RESUMEN
High-dimensional cytometry is a powerful technique for deciphering the immunopathological factors common to multiple individuals. However, rational comparisons of multiple batches of experiments performed on different occasions or at different sites are challenging because of batch effects. In this study, we describe the integration of multibatch cytometry datasets (iMUBAC), a flexible, scalable, and robust computational framework for unsupervised cell-type identification across multiple batches of high-dimensional cytometry datasets, even without technical replicates. After overlaying cells from multiple healthy controls across batches, iMUBAC learns batch-specific cell-type classification boundaries and identifies aberrant immunophenotypes in patient samples from multiple batches in a unified manner. We illustrate unbiased and streamlined immunophenotyping using both public and in-house mass cytometry and spectral flow cytometry datasets. The method is available as the R package iMUBAC (https://github.com/casanova-lab/iMUBAC).
Asunto(s)
Inmunofenotipificación/métodos , Leucocitos Mononucleares/fisiología , Programas Informáticos , Algoritmos , Biología Computacional , Citometría de Flujo , HumanosRESUMEN
BACKGROUND: Lymphocyte differentiation is regulated by coordinated actions of cytokines and signaling pathways. IL-21 activates STAT1, STAT3, and STAT5 and is fundamental for the differentiation of human B cells into memory cells and antibody-secreting cells. While STAT1 is largely nonessential and STAT3 is critical for this process, the role of STAT5 is unknown. OBJECTIVES: This study sought to delineate unique roles of STAT5 in activation and differentiation of human naive and memory B cells. METHODS: STAT activation was assessed by phospho-flow cytometry cell sorting. Differential gene expression was determined by RNA-sequencing and quantitative PCR. The requirement for STAT5B in B-cell and CD4+ T-cell differentiation was assessed using CRISPR-mediated STAT5B deletion from B-cell lines and investigating primary lymphocytes from individuals with germline STAT5B mutations. RESULTS: IL-21 activated STAT5 and strongly induced SOCS3 in human naive, but not memory, B cells. Deletion of STAT5B in B-cell lines diminished IL-21-mediated SOCS3 induction. PBMCs from STAT5B-null individuals contained expanded populations of immunoglobulin class-switched B cells, CD21loTbet+ B cells, and follicular T helper cells. IL-21 induced greater differentiation of STAT5B-deficient B cells into plasmablasts in vitro than B cells from healthy donors, correlating with higher expression levels of transcription factors promoting plasma cell formation. CONCLUSIONS: These findings reveal novel roles for STAT5B in regulating IL-21-induced human B-cell differentiation. This is achieved by inducing SOCS3 to attenuate IL-21 signaling, and BCL6 to repress class switching and plasma cell generation. Thus, STAT5B is critical for restraining IL-21-mediated B-cell differentiation. These findings provide insights into mechanisms underpinning B-cell responses during primary and subsequent antigen encounter and explain autoimmunity and dysfunctional humoral immunity in STAT5B deficiency.
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Citocinas , Factor de Transcripción STAT5 , Diferenciación Celular , Citocinas/metabolismo , Homeostasis , Humanos , Isotipos de Inmunoglobulinas/metabolismo , ARN , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismoRESUMEN
Inborn errors of the IL-17-mediated signaling have been associated with chronic mucocutaneous candidiasis (CMC). We describe a patient with CMC, atopic dermatitis, enamel dysplasia, and recurrent parotitis harboring a novel compound heterozygous mutation of TRAF3IP2, leading to autosomal recessive ACT1 deficiency and deficient IL-17 signaling.
