Monogenic early-onset lymphoproliferation and autoimmunity: Natural history of STAT3 gain-of-function syndrome.

BACKGROUND: In 2014, germline signal transducer and activator of transcription (STAT) 3 gain-of-function (GOF) mutations were first described to cause a novel multisystem disease of early-onset lymphoproliferation and autoimmunity. OBJECTIVE: This pivotal cohort study defines the scope, natural history, treatment, and overall survival of a large global cohort of patients with pathogenic STAT3 GOF variants. METHODS: We identified 191 patients from 33 countries with 72 unique mutations. Inclusion criteria included symptoms of immune dysregulation and a biochemically confirmed germline heterozygous GOF variant in STAT3. RESULTS: Overall survival was 88%, median age at onset of symptoms was 2.3 years, and median age at diagnosis was 12 years. Immune dysregulatory features were present in all patients: lymphoproliferation was the most common manifestation (73%); increased frequencies of double-negative (CD4-CD8-) T cells were found in 83% of patients tested. Autoimmune cytopenias were the second most common clinical manifestation (67%), followed by growth delay, enteropathy, skin disease, pulmonary disease, endocrinopathy, arthritis, autoimmune hepatitis, neurologic disease, vasculopathy, renal disease, and malignancy. Infections were reported in 72% of the cohort. A cellular and humoral immunodeficiency was observed in 37% and 51% of patients, respectively. Clinical symptoms dramatically improved in patients treated with JAK inhibitors, while a variety of other immunomodulatory treatment modalities were less efficacious. Thus far, 23 patients have undergone bone marrow transplantation, with a 62% survival rate. CONCLUSION: STAT3 GOF patients present with a wide array of immune-mediated disease including lymphoproliferation, autoimmune cytopenias, and multisystem autoimmunity. Patient care tends to be siloed, without a clear treatment strategy. Thus, early identification and prompt treatment implementation are lifesaving for STAT3 GOF syndrome.

STAT5b: A master regulator of key biological pathways.

The Signal Transducer and Activator of Transcription (STAT)-5 proteins are required in immune regulation and homeostasis and play a crucial role in the development and function of several hematopoietic cells. STAT5b activation is involved in the expression of genes that participate in cell development, proliferation, and survival. STAT5a and STAT5b are paralogs and only human mutations in STAT5B have been identified leading to immune dysregulation and hematopoietic malignant transformation. The inactivating STAT5B mutations cause impaired post-natal growth, recurrent infections and immune dysregulation, whereas gain of function somatic mutations cause dysregulated allergic inflammation. These mutations are rare, and they are associated with a wide spectrum of clinical manifestations which provide a disease model elucidating the biological mechanism of STAT5 by studying the consequences of perturbations in STAT5 activity. Further, the use of Jak inhibitors as therapy for a variety of autoimmune and malignant disorders has increased substantially heading relevant lessons for the consequences of Jak/STAT immunomodulation from the human model. This review summarizes the biology of the STAT5 proteins, human disease associate with molecular defects in STAT5b, and the connection between aberrant activation of STAT5b and the development of certain cancers.

Phenotypic and Functional Plasticity of CXCR6+ Peripheral Blood NK Cells.

Human NK cells are comprised of phenotypic subsets, whose potentially unique functions remain largely unexplored. C-X-C-motif-chemokine-receptor-6 (CXCR6) + NK cells have been identified as phenotypically immature tissue-resident NK cells in mice and humans. A small fraction of peripheral blood (PB)-NK cells also expresses CXCR6. However, prior reports about their phenotypic and functional plasticity are conflicting. In this study, we isolated, expanded, and phenotypically and functionally evaluated CXCR6+ and CXCR6- PB-NK cells, and contrasted results to bulk liver and spleen NK cells. We found that CXCR6+ and CXCR6- PB-NK cells preserved their distinct phenotypic profiles throughout 14 days of in vitro expansion ("day 14"), after which phenotypically immature CXCR6+ PB-NK cells became functionally equivalent to CXCR6- PB-NK cells. Despite a consistent reduction in CD16 expression and enhanced expression of the transcription factor Eomesodermin (Eomes), day 14 CXCR6+ PB-NK cells had superior antibody-dependent cellular cytotoxicity (ADCC) compared to CXCR6- PB-NK cells. Further, bulk liver NK cells responded to IL-15, but not IL-2 stimulation, with STAT-5 phosphorylation. In contrast, bulk splenic and PB-NK cells robustly responded to both cytokines. Our findings may allow for the selection of superior NK cell subsets for infusion products increasingly used to treat human diseases.

HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease.

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)-dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.

Novel Heterozygous Mutation in NFKB2 Is Associated With Early Onset CVID and a Functional Defect in NK Cells Complicated by Disseminated CMV Infection and Severe Nephrotic Syndrome.

