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1.
J Clin Immunol ; 44(4): 98, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38598033

RESUMO

Biallelic null or hypomorphic variants in JAK3 cause SCID and less frequently Omenn syndrome. We investigated homozygous hypomorphic JAK3 mutations in two patients, and expression and function of a novel JAK3R431P variant in Omenn syndrome. Immunophenotyping of PBMC from the patient with the novel JAK3R431P variant was undertaken, by flow cytometry and Phosflow after stimulation with IL-2, IL-7, and IL-15. JAK3 expression was investigated by Western blotting. We report two patients with homozygous hypomorphic JAK3 variants and clinical features of Omenn syndrome. One patient had a previously described JAK3R775H variant, and the second had a novel JAK3R431P variant. One patient with a novel JAK3R431P variant had normal expression of JAK3 in immortalised EBV-LCL cells but reduced phosphorylation of STAT5 after stimulation with IL-2, IL-7, and IL-15 consistent with impaired kinase activity. These results suggest the JAK3R431P variant to be hypomorphic. Both patients are alive and well after allogeneic haematopoietic stem cell transplantation. They have full donor chimerism, restitution of thymopoiesis and development of appropriate antibody responses following vaccination. We expand the phenotype of hypomorphic JAK3 deficiency and demonstrate the importance of functional testing of novel variants in disease-causing genes.


Assuntos
Janus Quinase 3 , Imunodeficiência Combinada Severa , Humanos , Lactente , Interleucina-15 , Interleucina-2 , Interleucina-7 , Janus Quinase 3/genética , Leucócitos Mononucleares , Imunodeficiência Combinada Severa/diagnóstico , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia
2.
J Allergy Clin Immunol ; 150(4): 955-964.e16, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35182547

RESUMO

BACKGROUND: Inflammatory phenomena such as hyperinflammation or hemophagocytic lymphohistiocytosis are a frequent yet paradoxical accompaniment to virus susceptibility in patients with impairment of type I interferon (IFN-I) signaling caused by deficiency of signal transducer and activator of transcription 2 (STAT2) or IFN regulatory factor 9 (IRF9). OBJECTIVE: We hypothesized that altered and/or prolonged IFN-I signaling contributes to inflammatory complications in these patients. METHODS: We explored the signaling kinetics and residual transcriptional responses of IFN-stimulated primary cells from individuals with complete loss of one of STAT1, STAT2, or IRF9 as well as gene-edited induced pluripotent stem cell-derived macrophages. RESULTS: Deficiency of any IFN-stimulated gene factor 3 component suppressed but did not abrogate IFN-I receptor signaling, which was abnormally prolonged, in keeping with insufficient induction of negative regulators such as ubiquitin-specific peptidase 18 (USP18). In cells lacking either STAT2 or IRF9, this late transcriptional response to IFN-α2b mimicked the effect of IFN-γ. CONCLUSION: Our data suggest a model wherein the failure of negative feedback of IFN-I signaling in STAT2 and IRF9 deficiency leads to immune dysregulation. Aberrant IFN-α receptor signaling in STAT2- and IRF9-deficient cells switches the transcriptional output to a prolonged, IFN-γ-like response and likely contributes to clinically overt inflammation in these individuals.


Assuntos
Interferon Tipo I , Fator IX , Humanos , Interferon Tipo I/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Interferon-alfa , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Ubiquitina Tiolesterase , Proteases Específicas de Ubiquitina
3.
Blood ; 136(9): 1055-1066, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32518946

RESUMO

Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.


Assuntos
Proteínas de Ligação a DNA/deficiência , Mutação em Linhagem Germinativa , Mutação com Perda de Função , Transtornos Linfoproliferativos/genética , Proteínas Proto-Oncogênicas/deficiência , Imunodeficiência Combinada Severa/genética , Aloenxertos , Apoptose , Subpopulações de Linfócitos B/patologia , Técnicas de Reprogramação Celular , Códon sem Sentido , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Dioxigenases , Evolução Fatal , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Recém-Nascido , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/patologia , Masculino , Mutação de Sentido Incorreto , Neoplasias Primárias Múltiplas/genética , Linhagem , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Imunodeficiência Combinada Severa/patologia , Subpopulações de Linfócitos T/patologia , Sequenciamento do Exoma
4.
Sci Immunol ; 9(95): eade5705, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38787962

RESUMO

Inborn errors of T cell development present a pediatric emergency in which timely curative therapy is informed by molecular diagnosis. In 11 affected patients across four consanguineous kindreds, we detected homozygosity for a single deleterious missense variant in the gene NudC domain-containing 3 (NUDCD3). Two infants had severe combined immunodeficiency with the complete absence of T and B cells (T -B- SCID), whereas nine showed classical features of Omenn syndrome (OS). Restricted antigen receptor gene usage by residual T lymphocytes suggested impaired V(D)J recombination. Patient cells showed reduced expression of NUDCD3 protein and diminished ability to support RAG-mediated recombination in vitro, which was associated with pathologic sequestration of RAG1 in the nucleoli. Although impaired V(D)J recombination in a mouse model bearing the homologous variant led to milder immunologic abnormalities, NUDCD3 is absolutely required for healthy T and B cell development in humans.