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Proteínas Adaptadoras Transductoras de Señales/genética , Candidiasis Mucocutánea Crónica/genética , Mutación/genética , Niño , Predisposición Genética a la Enfermedad/genética , Humanos , Interleucina-17/genética , MasculinoRESUMEN
BACKGROUND: Inherited chronic mucocutaneous candidiasis (CMC) is often caused by inborn errors of immunity, impairing the response to, or the production of IL-17A and IL-17F. About half of the cases carry STAT1 gain-of-function (GOF) mutations. Only few patients have been reported with mutations of TRAF3IP2, a gene encoding the adaptor ACT1 essential for IL-17 receptor(R) signaling. We investigated a 10-year-old girl with CMC, carrying a heterozygous variant of STAT1 and compound heterozygous variants of TRAF3IP2. METHODS: By flow cytometry, STAT1 levels and phosphorylation (CD14+) as well as IL-17A, IL-22, IFN-γ, and IL-4 production (memory CD4+ T cells) were determined. ACT1 expression and binding to IL-17RA were assessed by Western blot and co-immunoprecipitation in HEK-293T cells transfected with plasmids encoding wild-type or mutant HA-tagged ACT1 and Flag-IL-17RA. We evaluated IL-17A responses by measuring luciferase induction under a NF-κB-driven reporter system in HEK-293T cells and Gro-α secretion in fibroblasts. RESULTS: A STAT1 variant (c.1363G>A/p.V455I) was identified by next-generation sequencing and classified as likely non-pathogenic as functional testing revealed normal STAT1 expression and phosphorylation upon IFN-γ. We also found compound heterozygous variants (c.1325A>G/p.D451G and c.1335delA/p.K454fs11*) of TRAF3IP2. By overexpression, despite normal protein expression, and impaired (K454fs11*) or normal (D451G) interaction with IL-17RA, both mutant alleles resulted in impaired NF-κB activation. Patient's fibroblasts displayed abolished GRO-α secretion upon IL-17A stimulation. Finally, ex vivo CD4+ T cells showed increased IL-17A, IL-22, and IL-4 and normal low IFN-γ expression upon stimulation. CONCLUSION: We identify novel compound heterozygous variants of TRAFP3IP2 causing autosomal recessive ACT1 deficiency in a child with CMC and provide a review of the current literature.
Asunto(s)
Candidiasis Mucocutánea Crónica , Proteínas Adaptadoras Transductoras de Señales , Alelos , Candidiasis Mucocutánea Crónica/genética , Niño , Femenino , Humanos , Mutación , Fosforilación , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Gain-of-function (GOF) mutations in PIK3CD cause a primary immunodeficiency characterized by recurrent respiratory tract infections, susceptibility to herpesvirus infections, and impaired antibody responses. Previous work revealed defects in CD8+ T and B cells that contribute to this clinical phenotype, but less is understood about the role of CD4+ T cells in disease pathogenesis. OBJECTIVE: We sought to dissect the effects of increased phosphoinositide 3-kinase (PI3K) signaling on CD4+ T-cell function. METHODS: We performed detailed ex vivo, in vivo, and in vitro phenotypic and functional analyses of patients' CD4+ T cells and a novel murine disease model caused by overactive PI3K signaling. RESULTS: PI3K overactivation caused substantial increases in numbers of memory and follicular helper T (TFH) cells and dramatic changes in cytokine production in both patients and mice. Furthermore, PIK3CD GOF human TFH cells had dysregulated phenotype and function characterized by increased programmed cell death protein 1, CXCR3, and IFN-γ expression, the phenotype of a TFH cell subset with impaired B-helper function. This was confirmed in vivo in which Pik3cd GOF CD4+ T cells also acquired an aberrant TFH phenotype and provided poor help to support germinal center reactions and humoral immune responses by antigen-specific wild-type B cells. The increase in numbers of both memory and TFH cells was largely CD4+ T-cell extrinsic, whereas changes in cytokine production and TFH cell function were cell intrinsic. CONCLUSION: Our studies reveal that CD4+ T cells with overactive PI3K have aberrant activation and differentiation, thereby providing mechanistic insight into dysfunctional antibody responses in patients with PIK3CD GOF mutations.
