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2.
Artigo em Inglês | MEDLINE | ID: mdl-31600547

RESUMO

BACKGROUND: Patients with signal transducer and activator of transcription 5b (STAT5b) deficiency have impairment in T-cell homeostasis and natural killer (NK) cells which leads to autoimmunity, recurrent infections, and combined immune deficiency. OBJECTIVE: In this study we characterized the NK cell defect in STAT5b-deficient human NK cells, as well as Stat5b-/- mice. METHODS: We used multiparametric flow cytometry, functional NK cell assays, microscopy, and a Stat5b-/- mouse model to elucidate the effect of impaired and/or absent STAT5b on NK cell development and function. RESULTS: This alteration generated a nonfunctional CD56bright NK cell subset characterized by low cytokine production. The CD56dim NK cell subset had decreased expression of perforin and CD16 and a greater frequency of cells expressing markers of immature NK cells. We observed low NK cell numbers and impaired NK cell maturation, suggesting that STAT5b is involved in terminal NK cell maturation in Stat5b-/- mice. Furthermore, human STAT5b-deficient NK cells had low cytolytic capacity, and fixed-cell microscopy showed poor convergence of lytic granules. This was accompanied by decreased expression of costimulatory and activating receptors. Interestingly, granule convergence and cytolytic function were restored after IL-2 stimulation. CONCLUSIONS: Our results show that in addition to the impaired terminal maturation of NK cells, human STAT5b mutation leads to impairments in early activation events in NK cell lytic synapse formation. Our data provide further insight into NK cell defects caused by STAT5b deficiency.

4.
Nat Commun ; 10(1): 3106, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308374

RESUMO

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.

5.
J Allergy Clin Immunol ; 144(1): 294-303.e13, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30735686

RESUMO

BACKGROUND: Chronic HIV infection is known to trigger a population redistribution and alteration in the functional capacity of natural killer (NK) cells. Because of improved antiretroviral treatments, there are rising numbers of adolescents and young adults worldwide who are living with HIV infection since birth. OBJECTIVE: We sought to determine how NK-cell phenotypic and functional subsets are altered in treated pediatric patients. METHODS: NK cells were contrasted among 29 HIV-unexposed and uninfected controls (5-19 years), 23 HIV-exposed but uninfected patients (3-19 years), and 25 HIV-infected patients (3-19 years) using multiparametric flow cytometry. RESULTS: Although most NK-cell markers did not differ, activating receptors such as NKp46, DNAX accessory molecule-1, and NKG2C and stimulatory receptors such as CD2 and CD11c were expressed by a higher frequency of NK cells in HIV-infected patients than in controls. Interestingly, there were less differences between HIV-infected and HIV-exposed but uninfected children. There was an inverse relationship between CD4/CD8 T-cell ratio (as a marker of disease progression) and CD11c and NKG2C frequency and CD69 upregulation on stimulation among HIV-infected patients. CONCLUSIONS: A chronic NK-cell activation phenotype persists in HIV-infected children receiving antiretroviral therapy and is associated with declining CD4/CD8 T-cell ratios. A lower CD4/CD8 T-cell ratio was associated with higher baseline granzyme B (P = .0068; R2 = 0.29) and degranulation potential (P = .022; R2 = 0.22) in stimulated NK cells. Thus, NK cells in HIV-infected children receiving treatment have reduced functional potential and an activated phenotype that distinguishes them from uninfected children.

7.
Leuk Lymphoma ; 60(8): 2025-2033, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30648453

RESUMO

Germline mutations in GATA2 are associated with a complex immunodeficiency and cancer predisposition syndrome. Somatic GATA2mut in myeloid malignancies may impart a similar phenotype. We reviewed adult patients with a diagnosis of GATA2mut hematological malignancy who were referred to our HHMC for genetic testing, and identified to have somatic GATA2mut. Nine patients with a median age of 63 years were included. Six patients (66.7%) were males. Atypical CML and acute myeloid leukemia were the most common initial presentation. The median overall VAF was 47.14%. Monocytopenia was pronounced when the GATA2mut involved the C-terminal ZFD. GATA2 N-terminal ZFD mutations tend to be co-mutated with biCEBPAmut. Unlike germline GATA2 mutations, monocytopenia associated with somatic GATA2 mutations often resolved at remission. We concluded that similar to germline GATA2 mutations, a subset of somatic GATA2 mutations can impart a germline phenotype.

