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1.
BMC Pediatr ; 21(1): 181, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33865340

RESUMO

BACKGROUND: Early diagnostic indicators and the identification of possible progression to severe or critical COVID-19 in children are unknown. To investigate the immune characteristics of early SARS-CoV-2 infection in children and possible key prognostic factors for early identification of critical COVID-19, a retrospective study including 121 children with COVID-19 was conducted. Peripheral blood lymphocyte subset counts, T cell-derived cytokine concentrations, inflammatory factor concentrations, and routine blood counts were analyzed statistically at the initial presentation. RESULTS: The T lymphocyte subset and natural killer cell counts decreased with increasing disease severity. Group III (critical cases) had a higher Th/Tc ratio than groups I and II (common and severe cases); group I had a higher B cell count than groups II and III. IL-6, IL-10, IFN-γ, SAA, and procalcitonin levels increased with increasing disease severity. Hemoglobin concentration, and RBC and eosinophil counts decreased with increasing disease severity. Groups II and III had significantly lower lymphocyte counts than group I. T, Th, Tc, IL-6, IL-10, RBC, and hemoglobin had relatively high contribution and area under the curve values. CONCLUSIONS: Decreased T, Th, Tc, RBC, hemoglobin and increased IL-6 and IL-10 in early SARS-CoV-2 infection in children are valuable indices for early diagnosis of severe disease. The significantly reduced Th and Tc cells and significantly increased IL-6, IL-10, ferritin, procalcitonin, and SAA at this stage in children with critical COVID-19 may be closely associated with the systemic cytokine storm caused by immune dysregulation.


Assuntos
/diagnóstico , Síndrome de Resposta Inflamatória Sistêmica/diagnóstico , Adolescente , Linfócitos B/citologia , Criança , Pré-Escolar , Síndrome da Liberação de Citocina/virologia , Citocinas/sangue , Feminino , Humanos , Imunidade , Lactente , Células Matadoras Naturais/citologia , Contagem de Linfócitos , Masculino , Prognóstico , Estudos Retrospectivos , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/citologia
2.
Cell ; 184(7): 1836-1857.e22, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33713619

RESUMO

COVID-19 exhibits extensive patient-to-patient heterogeneity. To link immune response variation to disease severity and outcome over time, we longitudinally assessed circulating proteins as well as 188 surface protein markers, transcriptome, and T cell receptor sequence simultaneously in single peripheral immune cells from COVID-19 patients. Conditional-independence network analysis revealed primary correlates of disease severity, including gene expression signatures of apoptosis in plasmacytoid dendritic cells and attenuated inflammation but increased fatty acid metabolism in CD56dimCD16hi NK cells linked positively to circulating interleukin (IL)-15. CD8+ T cell activation was apparent without signs of exhaustion. Although cellular inflammation was depressed in severe patients early after hospitalization, it became elevated by days 17-23 post symptom onset, suggestive of a late wave of inflammatory responses. Furthermore, circulating protein trajectories at this time were divergent between and predictive of recovery versus fatal outcomes. Our findings stress the importance of timing in the analysis, clinical monitoring, and therapeutic intervention of COVID-19.


Assuntos
/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Expressão Gênica/imunologia , Células Matadoras Naturais/metabolismo , Índice de Gravidade de Doença , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Estudos de Casos e Controles , Células Dendríticas/citologia , Feminino , Humanos , Células Matadoras Naturais/citologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Transcriptoma/imunologia , Adulto Jovem
3.
Science ; 371(6536)2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33766856

RESUMO

The pathways that lead to the development of tissue-resident lymphocytes, including liver type 1 innate lymphoid cells (ILC1s), remain unclear. We show here that the adult mouse liver contains Lin-Sca-1+Mac-1+ hematopoietic stem cells derived from the fetal liver. This population includes Lin-CD122+CD49a+ progenitors that can generate liver ILC1s but not conventional natural killer cells. Interferon-γ (IFN-γ) production by the liver ILC1s themselves promotes the development of these cells in situ, through effects on their IFN-γR+ liver progenitors. Thus, an IFN-γ-dependent loop drives liver ILC1 development in situ, highlighting the contribution of extramedullary hematopoiesis to regional immune composition within the liver.