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Linfocitos T CD4-Positivos , Diferenciación Celular , Fosfatidilinositol 3-Quinasas/genética , Animales , Linfocitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Mutación con Ganancia de Función , Humanos , Ratones , FenotipoRESUMEN
Cytokine-mediated intracellular signaling pathways are fundamental for the development, activation, and differentiation of lymphocytes. These distinct processes underlie protection against infectious diseases after natural infection with pathogens or immunization, thereby providing the host with long-lived immunological memory. In contrast, aberrant cytokine signaling can also result in conditions of immune dysregulation, such as early-onset autoimmunity. Thus, balanced signals provided by distinct cytokines, and delivered to specific cell subsets, are critical for immune homeostasis. The essential roles of cytokines in human immunity have been elegantly and repeatedly revealed by the discovery of individuals with mutations in cytokine ligands, receptors, and downstream transcription factors that cause primary immunodeficiency or autoimmune conditions. In this article, we review how the discovery and characterization of such individuals has identified nonredundant, and often highly specialized, functions of specific cytokines and immune cell subsets in human lymphocyte biology, host defense against infections, and immune regulation.
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Diferenciación Celular , Citocinas/metabolismo , Síndromes de Inmunodeficiencia/inmunología , Infecciones/inmunología , Linfocitos/fisiología , Animales , Citocinas/inmunología , Homeostasis , Humanos , Inmunidad Innata , Inmunomodulación , Activación de Linfocitos , Transducción de SeñalRESUMEN
Increasing evidence suggests that NK cells act to promote effective T cell-based antitumor responses. Using the B16-OVA melanoma model and an optimized Gram-positive bacteria-dendritic cell (DC) vaccination strategy, we determined that in vivo depletion of NK cells at time of tumor challenge abolished the benefit of DC immunotherapy. The contribution of NK cells to DC immunotherapy was dependent on tumor Ag presentation by DC, suggesting that NK cells act as helper cells to prime or reactivate tumor-specific T cells. The absence of NK cells at tumor challenge resulted in greater attenuation of tumor immunity than observed with selective depletion of either CD4 or CD8 T cell subsets. Although successful DC immunotherapy required IFN-γ, perforin expression was dispensable. Closer examination of the role of NK cells as helper cells in enhancing antitumor responses will reveal new strategies for clinical interventions using DC-based immunotherapy.
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Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Células Dendríticas/trasplante , Inmunidad Celular , Células Asesinas Naturales/inmunología , Neoplasias/terapia , Vacunación , Animales , Presentación de Antígeno/genética , Antígenos de Neoplasias/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Células Dendríticas/inmunología , Células Dendríticas/patología , Interferón gamma/genética , Interferón gamma/inmunología , Células Asesinas Naturales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/inmunologíaRESUMEN
BACKGROUND: Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. OBJECTIVE: We sought to determine the signaling pathways and cellular interactions required for the development and function of TFH cells in human subjects. METHODS: Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cTFH) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS, or BTK. RESULTS: Loss-of-function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS, or BTK reduced cTFH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cTFH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cTFH cell function in vitro and in vivo, as corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1, and IL12RB1 LOF mutations. CONCLUSION: Specific mutations affect the quantity and quality of cTFH cells, highlighting the need to assess TFH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain TFH cell-induced B-cell differentiation. These findings shed new light on TFH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.
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Síndromes de Inmunodeficiencia/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Agammaglobulinemia Tirosina Quinasa , Linfocitos B/inmunología , Ligando de CD40/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Células Cultivadas , Humanos , Quinasa I-kappa B/genética , Inmunidad Humoral/genética , Síndromes de Inmunodeficiencia/genética , Memoria Inmunológica , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Interferón gamma/genética , Interferón gamma/metabolismo , Activación de Linfocitos , Mutación/genética , Proteínas Tirosina Quinasas/genética , Receptores de Citocinas/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genética , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Inborn errors of IFN-γ immunity can underlie tuberculosis (TB). We report three patients from two kindreds without EBV viremia or disease but with severe TB and inherited complete ITK deficiency, a condition associated with severe EBV disease that renders immunological studies challenging. They have CD4+ αß T lymphocytopenia with a concomitant expansion of CD4-CD8- double-negative (DN) αß and Vδ2- γδ T lymphocytes, both displaying a unique CD38+CD45RA+T-bet+EOMES- phenotype. Itk-deficient mice recapitulated an expansion of the γδ T and DN αß T lymphocyte populations in the thymus and spleen, respectively. Moreover, the patients' T lymphocytes secrete small amounts of IFN-γ in response to TCR crosslinking, mitogens, or forced synapse formation with autologous B lymphocytes. Finally, the patients' total lymphocytes secrete small amounts of IFN-γ, and CD4+, CD8+, DN αß T, Vδ2+ γδ T, and MAIT cells display impaired IFN-γ production in response to BCG. Inherited ITK deficiency undermines the development and function of various IFN-γ-producing T cell subsets, thereby underlying TB.
