Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome

Abstract

Background: Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers.

Objective: We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC.

Methods: We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations.

Results: We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation.

Conclusions: Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.

Figure 1. Clinical Phenotypes

(A): Patient 1, extensive candidiasis of nails, causing dystrophy and paronychia. (B): Patient 3, Multiple intracranial aneurysms in the Circle of Willis, as detected by CT angiogram and MR angiogram. (1C): Bilateral lower lobe bronchiectasis with endobronchial/peribronchial consolidation seen on chest CT. (1D): multiple calcifications in descending aorta detected by CT of patient 3.

Figure 1. Clinical Phenotypes

(A): Patient 1, extensive candidiasis of nails, causing dystrophy and paronychia. (B): Patient 3, Multiple intracranial aneurysms in the Circle of Willis, as detected by CT angiogram and MR angiogram. (1C): Bilateral lower lobe bronchiectasis with endobronchial/peribronchial consolidation seen on chest CT. (1D): multiple calcifications in descending aorta detected by CT of patient 3.

Figure 2. STAT1 staining

(A): Intracellular staining of phospho-tyrosine 701 STAT1 in CD3+ lymphocytes isolated from healthy controls and STAT1 gain of function mutants (patient 2 and 3) after stimulation with IFN-γ, IL-6, or IL-21 for 15 minutes. (B): Kinetics of the STAT1 phosphorylation were studied by intracellular phospho-tyrosine 701 STAT1 staining in U3A cells transfected with mutant or WT constructs and stimulated with IFN-γ. Stimulation indices at 60 minutes following stimulation (SI: MFI of pSTAT1 at 60′ after stimulation /MFI pSTAT1 at rest) were higher for mutants (SI: 2.6 for R210I, 4.0 for L358W, and 4.4 for T385M), than for WT STAT1 (SI of 0.9, p <0.05).

Figure 2. STAT1 staining

(A): Intracellular staining of phospho-tyrosine 701 STAT1 in CD3+ lymphocytes isolated from healthy controls and STAT1 gain of function mutants (patient 2 and 3) after stimulation with IFN-γ, IL-6, or IL-21 for 15 minutes. (B): Kinetics of the STAT1 phosphorylation were studied by intracellular phospho-tyrosine 701 STAT1 staining in U3A cells transfected with mutant or WT constructs and stimulated with IFN-γ. Stimulation indices at 60 minutes following stimulation (SI: MFI of pSTAT1 at 60′ after stimulation /MFI pSTAT1 at rest) were higher for mutants (SI: 2.6 for R210I, 4.0 for L358W, and 4.4 for T385M), than for WT STAT1 (SI of 0.9, p <0.05).

Figure 3. Treg subsets, FOXP3 expression, cytokine profiles and STAT5 signaling

(A): Percentage and level of FOXP3 and HELIOS expression within CD3⁺CD4⁺ gated T cells from patients 2 and 3 compared to healthy controls. (B): Induction of FOXP3 and absence of HELIOS expression in naïve T cells from healthy control and patients 2 and 3 on day 5 after anti-CD3/CD28 stimulation in the presence of IL-2 (NONE) only, + TGF-β1, + TGF-β1 and IFN-γ or + TGF-β1 and IL-21. (C): Comparison of cytokine expression between FOXP3⁺ and FOXP⁻CD4⁺ T cells within fresh PBMCs of healthy controls and patients 2 and 3 stimulated with PMA/ionomycin. (D): Normal IL-2 induced STAT5 phosphorylation in CD3⁺ lymphocytes of patient 3 (solid line histogram) compared to healthy control (dashed histogram) and unstimulated cells (shaded histogram). Patient 2 had similar expression profile (data not shown).