Assuntos
Imunodeficiência Combinada Severa , Recombinação V(D)J , Humanos , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Animais , Camundongos , Recombinação V(D)J/imunologia , Recombinação V(D)J/genética , Masculino , Feminino , Lactente , Linfócitos B/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Linfócitos T/imunologia , Pré-Escolar , Mutação de Sentido Incorreto
5.
Apoptosis ; 18(6): 739-50, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23456623

RESUMO

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic ligand from the TNF-alpha family that is under consideration, along with agonistic anti-TRAIL receptor antibodies, as a potential anti-tumor agent. However, most primary human tumors are resistant to monotherapy with TRAIL apoptogens, and thus the potential applicability of TRAIL in anti-tumor therapy ultimately depends on its rational combination with drugs targeting these resistances. In our high-throughput screening for novel agents/drugs that could sensitize TRAIL-resistant colorectal cancer cells to TRAIL-induced apoptosis, we found homoharringtonine (HHT), a cephalotaxus alkaloid and tested anti-leukemia drug, to be a very effective, low nanomolar enhancer of TRAIL-mediated apoptosis/growth suppression of these resistant cells. Co-treatment of TRAIL-resistant RKO or HT-29 cells with HHT and TRAIL led to the effective induction of apoptosis and the complete elimination of the treated cells. HHT suppressed the expression of the anti-apoptotic proteins Mcl-1 and cFLIP and enhanced the TRAIL-triggered activation of JNK and p38 kinases. The shRNA-mediated down-regulation of cFLIP or Mcl-1 in HT-29 or RKO cells variably enhanced their TRAIL-induced apoptosis but it did not markedly sensitize them to TRAIL-mediated growth suppression. However, with the notable exception of RKO/sh cFLIP cells, the downregulation of cFLIP or Mcl-1 significantly lowered the effective concentration of HHT in HHT + TRAIL co-treatment. Combined HHT + TRAIL therapy also led to the strong suppression of HT-29 tumors implanted into immunodeficient mice. Thus, HHT represents a very efficient enhancer of TRAIL-induced apoptosis with potential application in TRAIL-based, anti-cancer combination therapy.


Assuntos
Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Harringtoninas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Células HT29 , Mepesuccinato de Omacetaxina , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Transplante Heterólogo
6.
Front Pediatr ; 10: 809061, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35311038

RESUMO

Pulmonary severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children is generally described as mild, and SARS-CoV-2 infection in immunocompromised children are observed as generally mild as well. A small proportion of pediatric patients will become critically ill due to (cardio)respiratory failure and require intensive care treatment. We report the case of a teenager with Hodgkin's lymphoma who acquired SARS-CoV-2 (detected by PCR) on the day of her autologous stem cell transplant and developed acute respiratory distress syndrome, successfully treated with a combination of antivirals, immunomodulation with steroids and biologicals, and ECMO.

7.
Stem Cell Reports ; 17(7): 1699-1713, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35750043

RESUMO

Conjunctival epithelial cells, which express viral-entry receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine type 2 (TMPRSS2), constitute the largest exposed epithelium of the ocular surface tissue and may represent a relevant viral-entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of basal, suprabasal, and superficial epithelial cells, and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA sequencing (RNA-seq), with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-κB activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplantation.


Assuntos
COVID-19 , Células Epiteliais/metabolismo , Humanos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo , SARS-CoV-2
8.
J Exp Med ; 219(6)2022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35442417

RESUMO

Type I interferons (IFN-I) play a critical role in human antiviral immunity, as demonstrated by the exceptionally rare deleterious variants of IFNAR1 or IFNAR2. We investigated five children from Greenland, Canada, and Alaska presenting with viral diseases, including life-threatening COVID-19 or influenza, in addition to meningoencephalitis and/or hemophagocytic lymphohistiocytosis following live-attenuated viral vaccination. The affected individuals bore the same homozygous IFNAR2 c.157T>C, p.Ser53Pro missense variant. Although absent from reference databases, p.Ser53Pro occurred with a minor allele frequency of 0.034 in their Inuit ancestry. The serine to proline substitution prevented cell surface expression of IFNAR2 protein, small amounts of which persisted intracellularly in an aberrantly glycosylated state. Cells exclusively expressing the p.Ser53Pro variant lacked responses to recombinant IFN-I and displayed heightened vulnerability to multiple viruses in vitro-a phenotype rescued by wild-type IFNAR2 complementation. This novel form of autosomal recessive IFNAR2 deficiency reinforces the essential role of IFN-I in viral immunity. Further studies are warranted to assess the need for population screening.


