Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations

Abstract

Germline loss-of-function mutations in the transcription factor signal transducer and activator of transcription 3 (STAT3) cause immunodeficiency, whereas somatic gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here, we report on 13 individuals from 10 families with lymphoproliferation and early-onset solid-organ autoimmunity associated with 9 different germline heterozygous mutations in STAT3. Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multiorgan autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections, and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T-cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 led to clinical improvement in 1 patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly described disorder. Some patients for this study were enrolled in a trial registered at www.clinicaltrials.gov as #NCT00001350.

Figure 1

STAT3 mutations confer a GOF. (A) Schematic of human STAT3 protein showing the location of 9 different STAT3 missense mutations and the resulting amino acid changes. Mutations were located throughout the protein in the all alpha, DNA binding, SH2, and C-terminal (transactivation) protein domains. The numbers represent the amino acid location based on STAT3 transcript variant 1 (protein model based on the Pfam protein family database). The p.T716M variant was detected in 2 different families (patient 7 and family 2). (B) Western blot of STAT3 expression. WT, GOF, LOF, and the 9 different mutant STAT3 transcripts were expressed in the STAT3-deficient A4 cell line and expression of pSTAT3 and total STAT3 protein was determined using western blot without (top) and with (bottom) IL-6 (15 minutes, 10 ng/mL). IL-6–treated DLD cells (parental A4 line) are shown as a control. Expression of all mutants led to the detection of STAT3 protein, with the exception of p.A703T, a mutation near the binding site of the antibody used here (clone 79D7). This variant was detectable after IL-6 activation and with a different antibody clone (supplemental Figure 2). Mutant STAT3 proteins were not phosphorylated at baseline, but were phosphorylated after stimulation with IL-6. (C-D) STAT3 binding activity as measured by luciferase assay. A4 cells were transfected with WT or the indicated STAT3 mutants and STAT3-driven firefly luciferase and control renilla reporters. (C) Luciferase was assayed at 48 hours and STAT3 activity is shown as a ratio of firefly/control for each construct. All constructs with the exception of V353F had significantly increased activity at baseline compared with WT. Results represent the mean ± SEM of 5 to 10 independent experiments; the dotted line represents WT (**P < .01, ***P < .001, ****P < .0001). (D) STAT3 activity following 12-hour activation with IL-6 (10 ng/mL, white) or IFN-α (50 ng/mL, black). All mutations demonstrated significantly increased STAT3 activity after at least 1 of the cytokine stimulations (P < .05), typically after both unless indicated. Results shown represent fold-change vs WT for each cytokine stimulus; the dotted line represents a fold-change of 1 (no change from WT). Results represent the mean ± SEM of 5 independent experiments. ns, not significant; pSTAT3, phosphorylated STAT3.

Figure 2

Elevated expression of SOCS3 in patient-derived EBV-transformed cell lines. Expression of the STAT3-target SOCS3 was determined by quantitative RT-PCR in resting (A) and (B) IL-21–activated (50 ng/mL for 21 hours) cell lines. Increased SOCS3 transcript was detected in patient-derived cells compared with healthy controls (HC1 and HC2). Results represent the fold-increased expression in SOCS3 transcript compared with unstimulated healthy control 1 (HC1) and are normalized to β-actin. Results represent the mean ± SEM of 3 independent experiments. *P < .05; **P < .01; ***P < .001.

Figure 3

Increased STAT3 activity in patient-derived EBV-transformed cell lines leads to decreased phosphorylation of other STAT molecules. (A) Impaired STAT5 phosphorylation in EBV-transformed cell lines derived from GOF STAT3 patients. Cells from patients (red) and healthy controls (blue) were stimulated with IL-2 (80 U/mL) and pSTAT5 measured by flow cytometry after 20 minutes. Numbers indicate the difference in MFI of phospho-STAT5 obtained by subtracting the MFI of unstimulated from stimulated samples. Yellow lines represent unstimulated patient samples; filled histograms, unstimulated healthy control samples. (B) Impaired STAT1 phosphorylation in EBV-transformed cell lines derived from GOF STAT3 patients. Cells from patients (red) and healthy controls (blue) were stimulated with IFN-γ (500 ng/mL) and pSTAT1 measured by flow cytometry after 20 minutes. Numbers indicate the difference in MFI of phospho-STAT1 obtained by subtracting the MFI of unstimulated from stimulated samples. Yellow lines represent unstimulated patient samples and filled histograms unstimulated healthy control samples. MFI, mean fluorescence intensity.

Figure 4

STAT3 GOF patients have decreased Treg numbers and functional markers. (A) Flow cytometric analysis of FOXP3⁺CD25⁺ Treg populations from patient 4, gated on CD4⁺ T cells, compared with a healthy control. (B) Decreased Treg percentages in STAT3 GOF patients. Treg populations were determined by flow cytometry. The percentages of CD4⁺ T cells that were FOXP3⁺CD25⁺ Tregs from patients were decreased compared with 6 healthy controls and 2 patients with ALPS who had FAS defects (*P < .05). (C) Tregs from STAT3 GOF patients have decreased expression of CD25. Treg populations (CD4⁺ FOXP3⁺CD127^low) were determined by flow cytometry and the CD25 expression further analyzed by histogram. Tregs from patients demonstrate lower levels of CD25 expression than healthy controls.

Figure 5

Patient response to anti-IL6R monoclonal antibody therapy. Patient 1 received therapy with the anti-IL6R monoclonal antibody tocilizumab. He had notable improvement of his long-standing polyarthritis and skin tightening as shown in the photographs (top, 2 years before treatment; middle, at initiation of treatment; and bottom, after one year of treatment). Indicated are the number of fixed DIP (distal interphalangeal) contractures of both his right and left hands at the designated times with respect to anti-IL-6R therapy. L, left; R, right; yr, year.