Assuntos
Interferon gama/metabolismo , Fígado/citologia , Fígado/imunologia , Linfócitos/citologia , Linfócitos/imunologia , Animais , Hematopoese Extramedular , Imunidade Inata , Interferon gama/genética , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/metabolismo , Linfopoese , Camundongos , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Transdução de Sinais , Proteínas com Domínio T/metabolismo
4.
Food Chem ; 350: 129302, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33618089

RESUMO

Caseinate was glycated with oligochitosan via transglutaminase (TGase) action and then hydrolyzed by trypsin to generate glycated caseinate hydrolysate (GCNH) that was investigated for in vivo immune-promoting activity. Caseinate hydrolysate (CNH) containing glucosamine of 5.7 g/kg had amino acid compositions similar to GCNH. In normal BALB/c mice, GCNH at 100-400 mg/(kg d) showed higher immune-promoting activity than CNH via increasing serum IgM, IgA, and IgG by 1.5-24.5%, enhancing spleen and thymus indices by 9.7-26.2%, or increasing splenocyte lymphocyte proliferation and natural killer (NK) cell activity by 1.2-11.5%. GCNH also exerted higher activity than CNH in the suppressed BALB/c mice through increasing serum IgM, IgA, and IgG by 2.6-10.5%, enhancing spleen and thymus indices by 0.4-50.1%, or increasing splenocyte lymphocyte proliferation and NK cell activity by 3.4-18.9%. The results highlight that this TGase-type oligochitosan-glycation is potential to generate functional protein ingredients that possess improved immune-promoting activity once hydrolyzed by trypsin.


Assuntos
Caseínas/química , Caseínas/farmacologia , Quitina/análogos & derivados , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Transglutaminases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caseínas/metabolismo , Quitina/química , Glicosilação , Hidrólise , Fatores Imunológicos/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Relação Estrutura-Atividade
5.
Nat Commun ; 12(1): 1282, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627642

RESUMO

Natural killer (NK) cells play a critical understudied role during HIV infection in tissues. In a natural host of SIV, the African green monkey (AGM), NK cells mediate a strong control of SIVagm infection in secondary lymphoid tissues. We demonstrate that SIVagm infection induces the expansion of terminally differentiated NKG2alow NK cells in secondary lymphoid organs displaying an adaptive transcriptional profile and increased MHC-E-restricted cytotoxicity in response to SIV Env peptides while expressing little IFN-γ. Such NK cell differentiation was lacking in SIVmac-infected macaques. Adaptive NK cells displayed no increased NKG2C expression. This study reveals a previously unknown profile of NK cell adaptation to a viral infection, thus accelerating strategies toward NK-cell directed therapies and viral control in tissues.


Assuntos
Células Matadoras Naturais/metabolismo , Linfonodos/metabolismo , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Algoritmos , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Chlorocebus aethiops , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Células K562 , Células Matadoras Naturais/citologia , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Macaca , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/patogenicidade , Transcriptoma/genética
6.
Methods Mol Biol ; 2194: 255-290, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32926371

RESUMO

The field of flow cytometry has witnessed rapid technological advancements in the last few decades. While the founding principles of fluorescent detection on cells (or particles) within a uniform fluid stream remains largely unchanged, the availability more sensitive cytometers with the ability to multiplex more and more florescent signals has resulted in very complex high-order assays. This results in the co-use of fluorophores with increased levels of emission overlap and/or spillover spreading than in years past and thus requires careful and well thought out planning for flow cytometry assay development. As an example, we present the development of a large 18-color (20 parameter) flow cytometry assay designed to take an in depth analysis of effector lymphocyte phenotypes, with careful attention to assay controls and panel design.


Assuntos
Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Células Matadoras Naturais/imunologia , Células T Matadoras Naturais/imunologia , Anticorpos , Humanos , Células Matadoras Naturais/citologia , Células T Matadoras Naturais/citologia , Coloração e Rotulagem/métodos
7.
Nat Commun ; 11(1): 5224, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067467

RESUMO

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation tests before clinical use, the cells are cryopreserved to bridge the necessary evaluation time. Standard degranulation and chromium release cytotoxicity assays confirm the ability of cryopreserved NK cells to kill target cells. Here, we report that tumor cells embedded in a 3-dimensional collagen gel, however, are killed by cryopreserved NK cells at a 5.6-fold lower rate compared to fresh NK cells. This difference is mainly caused by a 6-fold decrease in the fraction of motile NK cells after cryopreservation. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.