Nuclear factor kappa-B subunit 2 (NF-κB2/p100/p52), encoded by NFKB2 (MIM: 164012) belongs to the NF-κB family of transcription factors that play a critical role in inflammation, immunity, cell proliferation, differentiation and survival. Heterozygous C-terminal mutations in NFKB2 have been associated with early-onset common variable immunodeficiency (CVID), central adrenal insufficiency and ectodermal dysplasia. Only two previously reported cases have documented decreased natural killer (NK) cell cytotoxicity, and little is known about the role of NF-κB2 in NK cell maturation and function. Here we report a 13-year-old female that presented at 6 years of age with a history of early onset recurrent sinopulmonary infections, progressive hair loss, and hypogamaglobulinemia consistent with a clinical diagnosis of CVID. At 9 years of age she had cytomegalovirus (CMV) pneumonia that responded to ganciclovir treatment. Functional NK cell testing demonstrated decreased NK cell cytotoxicity despite normal NK cell numbers, consistent with a greater susceptibility to systemic CMV infection. Research exome sequencing (ES) was performed and revealed a novel de novo heterozygous nonsense mutation in NFKB2 (c.2611C>T, p.Gln871*) that was not carried by either of her parents. The variant was Sanger sequenced and confirmed to be de novo in the patient. At age 12, she presented with a reactivation of the systemic CMV infection that was associated with severe and progressive nephrotic syndrome with histologic evidence of pedicellar effacement and negative immunofluorescence. To our knowledge, this is the third NF-κB2 deficient patient in which an abnormal NK cell function has been observed, suggesting a role for non-canonical NF-κB2 signaling in NK cell cytotoxicity. NK cell function should be assessed in patients with mutations in the non-canonical NF-κB pathway to explore the risk for systemic viral infections that may lead to severe complications and impact patient survival. Similarly NF-κB2 should be considered in patients with combined immunodeficiency who have aberrant NK cell function. Further studies are needed to characterize the role of NF-κB2 in NK cell cytotoxic function.

Gain-of-Function STAT1 Mutation With Familial Lymphadenopathy and Hodgkin Lymphoma.

In this report, we describe a novel T437N STAT1 mutation found in a mother and 3 of her 4 children which we demonstrate yields gain-of-function. All of the four patients with the T437N STAT1 mutation experienced lymphadenopathy. However, two of the children developed Nodular Lymphocyte Predominant Hodgkin Lymphoma (NHLPL) and have responded to chemotherapeutic regimens. The fourth sibling had neither the STAT1 variant nor lymphadenopathy or malignancy. To our knowledge this is the first description of a potential association between STAT1 GOF mutations and lymphoma development.

JAK/STAT proteins and their biological impact on NK cell development and function.

NK cells are important early effectors in the innate immune response to a variety of viral infections and for elimination of tumor cells. The JAK/STAT signaling cascade is critical for NK cell development, maturation, survival, and proliferation, therefore, it is important to understand the role of this pathway in NK cell biology. Many cytokines can activate multiple JAK/STAT protein family members, creating a severe phenotype when mutations impair their function or expression. Here we discuss the impact of defective JAK/STAT signaling pathways on NK cell development, activation and cytotoxicity.

The Impact of Immunodeficiency on NK Cell Maturation and Function.

PURPOSE OF REVIEW: Natural killer cells are innate lymphoid cells (ILCs) that play critical roles in human host defense and are especially useful in combating viral pathogens and malignancy. RECENT FINDINGS: The NK cell deficiency (NKD) is particularly underscored in patients with a congenital immunodeficiency in which NK cell development or function is affected. The classical NK cell deficiency (cNKD) is a result of absent or a profound decrease in the number of circulating NK cells. In contrast, functional NKD (fNKD) is characterized by abnormal NK cell function but with normal number of NK cells. The combined immune deficiencies with significant impact on NK cells are not considered classical or functional NK cell deficiencies. In these disorders, the impairment of NK cells represents an important aspect of the overall immunodeficiency. In turn, this leads to improved insights on the NK cell development and function. Here, we detail the NK cell biology based upon recent natural killer cell defects described in combined immune deficiencies.

Mutations in PI3K110δ cause impaired natural killer cell function partially rescued by rapamycin treatment.

BACKGROUND: Heterozygous gain-of-function mutations in PI3K110δ lead to lymphadenopathy, lymphoid hyperplasia, EBV and cytomegalovirus viremia, and sinopulmonary infections. OBJECTIVE: The known role of natural killer (NK) cell function in the control of EBV and cytomegalovirus prompted us to investigate the functional and phenotypic effects of PI3K110δ mutations on NK cell subsets and cytotoxic function. METHODS: Mutations in patients were identified by using whole-exome or targeted sequencing. We performed NK cell phenotyping and functional analysis of patients' cells using flow cytometry, standard Cr51 cytotoxicity assays, and quantitative confocal microscopy. RESULTS: PI3K110δ mutations led to an altered NK cell developmental phenotype and cytotoxic dysfunction. Impaired NK cell cytotoxicity was due to decreased conjugate formation with susceptible target cells and abrogated activation of cell machinery required for target cell killing. These defects were restored partially after initiation of treatment with rapamycin in 3 patients. CONCLUSION: We describe novel NK cell functional deficiency caused by PI3K110δ mutation, which is a likely contributor to the severe viremia observed in these patients. Rapamycin treatment partially restores NK cell function, providing a further rationale for its use in patients with this disease.

