNF-\selectlanguage{greek}α. \selectlanguage{english}All fibroblasts showed strong responses when
stimulated with IL-1\selectlanguage{greek}β.

\selectlanguage{english}Finally, proportions of ex vivo IL-17A-, IL-22-, IL-4- producing memory
CD4\textsuperscript{+} cells were raised in the patient, whereas
IFN-\selectlanguage{greek}γ-\selectlanguage{english}producing memory CD4\textsuperscript{+} cells were within the low
ranges of the controls \textbf{(Figure 5).}

\textbf{Discussion}

IL-17 immunity is critical for the induction of mucosal pro-inflammatory
cytokines and chemokines involved in mucocutaneous protection
against\emph{C. albicans} infections {[}6{]}. Patients with low
proportions of Th17 cells (e.g AD STAT3, AR ZNF341 or ROR-\selectlanguage{greek}γ/γ\selectlanguage{english}T
deficiency, or STAT1 GOF), or defects in IL-17 signaling (e.g. AR
IL-17RA, IL-17RC, ACT1, AD IL-17F, JNK1 deficiencies) are particularly
susceptible to CMC\textbf{(Figure 1)} {[}2-4, 7, 8{]}. Genetic analysis
of our index case revealed variants in \emph{STAT1} and \emph{TRAF3IP2}
\textbf{(Figure 2)} . Considering that STAT1 GOF mutations have been
widely described to cause CMC, we evaluated the functional impact of
this variant. High levels of STAT1 expression and/or STAT1
phosphorylation upon activation of the IFN pathway are typical
immunological features of STAT1 GOF patients {[}4, 17, 18{]}. Based on
the similar STAT1 and pSTAT1 levels found in patient's cells and healthy
controls, and considering that her father, harboring the same mutation,
was asymptomatic, we classified this variant as likely not pathogenic
\textbf{(Figure 2-3)} . We therefore studied the \emph{ACT1} variants
and their possible disease-causing impact. The ACT1 SEFIR domain allows
for the recruitment of ACT1 to the IL-17RA/IL-17RC upon IL-17A
stimulation {[}8, 10-12{]}. By overexpression, the patient's alleles
were expressed at similar levels as compared to the WT protein, with
p.K454Fsf11* leading to the expression of a truncated protein, impairing
its interaction with IL-17RA, whereas p.D451G showed a normal
interaction \textbf{(Figure 4A/B).} After ACT1-IL-17RA/IL-17RC
interaction upon IL-17A/IL-17F stimulation, the kinase TAK1 and the E3
ubiquitin ligase TRAF6 are recruited to the receptor to facilitate the
activation of the transcription factor NF-\selectlanguage{greek}κ\selectlanguage{english}B \textbf{(Figure 1)} {[}10,
11{]}. By luciferase, we showed that both patient's \emph{ACT1} alleles
strongly impaired constitutive and IL-17-dependent NF-\selectlanguage{greek}κ\selectlanguage{english}B
activation\textbf{(Figure 4C)} . Furthermore, patient's SV40-fibroblasts
displayed abolished responses to IL-17A, comparable to that of patients
with AR IL-17RA or IL-17RC deficiency \textbf{(Figure 4D)} {[}5, 7,
12{]}. In contrast, all of them retained the ability to respond to TNF-\selectlanguage{greek}α
\selectlanguage{english}and IL-1\selectlanguage{greek}β, \selectlanguage{english}suggesting that D451G and K454fs11* ACT1 mutations
specifically affect the IL-17A-induced ACT1-mediated GRO-\selectlanguage{greek}α \selectlanguage{english}production,
known to play a protective role in fungal infection through the
recruitment of neutrophils {[}19{]}. Finally, as previously reported
{[}12, 16{]}, the patient displayed enhanced proportions of IL-17A-and
IL-22-producing CD4\textsuperscript{+} cells upon stimulation whereas
baseline Th17 proportions were normal. This observation highlights the
importance of functional assays, as the pattern of T cell
differentiation is variable. Two previous reports {[}12, 16{]}, as well
as our case, revealed normal or slightly reduced IFN-\selectlanguage{greek}γ-\selectlanguage{english}producing memory
CD4\textsuperscript{+} cells whereas others observed reduced Th1
proportion {[}14{]}. Furthermore, and in contrast to previous cases
{[}14, 16{]}, levels of IL-4-producing Th2 cells were higher in our
patient than those found in relatives, and healthy controls
\textbf{(Figure 5)} .