Asunto(s)
Receptores de Antígenos de Linfocitos T gamma-delta , Tuberculosis , Animales , Humanos , Ratones , Interferón gamma , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Subgrupos de Linfocitos T , TimoRESUMEN
Patients with autosomal recessive (AR) IL-12p40 or IL-12Rß1 deficiency display Mendelian susceptibility to mycobacterial disease (MSMD) due to impaired IFN-γ production and, less commonly, chronic mucocutaneous candidiasis (CMC) due to impaired IL-17A/F production. We report six patients from four kindreds with AR IL-23R deficiency. These patients are homozygous for one of four different loss-of-function IL23R variants. All six patients have a history of MSMD, but only two suffered from CMC. We show that IL-23 induces IL-17A only in MAIT cells, possibly contributing to the incomplete penetrance of CMC in patients unresponsive to IL-23. By contrast, IL-23 is required for both baseline and Mycobacterium-inducible IFN-γ immunity in both Vδ2+ γδ T and MAIT cells, probably contributing to the higher penetrance of MSMD in these patients. Human IL-23 appears to contribute to IL-17A/F-dependent immunity to Candida in a single lymphocyte subset but is required for IFN-γ-dependent immunity to Mycobacterium in at least two lymphocyte subsets.
Asunto(s)
Interferón gamma , Interleucina-23 , Infecciones por Mycobacterium , Mycobacterium , Humanos , Predisposición Genética a la Enfermedad , Interleucina-17/genética , Interleucina-23/genética , Infecciones por Mycobacterium/inmunologíaAsunto(s)
Síndrome de Job/genética , Síndrome de Job/metabolismo , Mutación , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Sustitución de Aminoácidos , Genes Dominantes , Células HEK293 , Humanos , Síndrome de Job/inmunología , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Proteínas Mutantes/metabolismo , Fosforilación/genética , Multimerización de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT3/químicaRESUMEN
Most patients with autosomal dominant hyper-IgE syndrome (AD-HIES) carry rare heterozygous STAT3 variants. Only six of the 135 in-frame variants reported have been experimentally shown to be dominant negative (DN), and it has been recently suggested that eight out-of-frame variants operate by haploinsufficiency. We experimentally tested these 143 variants, 7 novel out-of-frame variants found in HIES patients, and other STAT3 variants from the general population. Strikingly, all 15 out-of-frame variants were DN via their encoded (1) truncated proteins, (2) neoproteins generated from a translation reinitiation codon, and (3) isoforms from alternative transcripts or a combination thereof. Moreover, 128 of the 135 in-frame variants (95%) were also DN. The patients carrying the seven non-DN STAT3 in-frame variants have not been studied for other genetic etiologies. Finally, none of the variants from the general population tested, including an out-of-frame variant, were DN. Overall, our findings show that heterozygous STAT3 variants, whether in or out of frame, underlie AD-HIES through negative dominance rather than haploinsufficiency.
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Genes Dominantes , Síndrome de Job/genética , Mutación/genética , Factor de Transcripción STAT3/genética , Adolescente , Adulto , Alelos , Empalme Alternativo/genética , Niño , Preescolar , Codón sin Sentido/genética , Evolución Molecular , Familia , Femenino , Mutación del Sistema de Lectura/genética , Genética de Población , Células HEK293 , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Linaje , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8+ T cells, memory CD4+ T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4+ T cells. In naive CD4+ T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient's naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.