8.
Immunol Rev ; 287(1): 202-225, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30565241

RESUMO

Human NK cells are innate immune effectors that play a critical roles in the control of viral infection and malignancy. The importance of their homeostasis and function can be demonstrated by the study of patients with primary immunodeficiencies (PIDs), which are part of the family of diseases known as inborn defects of immunity. While NK cells are affected in many PIDs in ways that may contribute to a patient's clinical phenotype, a small number of PIDs have an NK cell abnormality as their major immunological defect. These PIDs can be collectively referred to as NK cell deficiency (NKD) disorders and include effects upon NK cell numbers, subsets, and/or functions. The clinical impact of NKD can be severe including fatal viral infection, with particular susceptibility to herpesviral infections, such as cytomegalovirus, varicella zoster virus, and Epstein-Barr virus. While NKD is rare, studies of these diseases are important for defining specific requirements for human NK cell development and homeostasis. New themes in NK cell biology are emerging through the study of both known and novel NKD, particularly those affecting cell cycle and DNA damage repair, as well as broader PIDs having substantive impact upon NK cells. In addition, the discovery of NKD that affects other innate lymphoid cell (ILC) subsets opens new doors for better understanding the relationship between conventional NK cells and other ILC subsets. Here, we describe the biology underlying human NKD, particularly in the context of new insights into innate immune cell function, including a discussion of recently described NKD with accompanying effects on ILC subsets. Given the impact of these disorders upon human immunity with a common focus upon NK cells, the unifying message of a critical role for NK cells in human host defense singularly emerges.

10.
Immunity ; 49(3): 464-476.e4, 2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-30193847

RESUMO

According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34-CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34-CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.

11.
Mol Immunol ; 2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-30054012

RESUMO

Natural killer (NK) cell lines, including YTS, NK92, NK3.3, and NKL, represent excellent models for the study of human natural killer cells. While phenotypic and functional differences between these cell lines have been reported, a multi-parametric study, encompassing genomic, phenotypic, and functional assays, has not been performed. Here, using a combination of techniques including microarray and copy number analyses, flow cytometry, and functional assays, we provide in-depth genetic, functional, and phenotypic comparison of YTS, NK92, NK3.3, and NKL cell lines. Specifically, we found that while the cell lines shared similarities in enrichment of growth and survival pathways, they had differential expression of 557 genes, including genes related to NK cell development, survival, and function. In addition, we provide genetic and phenotypic analyses that demonstrate distinct developmental origins of NK92, YTS, and NKL cell lines. Specifically, NK92 has a phenotype associated with the CD56bright NK cell subset, while both YTS and NKL appear more CD56dim-like. Finally, by classifying cell lines based on their lytic potential, we identified genes differentially expressed between NK cell lines with high and low lytic function. Taken together, these data provide the first comprehensive genetic, phenotypic, and functional analyses of these commonly used NK cell lines and provides deeper understanding into their origins and function. This will ultimately improve their use as models for human NK cell biology.

14.
Am J Hum Genet ; 102(6): 1126-1142, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29805043

RESUMO

The proteasome processes proteins to facilitate immune recognition and host defense. When inherently defective, it can lead to aberrant immunity resulting in a dysregulated response that can cause autoimmunity and/or autoinflammation. Biallelic or digenic loss-of-function variants in some of the proteasome subunits have been described as causing a primary immunodeficiency disease that manifests as a severe dysregulatory syndrome: chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE). Proteasome maturation protein (POMP) is a chaperone for proteasome assembly and is critical for the incorporation of catalytic subunits into the proteasome. Here, we characterize and describe POMP-related autoinflammation and immune dysregulation disease (PRAID) discovered in two unrelated individuals with a unique constellation of early-onset combined immunodeficiency, inflammatory neutrophilic dermatosis, and autoimmunity. We also begin to delineate a complex genetic mechanism whereby de novo heterozygous frameshift variants in the penultimate exon of POMP escape nonsense-mediated mRNA decay (NMD) and result in a truncated protein that perturbs proteasome assembly by a dominant-negative mechanism. To our knowledge, this mechanism has not been reported in any primary immunodeficiencies, autoinflammatory syndromes, or autoimmune diseases. Here, we define a unique hypo- and hyper-immune phenotype and report an immune dysregulation syndrome caused by frameshift mutations that escape NMD.