Clinical and laboratory findings, and management of all eleven
previously reported patients with AR ACT1 deficiency, including our
report, are summarized in \textbf{Figure 6} and, in more detail,
in\textbf{supplementary Table 3} {[}12-16{]}. Of note, detailed
information was not available for all patients. The overall phenotype
was characterized by CMC in early childhood (9/11, before age 2 years)
requiring medical attention, treatment, or antifungal prophylaxis for a
prolonged time in most cases. Dermatologic manifestations were common,
and \emph{Staphylococcus aureus} was specifically reported in the
context of skin lesions in 5/11 patients. Treatment responses of CMC
were satisfactory when documented, and no fatal cases have been
described \textbf{(supplementary Table 3 and Figure 6)} . The clinical
course of our patient was similar, as she suffered from CMC, chronic
cheilitis, recurrent wheezing, and skin lesions \textbf{(supplementary
Table 3)} . Importantly, standard immune phenotyping and immunoglobulin
levels were unremarkable in most patients when reported. Raised Th17
percentages appear to be the most common finding (6/7) when tested. In
contrast hyper IgG (P1-P3), hyper IgE (P3, P4), or eosinophilia (P1, P4)
was found in only few cases.

In conclusion, we describe a patient with AR ACT1 deficiency, caused by
novel compound heterozygous (D451G and K454fs11*) variants
of\emph{TRAF3IP2} . Although only K454fs11* apparently affected ACT1
binding to IL-17RA, patient's cells were unresponsive to IL-17A,
explaining the clinical phenotype of CMC, further highlighting the
importance of performing detailed functional studies to understand the
clinical impact of genetic variants found in patients, particularly in
the field of immune dysregulation syndromes.

\textbf{Figure legends}

\textbf{Figure 1. Schematic representation of the cytokine pathways
tested in the patient.} Pathways evaluated in this manuscript include:

IFN-\selectlanguage{greek}γ-\selectlanguage{english}JAK-STAT pathway (purple): binding of IFN-\selectlanguage{greek}γ \selectlanguage{english}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-\selectlanguage{greek}κ\selectlanguage{english}B pathway: this pathway can be triggered by cytokines such as IL-17
(red), IL-1\selectlanguage{greek}β \selectlanguage{english}(green), and TNF-\selectlanguage{greek}α \selectlanguage{english}(pink), leading to the recruitment of
TRAF proteins to trigger downstream activation of NF-\selectlanguage{greek}κ\selectlanguage{english}B 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-\selectlanguage{greek}κ\selectlanguage{english}B activation upon IL-17. IL: interleukin; TNF:
tumor necrosis factor; TRAF: tumor necrosis factor receptor-associated
factor; NF-\selectlanguage{greek}κ\selectlanguage{english}B: nuclear factor \selectlanguage{greek}κ\selectlanguage{english}B; MAPK: mitogen-activated protein
kinase; AP-1: activator protein-1.

\textbf{Figure 2. 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.
\textbf{B)}Pedigree of the kindred showing the familial segregation of
\emph{STAT1}and \emph{ACT1} alleles. \textbf{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).

\selectlanguage{greek}\textbf{Figure 3. IFN-γ-dependent STAT1 activation in patient's
CD14\textsuperscript{+} monocytes:}\selectlanguage{english} Geometric mean fluorescence
intensity (gMFI) of total STAT1 \textbf{(A),} and pSTAT1
\textbf{(B),}evaluated by flow cytometry in non-stimulated (black), or
IFN-\selectlanguage{greek}γ-\selectlanguage{english}stimulated (gray) CD14\textsuperscript{+} monocytes from healthy
controls, patient's relatives (father, mother and sister), and the
patient.

\textbf{Figure 4. Patient's \emph{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.
\textbf{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. \textbf{C)}HEK-293T cells were co-transfected with WT
or mutant (D451G, K454fs11*, or T536I) ACT1-encoding plasmids along with
NF-\selectlanguage{greek}κ\selectlanguage{english}B--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). \textbf{D)}GRO-\selectlanguage{greek}α \selectlanguage{english}production, as measured by ELISA, after
stimulation (IL-17A, TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-17A+TNF-\selectlanguage{greek}α, \selectlanguage{english}or IL-1\selectlanguage{greek}β\selectlanguage{english}) of SV40-fibroblasts
from healthy individuals, patients with IL-17RA, IL-17RC, or ACT1
(ACT1\textsuperscript{T536I/T536I}) deficiency, and the patient under
study (D451G, K454fs11*).

\textbf{Figure 5. 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-\selectlanguage{greek}γ, \selectlanguage{english}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.

\textbf{Figure 6: Clinical (blue) and laboratory phenotype (red) and
therapeutic management (green) of previously reported patients with ACT1
deficiency (n=11).}

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30.\selectlanguage{english}
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