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Genes rel , Enfermedades de Inmunodeficiencia Primaria/genética , Enfermedades de Inmunodeficiencia Primaria/inmunología , Proteínas Proto-Oncogénicas c-rel/deficiencia , Proteínas Proto-Oncogénicas c-rel/genética , Inmunidad Adaptativa/genética , Inmunidad Adaptativa/inmunología , Niño , Consanguinidad , Femenino , Trasplante de Células Madre Hematopoyéticas , Homocigoto , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Activación de Linfocitos , Linfocitos/clasificación , Linfocitos/inmunología , Mutación , Células Mieloides/inmunología , Enfermedades de Inmunodeficiencia Primaria/terapia , Isoformas de ProteínasRESUMEN
Autosomal dominant (AD) NFKB1 deficiency is thought to be the most common genetic etiology of common variable immunodeficiency (CVID). However, the causal link between NFKB1 variants and CVID has not been demonstrated experimentally and genetically, and there has been insufficient biochemical characterization and enrichment analysis. We show that the cotransfection of NFKB1-deficient HEK293T cells (lacking both p105 and its cleaved form p50) with a κB reporter, NFKB1/p105, and a homodimerization-defective RELA/p65 mutant results in p50:p65 heterodimer-dependent and p65:p65 homodimer-independent transcriptional activation. We found that 59 of the 90 variants in patients with CVID or related conditions were loss of function or hypomorphic. By contrast, 258 of 260 variants in the general population or patients with unrelated conditions were neutral. None of the deleterious variants displayed negative dominance. The enrichment in deleterious NFKB1 variants of patients with CVID was selective and highly significant (P = 2.78 × 10-15). NFKB1 variants disrupting NFKB1/p50 transcriptional activity thus underlie AD CVID by haploinsufficiency, whereas neutral variants in this assay should not be considered causal.
Asunto(s)
Inmunodeficiencia Variable Común/genética , Subunidad p50 de NF-kappa B/genética , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Células HEK293 , Haploinsuficiencia/genética , Humanos , FN-kappa B/genética , Fenotipo , Activación Transcripcional/genéticaRESUMEN
The pathophysiology of adverse events following programmed cell death protein 1 (PD-1) blockade, including tuberculosis (TB) and autoimmunity, remains poorly characterized. We studied a patient with inherited PD-1 deficiency and TB who died of pulmonary autoimmunity. The patient's leukocytes did not express PD-1 or respond to PD-1-mediated suppression. The patient's lymphocytes produced only small amounts of interferon (IFN)-γ upon mycobacterial stimuli, similarly to patients with inborn errors of IFN-γ production who are vulnerable to TB. This phenotype resulted from a combined depletion of Vδ2+ γδ T, mucosal-associated invariant T and CD56bright natural killer lymphocytes and dysfunction of other T lymphocyte subsets. Moreover, the patient displayed hepatosplenomegaly and an expansion of total, activated and RORγT+ CD4-CD8- double-negative αß T cells, similar to patients with STAT3 gain-of-function mutations who display lymphoproliferative autoimmunity. This phenotype resulted from excessive amounts of STAT3-activating cytokines interleukin (IL)-6 and IL-23 produced by activated T lymphocytes and monocytes, and the STAT3-dependent expression of RORγT by activated T lymphocytes. Our work highlights the indispensable role of human PD-1 in governing both antimycobacterial immunity and self-tolerance, while identifying potentially actionable molecular targets for the diagnostic and therapeutic management of TB and autoimmunity in patients on PD-1 blockade.