Assuntos
Movimento Celular , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Técnicas de Cultura de Células , Sobrevivência Celular , Células Cultivadas , Criopreservação , Citotoxicidade Imunológica , Humanos , Células Matadoras Naturais/química
8.
Sci Rep ; 10(1): 17718, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077873

RESUMO

COVID-19 has been widely spreading. We aimed to examine adaptive immune cells in non-severe patients with persistent SARS-CoV-2 shedding. 37 non-severe patients with persistent SARS-CoV-2 presence that were transferred to Zhongnan hospital of Wuhan University were retrospectively recruited to the PP (persistently positive) group, which was further allocated to PPP group (n = 19) and PPN group (n = 18), according to their testing results after 7 days (N = negative). Epidemiological, demographic, clinical and laboratory data were collected and analyzed. Data from age- and sex-matched non-severe patients at disease onset (PA [positive on admission] patients, n = 37), and lymphocyte subpopulation measurements from matched 54 healthy subjects were extracted for comparison (HC). Compared with PA patients, PP patients had much improved laboratory findings. The absolute numbers of CD3+ T cells, CD4+ T cells, and NK cells were significantly higher in PP group than that in PA group, and were comparable to that in healthy controls. PPP subgroup had markedly reduced B cells and T cells compared to PPN group and healthy subjects. Finally, paired results of these lymphocyte subpopulations from 10 PPN patients demonstrated that the number of T cells and B cells significantly increased when the SARS-CoV-2 tests turned negative. Persistent SARS-CoV-2 presence in non-severe COVID-19 patients is associated with reduced numbers of adaptive immune cells. Monitoring lymphocyte subpopulations could be clinically meaningful in identifying fully recovered COVID-19 patients.


Assuntos
Linfócitos B/citologia , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Linfócitos T/citologia , Adulto , Linfócitos B/imunologia , Linfócitos B/metabolismo , Betacoronavirus/isolamento & purificação , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Linfócitos T/imunologia , Linfócitos T/metabolismo
9.
Cell ; 183(4): 982-995.e14, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-32991843

RESUMO

Initially, children were thought to be spared from disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, a month into the epidemic, a novel multisystem inflammatory syndrome in children (MIS-C) emerged. Herein, we report on the immune profiles of nine MIS-C cases. All MIS-C patients had evidence of prior SARS-CoV-2 exposure, mounting an antibody response with intact neutralization capability. Cytokine profiling identified elevated signatures of inflammation (IL-18 and IL-6), lymphocytic and myeloid chemotaxis and activation (CCL3, CCL4, and CDCP1), and mucosal immune dysregulation (IL-17A, CCL20, and CCL28). Immunophenotyping of peripheral blood revealed reductions of non-classical monocytes, and subsets of NK and T lymphocytes, suggesting extravasation to affected tissues. Finally, profiling the autoantigen reactivity of MIS-C plasma revealed both known disease-associated autoantibodies (anti-La) and novel candidates that recognize endothelial, gastrointestinal, and immune-cell antigens. All patients were treated with anti-IL-6R antibody and/or IVIG, which led to rapid disease resolution.


Assuntos
Inflamação/patologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Adolescente , Anticorpos Antivirais/sangue , Autoanticorpos/sangue , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Quimiocina CCL3/metabolismo , Criança , Pré-Escolar , Infecções por Coronavirus/complicações , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Imunidade Humoral , Lactente , Recém-Nascido , Inflamação/metabolismo , Interleucina-17/metabolismo , Interleucina-18/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Masculino , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Adulto Jovem
10.
PLoS One ; 15(9): e0238347, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32870938

RESUMO

Highly exposed seronegative (HESN) individuals present a unique setting to study mechanisms of protection against HIV acquisition. As natural killer (NK) cell activation and function have been implicated as a correlate of protection in HESN individuals, we sought to better understand the features of NK cells that may confer protection. We used mass cytometry to phenotypically profile NK cells from a cohort of Beninese sex workers and healthy controls. We found that NK cells from HESN women had increased expression of NKG2A, NKp30 and LILRB1, as well as the Fc receptor CD16, and decreased expression of DNAM-1, CD94, Siglec-7, and NKp44. Using functional assessments of NK cells from healthy donors against autologous HIV-infected CD4+ T cells, we observed that NKp30+ and Siglec-7+ cells had improved functional activity. Further, we found that NK cells from HESN women trended towards increased antibody-dependent cellular cytotoxicity (ADCC) activity; this activity correlated with increased CD16 expression. Overall, we identify features of NK cells in HESN women that may contribute to protection from HIV infection. Follow up studies with larger cohorts are warranted to confirm these findings.


Assuntos
Infecções por HIV/patologia , Células Matadoras Naturais/metabolismo , Adulto , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Diferenciação Mielomonocítica/metabolismo , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Genótipo , Infecções por HIV/imunologia , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Lectinas/metabolismo , Modelos Lineares , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Fenótipo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Profissionais do Sexo
11.
J Vis Exp ; (160)2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32628161

RESUMO

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFNγ) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.


