PMC6491627_01
Pulmonary imaging during an acute respiratory infection at age 5 years. (A) The patient had numerous, enhancing cavitary lung lesions (black arrow) noted using CT scan of the chest, which improved after drainage and prolonged antibiotic therapy. (B) Several of the pneumatoceles were fluid-filled (short white arrow) and a parapneumonic effusion was present (long white arrow).
fped-07-00130-g0001.jpg
PMC6491627_01_fped-07-00130-g0001.jpg
F1
At age 5 he was admitted to the hospital with pneumonia complicated by a parapneumonic effusion and multiple cavitary lung lesions (Figure 1).
A de novo mutation in STAT3 has decreased basal activity and leads to low Th17 cells. (A) Targeted Sanger sequencing revealed a STAT3 mutation, c.1934T>A (leading to p.L645Q), in the patient that was not present in his parents. (B) To measure basal levels of STAT3 activity a luciferase assay was performed. Wild-type (WT, black) or mutant (gray) STAT3 plasmids were co-transfected into STAT3-deficient cells along with a STAT3-responsive luciferase reporter. STAT3 activity is shown as a ratio of firefly/control normalized to WT. Data represent 3 experiments +- SEM, **p < 0.01 as it designates the level of significance (unpaired t-test). (C) Intracellular cytokine staining was performed to detect Th17 cells produced after stimulation. A gate was placed on CD3+CD4+ T-cells, then they were assessed for the ability to produce IL-17 and interferon-gamma (IFNgamma) as a positive control. The patient produced reduced levels of Th17 cells compared to his parents.
fped-07-00130-g0002.jpg
PMC6491627_01_fped-07-00130-g0002.jpg
F2
AD-HIES was suspected. A de novo, heterozygous missense mutation in STAT3 (c.1934T>A, p.L645Q) was detected (Figure 2A). Reduction of transcriptional activity of STAT3 p.L645Q to 85% of wild-type was confirmed using a luciferase assay (Figure 2B). The patient also had decreased CD3+CD4+ T cells expressing IL-17 (Figure 2C).
The SH2 domain STAT3 variant p.L645Q fails to phosphorylate normally after stimulation. Patient-derived cells were left unstimulated (solid black) or stimulated with IL-6 or interferon-alpha (IFNalpha) and assessed for phospho-STAT3 (p-STAT3) using flow cytometry. Cells derived from the patient (solid gray) fail to phosphorylate after stimulation compared to cells derived from the father (dashed gray) or mother (dotted gray).
fped-07-00130-g0003.jpg
PMC6491627_01_fped-07-00130-g0003.jpg
F3
Furthermore, STAT3 phosphorylation in patient-derived cells was impaired in response to cytokine stimulation (Figure 3), which has been reported in patients with AD-HIES and STAT3 mutations in the SH2 domain.