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Autoinmunidad/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Receptor de Muerte Celular Programada 1/genética , Factor de Transcripción STAT3/genética , Tuberculosis/inmunología , Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Antígeno CD56/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Niño , Humanos , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/efectos adversos , Interleucina-23/genética , Interleucina-6/genética , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/patología , Masculino , Mycobacterium tuberculosis/patogenicidad , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Neoplasias/mortalidad , Receptor de Muerte Celular Programada 1/deficiencia , Tuberculosis/genética , Tuberculosis/mortalidadRESUMEN
Autosomal inborn errors of type I IFN immunity and autoantibodies against these cytokines underlie at least 10% of critical COVID-19 pneumonia cases. We report very rare, biochemically deleterious X-linked TLR7 variants in 16 unrelated male individuals aged 7 to 71 years (mean: 36.7 years) from a cohort of 1,202 male patients aged 0.5 to 99 years (mean: 52.9 years) with unexplained critical COVID-19 pneumonia. None of the 331 asymptomatically or mildly infected male individuals aged 1.3 to 102 years (mean: 38.7 years) tested carry such TLR7 variants (p = 3.5 × 10-5). The phenotypes of five hemizygous relatives of index cases infected with SARS-CoV-2 include asymptomatic or mild infection (n=2, 5 and 38 years), or moderate (n=1, 5 years), severe (n=1, 27 years), or critical (n=1, 29 years) pneumonia. Two boys (aged 7 and 12 years) from a cohort of 262 male patients with severe COVID-19 pneumonia (mean: 51.0 years) are hemizygous for a deleterious TLR7 variant. The cumulative allele frequency for deleterious TLR7 variants in the male general population is < 6.5x10-4 We also show that blood B cell lines and myeloid cell subsets from the patients do not respond to TLR7 stimulation, a phenotype rescued by wild-type TLR7 The patients' blood plasmacytoid dendritic cells (pDCs) produce low levels of type I IFNs in response to SARS-CoV-2. Overall, X-linked recessive TLR7 deficiency is a highly penetrant genetic etiology of critical COVID-19 pneumonia, in about 1.8% of male patients below the age of 60 years. Human TLR7 and pDCs are essential for protective type I IFN immunity against SARS-CoV-2 in the respiratory tract.
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COVID-19/complicaciones , Enfermedades Genéticas Ligadas al Cromosoma X/complicaciones , Enfermedades del Sistema Inmune/complicaciones , Receptor Toll-Like 7/deficiencia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Niño , Preescolar , Humanos , Lactante , Masculino , Persona de Mediana Edad , Linaje , Penetrancia , Receptor Toll-Like 7/genética , Adulto JovenRESUMEN
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated downstream of many key cytokine receptors expressed by lymphocytes. As such, it plays a critical role in regulating B cells as well as CD4+ and CD8+ T cells. Patients with clinically significant immunodeficiency and immune dysregulation resulting from loss-of-function or gain-of-function mutations in STAT3 have been described. These individuals provide insight into the critical role of this transcription factor in the regulation of immune responses and the balance between effective immune protection and autoimmunity.
Asunto(s)
Linfocitos B/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Animales , Autoinmunidad/genética , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Mutación con Ganancia de Función , Humanos , Inmunomodulación/genética , Mutación con Pérdida de Función , Factor de Transcripción STAT3/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/inmunologíaRESUMEN
Heterozygosity for human signal transducer and activator of transcription 3 (STAT3) dominant-negative (DN) mutations underlies an autosomal dominant form of hyper-immunoglobulin E syndrome (HIES). We describe patients with an autosomal recessive form of HIES due to loss-of-function mutations of a previously uncharacterized gene, ZNF341 ZNF341 is a transcription factor that resides in the nucleus, where it binds a specific DNA motif present in various genes, including the STAT3 promoter. The patients' cells have low basal levels of STAT3 mRNA and protein. The autoinduction of STAT3 production, activation, and function by STAT3-activating cytokines is strongly impaired. Like patients with STAT3 DN mutations, ZNF341-deficient patients lack T helper 17 (TH17) cells, have an excess of TH2 cells, and have low memory B cells due to the tight dependence of STAT3 activity on ZNF341 in lymphocytes. Their milder extra-hematopoietic manifestations and stronger inflammatory responses reflect the lower ZNF341 dependence of STAT3 activity in other cell types. Human ZNF341 is essential for the STAT3 transcription-dependent autoinduction and sustained activity of STAT3.