Impaired selective cytokine production by CD4(+) T cells in Common Variable Immunodeficiency associated with the absence of memory B cells.

Common Variable Immunodeficiency (CVID) is a primary immunodeficiency characterized by B cell dysfunction and decreased serum immunoglobulin. CVID patients are classified by the absence or presence of memory B cells. In addition, T cell defects have been demonstrated in only a proportion of CVID patients. The aim of this study was to evaluate the function of CD4(+) T cells from CVID patients and its association with memory B cells. Patients were classified according to their Freiburg groups: group Ia and Ib, with decreased switched memory B cells (<0.4 of PBL), and group II, with normal B cell subsets. Their T cell function was evaluated after stimulation. We observed normal and even increased CD4(+) T cell proliferation in group Ia (p=0.0277). The proliferation positively correlated with the clinical severity score (r=0.4796). We observed lower levels of IL-17A and IL-10 in group Ia (p=0.0177, 0.0109) and Ib (p=0.0009, 0.0084) patients. Group Ib patients also had low levels of IL-13 and IL-9 (p=0.0169, 0.010). Group II patients had similar cytokine production to that of the controls. BAFFR expression was reduced in groups Ia (p=0.0001) and Ib (p=0.0002) and showed an inverse correlation with the severity score (p=0.0262; r=0.5371). ICOS expression was reduced in group Ia (p=0.0364), and PD-1 was increased in group Ib (p=0.0432) patients. This study shows a selective impairment in cytokine production in group Ia patients, which was more extensive than in group Ib patients. The impairment was associated with BAFFR expression in B cells, with ICOS and PD-1 in T cells and, remarkably, with the absence of memory B cells and with the disease severity. Our results suggest that the evaluation of cytokine expression by T cells in combination with the study of B cell memory could be important for understand the pathogenesis of CVID patients.

A novel CD40LG deletion causes the hyper-IgM syndrome with normal CD40L expression in a 6-month-old child.

The X-linked hyper-IgM syndrome (XHIGM) is the most common form of HIGM. Patients are clinically diagnosed on the basis of recurrent sinopulmonary infections, accompanied with low levels of IgG and IgA, normal to elevated levels of IgM, and the presence of peripheral B cells. Here, we have reported a novel deletion of four nucleotides in CD40LG exon 3, c.375_378delCAAA, which led to a frameshift mutation with a premature stop codon, p.Asn101*126. The deletion resulted in a truncated protein, in which majority of the extracellular domain was lost. However, detection of surface CD40L was still possible as the intracellular, transmembrane, and part of the extracellular domains were not affected. This indicated that this mutation did not affect protein stability and that immunodetection of CD40L expression is not enough for the diagnosis of XHIGM. Our study strongly suggests that genetic diagnosis for XHIGM should always be performed when clinical data support this diagnosis and CD40L protein is present.

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.

First report of the Hyper-IgM syndrome Registry of the Latin American Society for Immunodeficiencies: novel mutations, unique infections, and outcomes.

Hyper-IgM (HIGM) syndrome is a heterogeneous group of disorders characterized by normal or elevated serum IgM levels associated with absent or decreased IgG, IgA and IgE. Here we summarize data from the HIGM syndrome Registry of the Latin American Society for Immunodeficiencies (LASID). Of the 58 patients from 51 families reported to the registry with the clinical phenotype of HIGM syndrome, molecular defects were identified in 37 patients thus far. We retrospectively analyzed the clinical, immunological and molecular data from these 37 patients. CD40 ligand (CD40L) deficiency was found in 35 patients from 25 families and activation-induced cytidine deaminase (AID) deficiency in 2 unrelated patients. Five previously unreported mutations were identified in the CD40L gene (CD40LG). Respiratory tract infections, mainly pneumonia, were the most frequent clinical manifestation. Previously undescribed fungal and opportunistic infections were observed in CD40L-deficient patients but not in the two patients with AID deficiency. These include the first cases of pneumonia caused by Mycoplasma pneumoniae, Serratia marcescens or Aspergillus sp. and diarrhea caused by Microsporidium sp. or Isospora belli. Except for four CD40L-deficient patients who died from complications of presumptive central nervous system infections or sepsis, all patients reported in this study are alive. Four CD40L-deficient patients underwent successful bone marrow transplantation. This report characterizes the clinical and genetic spectrum of HIGM syndrome in Latin America and expands the understanding of the genotype and phenotype of this syndrome in tropical areas.