Figure legends
Figure 1. 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.
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. B)Pedigree of the kindred showing the familial segregation of STAT1and 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).
Figure 3. 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.
Figure 4. 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*).
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-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.