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Síndrome de Job/genética , Factor de Transcripción STAT3/genética , Factores de Transcripción/genética , Transcripción Genética/inmunología , Adolescente , Adulto , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Núcleo Celular/metabolismo , Consanguinidad , Citocinas/inmunología , Citocinas/metabolismo , Exones/genética , Femenino , Genes Recesivos/genética , Genes Recesivos/inmunología , Homocigoto , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Síndrome de Job/sangre , Síndrome de Job/inmunología , Mutación con Pérdida de Función , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Linaje , Regiones Promotoras Genéticas/genética , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT3/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Secuenciación del Exoma , Adulto Joven , Dedos de Zinc/genéticaRESUMEN
BACKGROUND: Colorectal cancer is responsible for almost 700,000 deaths annually worldwide. Therapeutic vaccination is a promising alternative to conventional treatment for colorectal cancer, using vaccines to induce targeted immune responses against tumour-associated antigens. In this study, we have developed chimaeric virus-like particles (VLP), a form of non-infectious non-replicative subunit vaccine consisting of rabbit haemorrhagic disease virus (RHDV) VP60 capsid proteins containing recombinantly inserted epitopes from murine topoisomerase IIα and survivin. These vaccines were developed in mono- (T.VP60, S.VP60) and multi-target (TS.VP60) forms, aiming to elucidate the potential benefits from multi-target vaccination. METHODS: Chimaeric RHDV VLP were developed by recombinantly inserting immune epitopes at the N-terminus of VP60. Vaccines were tested against a murine model of colorectal cancer by establishing MC38-OVA tumours subcutaneously. Unmethylated CpG DNA oligonucleotides (CpGs) were used as a vaccine adjuvant. Statistical tests employed included the Mantel-Cox log-rank test, ANOVA and unpaired t-tests depending on the data analysed, with a post hoc Bonferroni adjustment for multiple measures. RESULTS: Chimaeric RHDV VLP were found to form a composite particle in the presence of CpGs. Overall survival was significantly improved amongst mice bearing MC38-OVA tumours following vaccination with T.VP60 (60%, 9/15), S.VP60 (60%, 9/15) or TS.VP60 (73%, 11/15). TS.VP60 significantly prolonged the vaccine-induced remission period in comparison to each mono-therapy. CONCLUSIONS: Chimaeric VLP containing multiple epitopes were found to confer an advantage for therapeutic vaccination in a model of colorectal cancer based on the prolongation of remission prior to tumour escape.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , ADN-Topoisomerasas de Tipo II/química , Proteínas Inhibidoras de la Apoptosis/química , Oligodesoxirribonucleótidos/administración & dosificación , Proteínas Represoras/química , Vacunas de Partículas Similares a Virus/administración & dosificación , Proteínas Estructurales Virales/genética , Animales , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Quimioterapia Adyuvante , Neoplasias Colorrectales/inmunología , Islas de CpG , Virus de la Enfermedad Hemorrágica del Conejo/genética , Virus de la Enfermedad Hemorrágica del Conejo/metabolismo , Virus de la Enfermedad Hemorrágica del Conejo/fisiología , Ratones , Oligodesoxirribonucleótidos/uso terapéutico , Survivin , Resultado del Tratamiento , Vacunas de Partículas Similares a Virus/uso terapéutico , Proteínas Estructurales Virales/metabolismo , Vacunas Virales/administración & dosificación , Vacunas Virales/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Virus-like particles (VLPs) are an effective means of establishing both prophylactic and therapeutic immunity against their source virus or heterologous antigens. The particulate nature and repetitive structure of VLPs makes them ideal for stimulating potent immune responses. Epitopes delivered by VLPs can be presented on MHC-II for stimulation of a humoral immune response, or cross-presented onto MHC-I leading to cell-mediated immunity. VLPs as particulate subunit vaccine carriers are showing promise in preclinical and clinical trials for the treatment of many conditions including cancer, autoimmunity, allergies and addiction. Supporting the delivery of almost any form of antigenic material, VLPs are ideal candidate vectors for development of future vaccines.