The patient was a 1-year-old boy of non-consanguineous Chinese parents. He was born at full-term with uneventful perinatal history. There was no family history of lung disease or primary immunodeficiency. He had no adverse reactions to vaccination with bacille Calmette-Guerin and hepatitis B. At 4 months of age, he was brought to the hospital for eczematous dermatitis ( Figure 1A ), mild cough, and a lump behind the left ear. His respiratory symptoms gradually worsened, and he developed respiratory distress and hypoxemia with possible cytomegalovirus (CMV) infection as the triggering event. Bronchoalveolar lavage and blood tests for CMV DNA were positive. He also suffered from repeated respiratory infections with positive sputum cultures of Acinetobacter baumanni, Klebsiella pneumonia, Pseudomonas aeringinosa and Stentophomonas maltophilia, and even twice respiratory failures requiring intubation and mechanical ventilation. Multiple courses of antibiotics have been required since then. He responded to the antibiotics with decreased inflammatory markers over 2-3 weeks, but fever recurred while on broad-spectrum antibiotics. At 6 months old, chest computed tomography suggested interstitial lung disease with diffuse ground-glass opacity ( Figure 1B ). The histology of lung biopsy showed chronic interstitial lung disease with diffuse thickening of alveolar membranes and hyperplastic type 2 pneumocytes ( Figure 1C ). The alveolar membranes contained some stromal cells and mild to moderate infiltrate of lymphocytes, some plasma cells, and macrophages. There was no proteinosis nor significant established interstitial fibrosis. Microbiological investigations of Lung biopsy including bacterial culture, fungal culture, acid-fast bacillus staining and PCR, and CMV were negative. Bronchoalveolar lavage pneumocystis pneumonia staining, blood tests for CMV, Epstein-Barr virus, human immunodeficiency virus, beta-glucan and QuantiFERON-TB were negative. He was treated empirically with prednisolone (1.8/mg/kg/day) with gradual tapering according to the disease status. Hydroxychloroquine (6 mg/kg/day) was also added. The respiratory symptoms were partially improved. When chest symptoms were relatively stable, he also exhibited recurrent diarrhea with elevated white blood cells and neutrophils ( Table 1 ) and one episode of stool culture positive for Clostridium difficile. At the age of 11months, he experienced increasing diarrhea, poor feeding, and septic shock with metabolic acidosis and elevated inflammatory markers. Unfortunately, he eventually died of respiratory failure at 1 year old.
Based on the clinical phenotype, congenital lung disease was considered for the patient. Genetic tests were done for the known related genes including SFTPC, SFTPB, ABCA3, CSF2RA, CSF2RB, NKX2.1, FOXF1 and SLC7A7, but no meaningful mutation was revealed. Therefore, the next-generation sequencing was performed for the patient and his parents. No severe combined immunodeficiency-related genetic mutation was found. Instead, a de novo heterozygous missense mutation of the STAT3 gene (c.989C>G, p.P330R) was identified in the patient ( Figures 2A, B ). The mutation was not found in the gnomAD (v2.1.1), ClinVar or Human Gene Mutation Database. This mutation was located within the DNA-binding domain (DBD), resulting in a proline to arginine substitution at position 330 ( Figure 2C ). The arginine residue was not observed at this position in homologous sequences ( Figure 2D ), suggesting highly conserved among different species.
Structurally, the proline is very rigid and therefore may induce a required exceptional backbone conformation at this position. To evaluate the impacts of this mutation on STAT3 structure, in silico studies were performed. The mutant residue lay on the surface of the STAT3 protein ( Figure 2E ) and decreased the hydrophobic interactions in this region ( Figure 2F ). We next performed the MD simulation of the WT and P330R STAT3 to estimate the conformational changes. The root-mean-square deviation (RMSD) for the P330R system showed higher fluctuations than the WT system ( Figure 2G ), suggesting an unstable protein structure. Beyond that, according to the electrostatic potential calculation, the charge changed from neutral (P) to positive (R) ( Figure 2H ), which may increase the interaction with negatively charged nucleic acids. These results strongly indicated that the mutation might alter the STAT3 structure and function.
The functional alterations of mutant STAT3 were investigated. STAT3 can be activated via phosphorylation upon different cytokine stimulations, such as IL-6. It was demonstrated that, upon IL-6 stimulation, the patient cells have much higher phosphorylation of STAT3 compared to normal controls. In addition, the mutation also activated STAT3 under the steady-state ( Figure 3A ). Next, we further performed functional validation by transfecting either WT or mutant constructs into HEK293 cells. Like the findings in patient cells, the phosphorylated STAT3 of mutant STAT3-transfected cells was significantly enhanced than that of the WT after IL-6 stimulation. The STAT3P330R mutant also displayed high phosphorylation in the basal condition ( Figure 3B ), suggesting constitutive activation. Besides, the P330R mutation also impaired the dephosphorylation of phosphorylated STAT3 on IL-6 deprivation ( Figure 3C ).
To detect the transcriptional activity of the P330R mutation, a STAT3-driven luciferase reporter assay was performed. We observed enhanced transcriptional activity of STAT3P330R under the steady-state ( Figure 3D ), further suggesting constitutive activation of mutant STAT3. The mutant also showed a severalfold increase in the luciferase activity compared with STAT3WT after being stimulated with IL-6 ( Figure 3E ). Collectively, these results suggested that the STAT3P330R represented a GOF mutation leading to both increased phosphorylation and transcriptional activity of STAT3.
 STAT3 plays an irreplaceable role in regulating the immune signaling pathways, including inhibiting Tregs and enhancing Th17 cell determination. Given this, we investigated whether the P330R mutation would cause Th17 and Treg cells dysregulation. PMA/ionomycin-activated PBMCs of the patient showed increased IL-17A levels in the T cells compared to the normal controls ( Figures 4A, B ), suggesting the over-activation of the Th17 pathway. However, the percentage of Treg cells was within a normal range in the patient ( Figure 4C ). The levels of immunoglobulin G, A and M were not reduced. Lymphocyte subpopulation analysis indicated decreased NK cell percentage but normal T and B cell levels in the patient's PBMCs ( Table 1 ). The proportions of effector memory (EM) CD4+ T, EM CD8+ T, naive CD4+ T and naive CD8+ T cells were also normal ( Figure 4D ). Taken together, these data suggested that the STAT3P330R gain-of-function mutation would contribute to activating the Th17 pathway.
STAT3 is activated by JAK-mediated phosphorylation, so targeting upstream JAK kinases could be a potential approach to inhibit STAT3 activation. We thus performed functional experiments to determine whether JAK inhibitors could suppress STAT3P330R activation. It was found that both ruxolitinib and tofacitinib significantly reduced the phosphorylation of mutant STAT3 induced by IL-6 stimulation ( Figure 5A ). Given the broad suppressive effect of JAK inhibitors, we sought to test the ability of a direct STAT3 selective inhibitor (Stattic) in pharmacologic inhibition of STAT3 GOF mutation. Similar to JAK inhibitors, the STAT3 inhibitor also markedly suppressed the phosphorylation of mutant STAT3 in the steady-state, as well as in IL-6-stimulated condition ( Figures 5B, C ). In addition, the inhibitory effect was dose-dependent. The more amount of inhibitor used, the more inhibition of STAT3P330R phosphorylation seen. These data indicated that selective inhibition of STAT3 represents a potential therapeutic approach for STAT3 GOF syndrome.