Assuntos
COVID-19 , Interferon Tipo I , Antivirais/metabolismo , Criança , Humanos , Padrões de Herança , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Receptor de Interferon alfa e beta
9.
Lancet Microbe ; 3(1): e21-e31, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34778853

RESUMO

BACKGROUND: Previous infection with SARS-CoV-2 affects the immune response to the first dose of the SARS-CoV-2 vaccine. We aimed to compare SARS-CoV-2-specific T-cell and antibody responses in health-care workers with and without previous SARS-CoV-2 infection following a single dose of the BNT162b2 (tozinameran; Pfizer-BioNTech) mRNA vaccine. METHODS: We sampled health-care workers enrolled in the PITCH study across four hospital sites in the UK (Oxford, Liverpool, Newcastle, and Sheffield). All health-care workers aged 18 years or older consenting to participate in this prospective cohort study were included, with no exclusion criteria applied. Blood samples were collected where possible before vaccination and 28 (±7) days following one or two doses (given 3-4 weeks apart) of the BNT162b2 vaccine. Previous infection was determined by a documented SARS-CoV-2-positive RT-PCR result or the presence of positive anti-SARS-CoV-2 nucleocapsid antibodies. We measured spike-specific IgG antibodies and quantified T-cell responses by interferon-γ enzyme-linked immunospot assay in all participants where samples were available at the time of analysis, comparing SARS-CoV-2-naive individuals to those with previous infection. FINDINGS: Between Dec 9, 2020, and Feb 9, 2021, 119 SARS-CoV-2-naive and 145 previously infected health-care workers received one dose, and 25 SARS-CoV-2-naive health-care workers received two doses, of the BNT162b2 vaccine. In previously infected health-care workers, the median time from previous infection to vaccination was 268 days (IQR 232-285). At 28 days (IQR 27-33) after a single dose, the spike-specific T-cell response measured in fresh peripheral blood mononuclear cells (PBMCs) was higher in previously infected (n=76) than in infection-naive (n=45) health-care workers (median 284 [IQR 150-461] vs 55 [IQR 24-132] spot-forming units [SFUs] per 106 PBMCs; p<0·0001). With cryopreserved PBMCs, the T-cell response in previously infected individuals (n=52) after one vaccine dose was equivalent to that of infection-naive individuals (n=19) after receiving two vaccine doses (median 152 [IQR 119-275] vs 162 [104-258] SFUs/106 PBMCs; p=1·00). Anti-spike IgG antibody responses following a single dose in 142 previously infected health-care workers (median 270 373 [IQR 203 461-535 188] antibody units [AU] per mL) were higher than in 111 infection-naive health-care workers following one dose (35 001 [17 099-55 341] AU/mL; p<0·0001) and higher than in 25 infection-naive individuals given two doses (180 904 [108 221-242 467] AU/mL; p<0·0001). INTERPRETATION: A single dose of the BNT162b2 vaccine is likely to provide greater protection against SARS-CoV-2 infection in individuals with previous SARS-CoV-2 infection, than in SARS-CoV-2-naive individuals, including against variants of concern. Future studies should determine the additional benefit of a second dose on the magnitude and durability of immune responses in individuals vaccinated following infection, alongside evaluation of the impact of extending the interval between vaccine doses. FUNDING: UK Department of Health and Social Care, and UK Coronavirus Immunology Consortium.


Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Formação de Anticorpos , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunoglobulina G , Leucócitos Mononucleares , Estudos Prospectivos , Linfócitos T , Reino Unido/epidemiologia , Vacinas Sintéticas , Vacinas de mRNA
10.
Nat Commun ; 12(1): 7092, 2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34876592

RESUMO

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNß or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.


Assuntos
Células Epiteliais/virologia , Interferon Tipo I/imunologia , Interferons/imunologia , Mucosa Nasal/virologia , SARS-CoV-2/fisiologia , Antivirais/imunologia , Antivirais/farmacologia , COVID-19/imunologia , COVID-19/virologia , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/imunologia , Humanos , Imunidade Inata , Cinética , Mucosa Nasal/citologia , Mucosa Nasal/imunologia , SARS-CoV-2/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tropismo Viral , Replicação Viral/efeitos dos fármacos , Interferon lambda
11.
Nat Med ; 27(5): 904-916, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33879890

RESUMO

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.


Assuntos
COVID-19/imunologia , Proteoma , SARS-CoV-2/imunologia , Análise de Célula Única/métodos , Transcriptoma , Estudos Transversais , Humanos , Monócitos/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia
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