15.
J Immunol ; 201(1): 124-133, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29752313

RESUMO

Epigenetic mechanisms, such as DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth coinfections, we evaluated the longitudinal effect of ascariasis and schistosomiasis infection on CD4+ T cell DNA methylation and the downstream tuberculosis (TB)-specific and bacillus Calmette-Guérin-induced immune phenotype. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared with age-matched uninfected controls, children with active Schistosoma haematobium and Ascaris lumbricoides infection had 751 differentially DNA-methylated genes, with 72% hypermethylated. Gene ontology pathway analysis identified inhibition of IFN-γ signaling, cellular proliferation, and the Th1 pathway. Targeted real-time quantitative PCR after methyl-specific endonuclease digestion confirmed DNA hypermethylation of the transcription factors BATF3, ID2, STAT5A, IRF5, PPARg, RUNX2, IRF4, and NFATC1 and cytokines or cytokine receptors IFNGR1, TNFS11, RELT (TNF receptor), IL12RB2, and IL12B (p < 0.001; Sidak-Bonferroni). Functional blockage of the IFN-γ signaling pathway was confirmed, with helminth-infected individuals having decreased upregulation of IFN-γ-inducible genes (Mann-Whitney p < 0.05). Hypomethylation of the IL-4 pathway and DNA hypermethylation of the Th1 pathway was confirmed by Ag-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-γ and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed-rank p < 0.05). In S. haematobium-infected individuals, these DNA methylation and immune phenotypic changes persisted at least 6 mo after successful deworming. This work demonstrates that helminth infection induces DNA methylation and immune perturbations that inhibit TB-specific immune control and that the duration of these changes are helminth specific.

16.
Front Immunol ; 9: 567, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29666621

RESUMO

Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a primary immunodeficiency caused by mutations in the autoimmune regulator gene (AIRE). Patients with AIRE mutations are susceptible to Candida albicans infection and present with autoimmune disorders. We previously demonstrated that cytoplasmic AIRE regulates the Syk-dependent Dectin-1 pathway. In this study, we further evaluated direct contact with fungal elements, synapse formation, and the response of macrophage-like THP-1 cells to C. albicans hyphae to determine the role of AIRE upon Dectin receptors function and signaling. We examined the fungal synapse (FS) formation in wild-type and AIRE-knockdown THP-1 cells differentiated to macrophages, as well as monocyte-derived macrophages from APECED patients. We evaluated Dectin-2 receptor signaling, phagocytosis, and cytokine secretion upon hyphal stimulation. AIRE co-localized with Dectin-2 and Syk at the FS upon hyphal stimulation of macrophage-like THP-1 cells. AIRE-knockdown macrophage-like THP-1 cells exhibited less Dectin-1 and Dectin-2 receptors accumulation, decreased signaling pathway activity at the FS, lower C. albicans phagocytosis, and less lysosome formation. Furthermore, IL-1ß, IL-6, or TNF-α secretion by AIRE-knockdown macrophage-like THP-1 cells and AIRE-deficient patient macrophages was decreased compared to control cells. Our results suggest that AIRE modulates the FS formation and hyphal recognition and help to orchestrate an effective immune response against C. albicans.

17.
Blood ; 132(1): 89-100, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29632024

RESUMO

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.

18.
Front Immunol ; 9: 445, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29563913

RESUMO

Human natural killer (NK) cells play a critical role in the control of viral infections and malignancy. Their importance in human health and disease is illustrated by severe viral infections in patients with primary immunodeficiencies that affect NK cell function and/or development. The recent identification of patients with phosphoinositide-3-kinase (PI3K)-signaling pathway mutations that can cause primary immunodeficiency provides valuable insight into the role that PI3K signaling plays in human NK cell maturation and lytic function. There is a rich literature that demonstrates a requirement for PI3K in multiple key aspects of NK cell biology, including development/maturation, homing, priming, and function. Here, I briefly review these previous studies and place them in context with recent findings from the study of primary immunodeficiency patients, particularly those with hyperactivating mutations in PI3Kδ signaling.

19.
Curr Biol ; 28(4): 489-502.e9, 2018 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-29398219

RESUMO

Natural killer (NK) cells are innate immune effectors that lyse virally infected and tumorigenic cells through the formation of an immunological synapse. Actin remodeling at the lytic immunological synapse is a critical requirement for multiple facets of cytotoxic function. Activating receptor and integrin signaling leads to the regulated turnover and remodeling of actin, which is required for adhesion, sustained receptor signaling, and ultimately exocytosis. NK cells undergo lytic granule exocytosis in hypodense regions of a pervasive actin network. Although these requirements have been well demonstrated, neither the dynamic regulation of synaptic actin nor its specific function, however, has been determined at a nanoscale level. Here, live-cell super-resolution microscopy demonstrates nanoscale filamentous actin dynamism in NK cell lytic granule secretion. Following cell spreading, the overall content of the branched actin network at an immune synapse is stable over time and contains branched actin fibers and discrete actin foci. Similar actin architecture is generated in cytolytic T cells, although the timescale differs from that of NK cells. Individual filament displacement leads to stochastic clearance formation and disappearance, which are independent of lytic granule positioning. Actin dynamism is dependent upon branched network formation mediated by Arp2/3 and contractility generated by myosin IIA. Importantly, the use of small-molecule inhibitors demonstrates that actin dynamism is ultimately needed for granule secretion. Thus, we describe a requirement for nanoscale actin fiber rearrangement in generating the complex actin architecture that enables lytic granule secretion.

20.
J Allergy Clin Immunol ; 142(2): 605-617.e7, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29330011

RESUMO

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.

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