Assuntos
Proliferação de Células , Interferon gama/biossíntese , Interleucina-15/metabolismo , Células Matadoras Naturais/fisiologia , Ativação Linfocitária/imunologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia
12.
Life Sci ; 257: 118064, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32652136

RESUMO

AIMS: Exosomes contain functional molecules from their cells of origin and can enter recipient cells for intercellular communication. Interferon ß (IFNß) has been shown to induce some lncRNAs to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (NK) cells. We aim to clarify whether IFNß induced exosomes can regulate the cytotoxicity of NK cells by transferring specific lncRNAs into NK cells. MAIN METHODS: Exosomes were isolated from the supernatants of A549 cells with or without IFNß treatment. Co-culture and ELISA assay were used to analyze the effect of exosomes on the cytotoxicity of NK cells. Human transcriptome array (HTA) was performed to analyze the profiling of RNAs wrapped in exosomes. Then subcellular location, qPCR, western blotting, dual-luciferase reporter assay and ELISA were used to determine long noncoding RNAs (lcnRNAs) location, sponge absorb effects, the expression of NKp46 and cytotoxicity of NK cells. KEY FINDINGS: ELISA assay showed IFNß induced exosomes can strengthen the cytotoxicity of NK cells. Through HTA we found the expression levels of 69 lncRNAs were significantly changed within IFNß induced exosomes. Additionally, we found a specific exosomal cargo, linc-EPHA6-1, acted as a competing endogenous RNA (ceRNA) for hsa-miR-4485-5p which subsequently up-regulate one of the natural cytotoxicity receptors (NKp46) expression. Furthermore, we verified over-expression of linc-EPHA6-1 significantly enhances the cytotoxicity of NK cells against A549 cells and Zika virus infected A549 cells. SIGNIFICANCE: Our results demonstrated that IFNß-induced exosomal linc-EPHA6-1 can regulate the cytotoxicity of NK cells.


Assuntos
Células Matadoras Naturais/citologia , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor EphA6/genética , Infecção por Zika virus/virologia , Células A549 , Exossomos/metabolismo , Humanos , Interferon beta/administração & dosagem , Interferon beta/metabolismo , RNA Longo não Codificante/genética , Regulação para Cima
13.
Nature ; 583(7817): 609-614, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32581358

RESUMO

Cytokines were the first modern immunotherapies to produce durable responses in patients with advanced cancer, but they have only modest efficacy and limited tolerability1,2. In an effort to identify alternative cytokine pathways for immunotherapy, we found that components of the interleukin-18 (IL-18) pathway are upregulated on tumour-infiltrating lymphocytes, suggesting that IL-18 therapy could enhance anti-tumour immunity. However, recombinant IL-18 previously did not demonstrate efficacy in clinical trials3. Here we show that IL-18BP, a high-affinity IL-18 decoy receptor, is frequently upregulated in diverse human and mouse tumours and limits the anti-tumour activity of IL-18 in mice. Using directed evolution, we engineered a 'decoy-resistant' IL-18 (DR-18) that maintains signalling potential but is impervious to inhibition by IL-18BP. Unlike wild-type IL-18, DR-18 exerted potent anti-tumour effects in mouse tumour models by promoting the development of poly-functional effector CD8+ T cells, decreasing the prevalence of exhausted CD8+ T cells that express the transcriptional regulator of exhaustion TOX, and expanding the pool of stem-like TCF1+ precursor CD8+ T cells. DR-18 also enhanced the activity and maturation of natural killer cells to effectively treat anti-PD-1 resistant tumours that have lost surface expression of major histocompatibility complex class I molecules. These results highlight the potential of the IL-18 pathway for immunotherapeutic intervention and implicate IL-18BP as a major therapeutic barrier.


Assuntos
Imunoterapia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-18/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Estimativa de Kaplan-Meier , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Camundongos , Receptores de Interleucina-18/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
14.
J Vis Exp ; (159)2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32510494

RESUMO

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by FcγRIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the FcγRIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.


