Severe allergic dysregulation due to a gain of function mutation in the transcription factor STAT6

Abstract

Background: Inborn errors of immunity have been implicated in causing immune dysregulation, including allergic diseases. STAT6 is a key regulator of allergic responses.

Objectives: This study sought to characterize a novel gain-of-function STAT6 mutation identified in a child with severe allergic manifestations.

Methods: Whole-exome and targeted gene sequencing, lymphocyte characterization, and molecular and functional analyses of mutated STAT6 were performed.

Results: This study reports a child with a missense mutation in the DNA binding domain of STAT6 (c.1114G>A, p.E372K) who presented with severe atopic dermatitis, eosinophilia, and elevated IgE. Naive lymphocytes from the affected patient displayed increased T_H2- and suppressed T_H1- and T_H17-cell responses. The mutation augmented both basal and cytokine-induced STAT6 phosphorylation without affecting dephosphorylation kinetics. Treatment with the Janus kinase 1/2 inhibitor ruxolitinib reversed STAT6 hyperresponsiveness to IL-4, normalized T_H1 and T_H17 cells, suppressed the eosinophilia, and improved the patient's atopic dermatitis.

Conclusions: This study identified a novel inborn error of immunity due to a STAT6 gain-of-function mutation that gave rise to severe allergic dysregulation. Janus kinase inhibitor therapy could represent an effective targeted treatment for this disorder.

Keywords: Inborn errors of immunity; Jakinibs; Janus kinase inhibitors; STAT6; gain-of-function mutation; primary atopic disorders.

Figure 1.. Identification of a STAT6^E372K mutation in a child with severe allergy dysregulation.

(A) Scattered eosinophils in dermis, hematoxylin and eosin staining (H&E), 40X (a). Parakeratosis, spongiosis and acanthosis of epidermis (black arrows), H&E, 20X (b). Mucin accumulation dissociating epithelial cells of follicular epithelium, dilatation of follicle (black arrows), periodic acid schiff and Alcian Blue 2.5 staining, 10X (c). Gastrointestinal pathology shows esophagitis and scattered eosinophils with marked fibrosis in the subepithelial area of the esophagus (black arrows), H&E; 20X, 40X and trichrome staining; 40X, respectively (d, e, f). Inflammation with eosinophils in lamina propria, intraepithelial and intravascular areas of sigmoid colon, (H&E); 20X, 40X, 40X, respectively (g, h, i). (B) Pedigree of STAT6 GOF patient. Double lines indicate consanguinity; a filled black square depicts the patient. Males and females are distinguished by squares and circles, respectively. (C) Sanger sequencing analysis of the STAT6 c.1114G>A mutation in the index patient versus a healthy control. (D) Schematic diagram of STAT6 protein domains. The depicted domains are the α-Helix domain (α-H), DNA binding domain (DBD), linker domain (LD), SH2 Src homology 2 domain (SH2), and a transactivation domain (TAD). The heterozygous mutation localizes to the DBD. Multiple sequence alignment analysis of STAT6 proteins of different species demonstrates conservation of the STAT6E372 residue. (E, F) Structural modeling of the interaction of the STAT6^E372K and STAT3^N420K with DNA.

Figure 2.. The STAT6^E372K mutation is associated with skewed T_H2 responses.

(A) Flow cytometric analysis and graphical representation of naïve (CD3⁺CD4⁺CD45RO⁻CD45RA⁺) and memory (CD3⁺CD4⁺CD45RO⁺CD45RA⁻) Teff cells in Healthy controls and the patient with STAT6^E372K mutation. (B) Flow cytometric analysis and graphical representation of T_H2 cells (CD3⁺CD4⁺GATA3⁺) in the respective groups. (C) Cells frequencies of CD4⁺CRTH2⁺, CD4⁺CCR4⁺, CD4⁺RORγ⁺, CD4⁺CCR6⁺, CD4⁺CXCR3⁺ T cells in the respective groups. (D) Flow cytometric analysis and graphical representation of Treg GATA3⁺ cells (CD3⁺CD4⁺CD127⁻Foxp3⁺GATA3⁺) in the respective groups. (E) Cells frequencies of Treg CRTH2⁺, Treg CCR4⁺, Treg RORγT⁺, Treg CCR6⁺, and Treg CXCR3⁺ T cells in the respective groups. (F) Cells frequencies of circulating T follicular helper (cT_FH), T follicular helper T_H2 (CD4⁺CXCR5⁺CD45RA⁻CXCR3⁻CCR6⁻), T follicular helper T_H17 (CD4⁺CXCR5⁺CD45RA⁻CXCR3⁻CCR6⁺), and T follicular regulatory cells in the respective groups. Each symbol represents one subject. Numbers in flow plots indicate percentages. Error bars indicate SEM.

Figure 3.. The STAT6^E372K mutation is associated with increased circulating CD23⁺ and IgE⁺ memory B cells.