Figure 3. Treg subsets, FOXP3 expression, cytokine profiles and STAT5 signaling

(A): Percentage and level of FOXP3 and HELIOS expression within CD3⁺CD4⁺ gated T cells from patients 2 and 3 compared to healthy controls. (B): Induction of FOXP3 and absence of HELIOS expression in naïve T cells from healthy control and patients 2 and 3 on day 5 after anti-CD3/CD28 stimulation in the presence of IL-2 (NONE) only, + TGF-β1, + TGF-β1 and IFN-γ or + TGF-β1 and IL-21. (C): Comparison of cytokine expression between FOXP3⁺ and FOXP⁻CD4⁺ T cells within fresh PBMCs of healthy controls and patients 2 and 3 stimulated with PMA/ionomycin. (D): Normal IL-2 induced STAT5 phosphorylation in CD3⁺ lymphocytes of patient 3 (solid line histogram) compared to healthy control (dashed histogram) and unstimulated cells (shaded histogram). Patient 2 had similar expression profile (data not shown).

Figure 3. Treg subsets, FOXP3 expression, cytokine profiles and STAT5 signaling

(A): Percentage and level of FOXP3 and HELIOS expression within CD3⁺CD4⁺ gated T cells from patients 2 and 3 compared to healthy controls. (B): Induction of FOXP3 and absence of HELIOS expression in naïve T cells from healthy control and patients 2 and 3 on day 5 after anti-CD3/CD28 stimulation in the presence of IL-2 (NONE) only, + TGF-β1, + TGF-β1 and IFN-γ or + TGF-β1 and IL-21. (C): Comparison of cytokine expression between FOXP3⁺ and FOXP⁻CD4⁺ T cells within fresh PBMCs of healthy controls and patients 2 and 3 stimulated with PMA/ionomycin. (D): Normal IL-2 induced STAT5 phosphorylation in CD3⁺ lymphocytes of patient 3 (solid line histogram) compared to healthy control (dashed histogram) and unstimulated cells (shaded histogram). Patient 2 had similar expression profile (data not shown).

Figure 3. Treg subsets, FOXP3 expression, cytokine profiles and STAT5 signaling

(A): Percentage and level of FOXP3 and HELIOS expression within CD3⁺CD4⁺ gated T cells from patients 2 and 3 compared to healthy controls. (B): Induction of FOXP3 and absence of HELIOS expression in naïve T cells from healthy control and patients 2 and 3 on day 5 after anti-CD3/CD28 stimulation in the presence of IL-2 (NONE) only, + TGF-β1, + TGF-β1 and IFN-γ or + TGF-β1 and IL-21. (C): Comparison of cytokine expression between FOXP3⁺ and FOXP⁻CD4⁺ T cells within fresh PBMCs of healthy controls and patients 2 and 3 stimulated with PMA/ionomycin. (D): Normal IL-2 induced STAT5 phosphorylation in CD3⁺ lymphocytes of patient 3 (solid line histogram) compared to healthy control (dashed histogram) and unstimulated cells (shaded histogram). Patient 2 had similar expression profile (data not shown).

Figure 4. Tregs suppressive function

(A) : Responders only histogram represents CFSE dilution on day 4 within CD4⁺ T cells. The histograms in the two right columns demonstrate the level of proliferative suppression within CFSE⁺ cells in the presence of 1/2 and 1/4 the numbers of Tregs and Teffs relative to responders. Left FACS plot column shows the level of FOXP3 expression within CFS⁻ Tregs/Teffs and CFSE⁺ responders on day 4. Patient 2 and control 1 are allogeneic and control 2 is autologous to responders and HLA-DR⁺ APCs. (B) Suppressive function of Tregs from patient 3 and healthy controls. Patient 3 and control 1 are allogeneic and control 2 is autologous to responders and HLA-DR⁺ APCs.

Figure 4. Tregs suppressive function

(A) : Responders only histogram represents CFSE dilution on day 4 within CD4⁺ T cells. The histograms in the two right columns demonstrate the level of proliferative suppression within CFSE⁺ cells in the presence of 1/2 and 1/4 the numbers of Tregs and Teffs relative to responders. Left FACS plot column shows the level of FOXP3 expression within CFS⁻ Tregs/Teffs and CFSE⁺ responders on day 4. Patient 2 and control 1 are allogeneic and control 2 is autologous to responders and HLA-DR⁺ APCs. (B) Suppressive function of Tregs from patient 3 and healthy controls. Patient 3 and control 1 are allogeneic and control 2 is autologous to responders and HLA-DR⁺ APCs.