Tiphanie P. Vogel

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

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.

Activation-Specific Metabolic Requirements for NK Cell IFN-γ Production

There has been increasing recognition of the importance of cellular metabolism and metabolic substrates for the function and differentiation of immune cells. In this study, for the first time to our knowledge, we investigate the metabolic requirements for production of IFN-γ by freshly isolated NK cells. Primary murine NK cells mainly use mitochondrial oxidative phosphorylation at rest and with short-term activation. Remarkably, we discovered significant differences in the metabolic requirements of murine NK cell IFN-γ production depending upon the activation signal. Stimulation of NK cell IFN-γ production was independent of glycolysis or mitochondrial oxidative phosphorylation when cells were activated with IL-12 plus IL-18. By contrast, stimulation via activating NK receptors required glucose-driven oxidative phosphorylation. Prolonged treatment with high-dose, but not low-dose, IL-15 eliminated the metabolic requirement for receptor stimulation. In summary, this study demonstrates that metabolism provides an essential second signal for induction of IFN-γ production by activating NK cell receptors that can be reversed with prolonged high-dose IL-15 treatment.

The Ying and Yang of STAT3 in Human Disease

The transcription factor signal transducer and activator of transcription 3 (STAT3) is a critical regulator of multiple, diverse cellular processes. Heterozgyous, germline, loss-of-function mutations in STAT3 lead to the primary immune deficiency Hyper-IgE syndrome. Heterozygous, somatic, gain-of-function mutations in STAT3 have been reported in malignancy. Recently, germline, heterozygous mutations in STAT3 that confer a gain-of-function have been discovered and result in early-onset, multi-organ autoimmunity. This review summarizes what is known about the role of STAT3 in human disease.

Somatic mutations and clonal hematopoiesis in congenital neutropenia

Severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome (SDS) are congenital neutropenia syndromes with a high rate of leukemic transformation. Hematopoietic stressors may contribute to leukemic transformation by increasing the mutation rate in hematopoietic stem/progenitor cells (HSPCs) and/or by promoting clonal hematopoiesis. We sequenced the exome of individual hematopoietic colonies derived from 13 patients with congenital neutropenia to measure total mutation burden and performed error-corrected sequencing on a panel of 46 genes on 80 patients with congenital neutropenia to assess for clonal hematopoiesis. An average of 3.6 ± 1.2 somatic mutations per exome was identified in HSPCs from patients with SCN compared with 3.9 ± 0.4 for healthy controls (P = NS). Clonal hematopoiesis due to mutations in TP53 was present in 48% (13/27) of patients with SDS but was not seen in healthy controls (0/17, P < .001) or patients with SCN (0/40, P < .001). Our SDS cohort was young (median age 6.3 years), and many of the patients had multiple TP53 mutations. Conversely, clonal hematopoiesis due to mutations of CSF3R was present in patients with SCN but was not detected in healthy controls or patients with SDS. These data show that hematopoietic stress, including granulocyte colony-stimulating factor, do not increase the mutation burden in HSPCs in congenital neutropenia. Rather, distinct hematopoietic stressors result in the selective expansion of HSPCs carrying specific gene mutations. In particular, in SDS there is enormous selective pressure to expand TP53-mutated HSPCs, suggesting that acquisition of TP53 mutations is an early, likely initiating event, in the transformation to myelodysplastic syndrome/acute myeloid leukemia in patients with SDS.

Up, Down, and All Around: Diagnosis and Treatment of Novel STAT3 Variant

The number of identified monogenic causes of childhood-onset autoimmunity due to nodal and extranodal lymphoproliferation has increased. These pathogenic genetic variants provide the potential for pathway-specific treatment. Novel variants also require pathway-specific verification. In this report, we describe a 14-year-old patient with a novel variant in STAT3. We report clinical and laboratory findings that support STAT3 p.G419R as a novel pathogenic STAT3 gain-of-function variant.

Genetic and mechanistic diversity in pediatric hemophagocytic lymphohistiocytosis

The HLH-2004 criteria are used to diagnose hemophagocytic lymphohistiocytosis (HLH), yet concern exists for their misapplication, resulting in suboptimal treatment of some patients. We sought to define the genomic spectrum and associated outcomes of a diverse cohort of children who met the HLH-2004 criteria. Genetic testing was performed clinically or through research-based whole-exome sequencing. Clinical metrics were analyzed with respect to genomic results. Of 122 subjects enrolled over the course of 17 years, 101 subjects received genetic testing. Biallelic familial HLH (fHLH) gene defects were identified in only 19 (19%) and correlated with presentation at younger than 1 year of age (P < .0001). Digenic fHLH variants were observed but lacked statistical support for disease association. In 28 (58%) of 48 subjects, research whole-exome sequencing analyses successfully identified likely molecular explanations, including underlying primary immunodeficiency diseases, dysregulated immune activation and proliferation disorders, and potentially novel genetic conditions. Two-thirds of patients identified by the HLH-2004 criteria had underlying etiologies for HLH, including genetic defects, autoimmunity, and malignancy. Overall survival was 45%, and increased mortality correlated with HLH triggered by infection or malignancy (P < .05). Differences in survival did not correlate with genetic profile or extent of therapy. HLH should be conceptualized as a phenotype of critical illness characterized by toxic activation of immune cells from different underlying mechanisms. In most patients with HLH, targeted sequencing of fHLH genes remains insufficient for identifying pathogenic mechanisms. Whole-exome sequencing, however, may identify specific therapeutic opportunities and affect hematopoietic stem cell transplantation options for these patients.