(A) Gating strategy for B cells analysis. (B) Cell Frequencies of Class switched memory B cells (CD19⁺, CD27⁺ IgD⁻). (C) Flow cytometric analysis and graphical representation of Plasmablasts (CD19⁺ CD20⁻ CD38⁺) in healthy controls and in the patient with STAT6^E372K mutation. (D) Flow cytometric analysis and graphical representation of IgE⁺ circulating B cells in healthy controls and in the patient with the STAT6^E372K mutation. (E, F) Flow cytometric analysis and graphical representation of CD23⁺ (E) and CD23–IgE+ memory (IgD⁻ CD27⁺) B cells (F) in healthy controls and in the patient with STAT6^E372K mutation. Each symbol represents one subject. Numbers in flow plots indicate percentages. Error bars indicate SEM.

Figure 4.. The STAT6^E372K mutation is associated with enhanced B cell class switching to IgE.

(A) Gating strategy for cell-sorting of naïve B cells (CD19⁺IgD⁺CD27⁻CD23⁻IgE⁻). (B) Purity check after cell sorting. (C, D) Flow cytometric analysis, frequencies of IgE⁺ B cells (C), and total IgE in the culture supernatants (D) in cultures of the respective sorted B cell populations that were treated with an isotype control mAb or with anti-CD40 mAb+IL-4. Each symbol represents one subject. Numbers in flow plots indicate percentages. Error bars indicate SEM.

Figure 5.. STAT6^E372K is a GOF mutation.

(A) Flow cytometric analysis and mean fluorescence intensity (MFI) of STAT6 in CD4⁺ T cells of Healthy controls and the patient with STAT6^E372K mutation. (B) Flow cytometric analysis and MFI of p-STAT6 (top) and frequencies of p-STAT6 cells (bottom) in CD4⁺ T cells of Healthy controls and the patient with STAT6^E372K mutation after stimulation with IL-4 (20ng/ml) for 5 to 30 minutes (Black line: Healthy control unstimulated, Red line: STAT6^E372K patient unstimulated, Blue: Healthy control stimulated with IL-4 and Red: STAT6^E372K patient stimulated with IL-4). (C) Flow cytometric analysis and MFI of p-STAT5 (top) and of p-STAT3 (bottom) in CD4⁺ T cells of Healthy controls and the patient with STAT6^E372K mutation after stimulation with IL-2 (20ng/ml) or IL-6 (20ng/ml), respectively for 5 to 30 minutes (D) Dephosphorylation kinetics of phospho-STAT6 in response to deprivation of IL-4 and in CD4⁺ T cells represented as absolute MFI (left) and normalized to maximum expression before deprivation (right). (E) MFI expression of STAT6 in HEK293 cell transfected with either STAT6^WT or STAT6^E372K proteins. (F) MFI expression of p-STAT6 in HEK293 cell transfected with either STAT6^WT or STAT6^E372K proteins after IL-4 (20ng/ml) stimulation for 5 to 30 minutes. (G) STAT6 response element-driven luciferase reporter activation by STAT6^WT or STAT6^E372K transfected into HEK293 cells at baseline and following IL-4 treatment for 1 and 4 hr. RLU: Relative Luciferase Unit. (H, I) Confocal microscopic analysis of total STAT6, p-STAT6 and DAPI in HEK293 cells transfected with either STAT6^WT or STAT6^E372K and then either sham-treated or stimulated with IL-4 for 1 and 4 hr, respectively. Numbers in flow plots indicate MFI. Error bars indicate SEM. Statistical tests: *P<0.05, **P<0.01, ****P<0.0001 by two-way ANOVA with Dunnett’s post hoc analysis (F; G, I).

Figure 6.. Ruxolitinib therapy suppresses the allergic dysregulation and inhibits the T_H2 skewing and STAT6 hyperactivation.

(A) Response of patient atopic dermatitis to ruxolitinib therapy. (a-e), Severe atopic dermatitis before ruxolitinib therapy covering the face (a), body (b, c), elbow and hand (d, e) of the patient. (f-i), control of atopic dermatitis after ruxolitinib treatment. (B) Flow cytometric analysis of naïve (CD3⁺CD4⁺CD45RO⁻CD45RA⁺) and memory (CD3⁺CD4⁺CD45RO⁺CD45RA⁻) Teff cells in healthy controls and the patient with STAT6^E372K mutation pre- and post-therapy with ruxolitinib. (C, D) Flow cytometric analysis of CD4⁺CCR4⁺(C) and CD4⁺CRTH2⁺(D) T cells in the respective groups. (E, F) Cells frequencies of CD4⁺CCR6⁺(E) and CD4⁺CXCR3⁺(F) T cells in the respective groups. (G, H) Flow cytometric analysis with representative plots of IL-4-, IL-17-, IFN-γ- and IL-10-producing circulating CD4⁺ T cells pre-and post-ruxolitinib therapy compared with healthy controls. (I) Flow cytometric analysis and MFI of p-STAT6 (left) and p-STAT6 fold change (right) in CD4⁺ T cells of healthy controls and the patient with STAT6^E372K mutation pre- and post-treated with ruxolitinib after stimulation with IL-4 (20ng/ml) for 5 to 30 minutes (Red line: STAT6^E372K patient pre-treatment unstimulated, Red: STAT6^E372K patient stimulated with IL-4, Orange line: STAT6^E372K patient post-treatment with ruxolitinib and orange: STAT6^E372K patient post-treated with ruxolitinib stimulated with IL-4). Each symbol represents one subject. Numbers in flow plots indicate percentages. Error bars indicate SEM.