The Heart and Pediatric Rheumatology

Recent advances in Kawasaki disease have included attempts to define genes involved in its pathogenesis. There have been recent advances in the studies of rheumatic carditis, leading to a better understanding of the mechanism of the disease. Histologic evaluation of patients with neonatal lupus erythematosus has revealed fibrosis with collagen deposition and calcification of the atrioventricular node. Therapy for cardiac involvement in systemic juvenile idiopathic arthritis should involve treatment of the underlying disease and systemic inflammatory state, and typically includes nonsteroidal antiinflammatory drugs, corticosteroids, disease-modifying drugs, and biologic therapies targeting tumor necrosis factor-alpha, interleukin-1, and interleukin-6.

Jakinibs for the treatment of immune dysregulation in patients with gain-of-function signal transducer and activator of transcription 1 (STAT1) or STAT3 mutations

Treatment of the autoimmune and immune-dysregulatory features of patients with STAT1 GOF or STAT3 GOF disease remains challenging. Jakinibs have been used to treat the severe immune-dysregulation in patients with either STAT1 GOF or STAT3 GOF mutations.

STAT3 Gain of Function: A New Kid on the Block in Interstitial Lung Diseases

A 5-year-old girl with failure to thrive and multiorgan disease was referred to our center for chronic hypoxemia. On evaluation, we noted tachypnea (respiratory rate 35/min), supraclavicular retractions, median diurnal oxygen saturation as measured by pulse oximetry (Sp O 2) = 91.7% at rest, percentage of time below Sp O 2 90% at 26% during sleep, and clubbing. A computed tomography scan showed diffuse interstitial lung disease (Figure 1). Spirometry was normal (TLC, 83% of predicted; FEV 1 , 83% of predicted; FEV 1 /FVC, 98%; and forced expiratory flow, midexpiratory phase, 142% of predicted), but it was not possible to measure DL CO .

Azathioprine-Associated Complete NK Cell Deficiency

To the editor, We report the case of a 5-year-old Caucasian girl with very early-onset inflammatory bowel disease (VOIBD) who had inadequate response to initial therapy with azathioprine. Immune system evaluation revealed an isolated complete natural killer cell (NK cell) deficiency, which resolved with discontinuation of the drug. The patient first presented at age 4 with a history of angular cheilitis and oral ulcerations of the soft palate. She had notable findings of microcytic anemia (Hb 10.7 g/dL, MCV 73.2 fL), elevated sedimentation rate of 72 mm/hr, and elevated fecal calprotectin of 396 mcg/g (normal less than 50 mcg/g). Colonoscopy showed segmental moderate inflammation with congestion, erosions, friability, and scarring throughout the entire colon with pathology demonstrating focal active ileitis and non-necrotizing granulomas in the terminal ileum compatible with Crohn’s disease. Due to the young age of onset, additional immunologic screening was performed including enumeration of T, B, and natural killer cells, immunoglobulins, pneumococcal and tetanus titers, and testing for chronic granulomatous disease which was normal or negative, respectively. She was subsequently treated with corticosteroids and azathioprine, with testing demonstrating wildtype thiopurine methyltransferase genotype, predictive of normal azathioprine metabolism. Over the ensuing 12 months, she continued to have active disease and azathioprine was incrementally increased to 3 mg/kg. Drug levels at this dose showed sub-therapeutic 6-TGN at 229 pmol/RBC (range 230–400), below risk levels for myelotoxicity with no elevation of the hepatotoxic metabolite 6-MMPN. Additional immunologic evaluation at that time revealed normal T cell phenotyping and lymphocyte proliferation to mitogens. Repeat lymphocyte subpopulations revealed an isolated absence of NK cells by clinical testing in two separate samples 2 days apart (Fig. 1a). Additional research-based phenotyping did not reveal the presence of any CD56-negative NK cell-like population expressing markers such as CD122, CD16, or NK cell receptors including killer immunoglobulin-like (KIR) or NKG2 molecules, confirming an absence of peripheral blood NK cells. Functional NK cell testing showed absent NK cell cytotoxicity of K562 target cells (Fig. 1b). Infectious testing included negative blood PCR for EBVandCMVwith no serologic evidence of infection for EBV, CMV, HSV, and VZV. Azathioprine was discontinued due to inadequate disease control and concern about its effects on NK cell compartment. After 3 weeks, NK cells returned and by 6 weeks had normalized in number (Fig. 1a and c), and NK cell cytotoxicity against K562 target cells was normal based on lytic units (Fig. 1b). Phenotyping of the patient’s NK cells after stopping azathioprine revealed a relative decrease in the percentage of mature NK cells, defined as CD56CD16 cells expressing CD57, while there was a corresponding increased percentage of immature CD56CD57NKG2A cells compared to a healthy agematched control (Fig. 1d and e). The CD56 NK cells had high expression of NKG2A, a receptor typically associated with the less mature CD56 subset, but did express KIR (not shown) * Megan A. Cooper Cooper_m@wustl.edu