Assuntos
Anticorpos/uso terapêutico , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores de IgG/metabolismo , Anticorpos/imunologia , Genótipo , Humanos , Células Matadoras Naturais/citologia , Ativação Linfocitária , Transdução de Sinais
15.
Cytometry A ; 97(8): 772-776, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32542842

RESUMO

A reduced peripheral blood absolute lymphocyte count with an elevated neutrophil count has been a consistent observation in hospitalized coronavirus disease 2019 (COVID-19) patients. In this brief meta-analysis, the reduction of lymphocyte subset counts in COVID-19 patients was investigated across 20 peer-reviewed studies meeting criteria for reporting lymphocyte subset counts and COVID-19 disease severity. CD4+ T cell, CD8+ T cell, B cell, NK cell, and total lymphocyte cell counts all showed statistically significant reduction in patients with severe/critical COVID-19 disease compared to mild/moderate disease. T-cell subsets showed the largest standardized magnitude of change. In some studies, multivariate analysis has shown that CD4 and/or CD8 T-cells counts are independently predictive of patient outcomes. © 2020 International Society for Advancement of Cytometry.


Assuntos
Linfócitos B/citologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Infecções por Coronavirus/sangue , Células Matadoras Naturais/citologia , Pneumonia Viral/sangue , Subpopulações de Linfócitos T/citologia , Betacoronavirus , Humanos , Contagem de Linfócitos , Neutrófilos/citologia , Pandemias
16.
Hum Cell ; 33(3): 582-589, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32449114

RESUMO

Activation of hepatic stellate cells (HSCs) is a prominent driver of liver fibrosis. This study was designed to investigate the effect of exosomes derived from natural killer (NK) cells on HSC activation and liver fibrosis. The exosomes were isolated from NK-92MI cells (NK-Exo) and identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot. Then NK-Exo was administered into TGF-ß1-treated LX-2 cells (human HSC line) and mice with CCl4-induced liver fibrosis. LX-2 cell proliferation was determined by CCK-8 assay. The levels of α-SMA and CoL1A1 were measured by qRT-PCR and western blot to evaluate HSC activation. Serum levels of AST and ALT were measured. Hematoxylin-eosin, Masson staining, and Sirius Red staining were performed to assess the pathological changes and collagen deposition. Cell supernatant derived from NK-92MI cells inhibited TGF-ß1-induced HSC proliferation and activation in LX-2 cells, and this effect was counteracted by the exosome inhibitor GW4869. Further assays confirmed that NK-Exo treatment significantly inhibited TGF-ß1-induced HSC proliferation and activation. Moreover, NK-Exo administration alleviated CCl4-induced liver fibrosis in mice. NK-Exo inhibited TGF-ß1-induced HSC activation and CCl4-induced liver fibrosis.


Assuntos
Exossomos/fisiologia , Células Estreladas do Fígado/patologia , Células Matadoras Naturais/citologia , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Cirrose Hepática/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fator de Crescimento Transformador beta1/efeitos adversos
17.
Cytotherapy ; 22(6): 322-328, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32278551

RESUMO

BACKGROUND: Optimal expansion of therapeutic natural killer (NK) cell products has required media supplementation with human or fetal bovine serum, which raises safety and regulatory concerns for clinical manufacturing. Serum-free media (SFM) have been optimized for T-cell expansion, but few SFM systems have been developed for NK cells. Here, we compare six commercial clinical-grade SFM with our standard fetal bovine serum-containing medium for their ability to support NK cell expansion and function. METHODS: Human peripheral blood NK cells were expanded in selected media by recursive weekly stimulation with K562-based feeder cells expressing membrane-bound interleukin-21 and CD137L. Expansion was the primary readout, and the best-performing SFM was then compared with standard medium for cytotoxicity, phenotype, degranulation and cytokine secretion. Multiple lots were compared for consistency, and media was analyzed throughout for nutrient consumption and metabolic byproducts. RESULTS: TexMACS, OpTmizer, SCGM, ABS-001 and StemXVivo demonstrated equal or inferior NK cell expansion kinetics compared with standard medium, but expansion was markedly superior with AIM V + 5% Immune Cell Serum Replacement (ICSR; mean 5448 vs. 2621-fold expansion in 14 days). Surprisingly, NK cells expanded in AIM V + ICSR also showed increased cytotoxicity, tumor necrosis factor α secretion and DNAM-1, NKG2D, NKp30, FasL, granzyme B and perforin expression. Lot-to-lot variability was minimal. Glucose and glutamine consumption were inversely related to lactate and ammonia production. DISCUSSION: The AIM V + ICSR SFM system supports excellent ex vivo expansion of clinical-grade NK cells with the phenotype and function needed for adoptive immunotherapy.


Assuntos
Meios de Cultura Livres de Soro/farmacologia , Células Alimentadoras/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Antígenos de Diferenciação de Linfócitos T/metabolismo , Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , Meios de Cultura Livres de Soro/química , Citotoxicidade Imunológica , Proteína Ligante Fas/metabolismo , Humanos , Células K562 , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
18.
Sci Rep ; 10(1): 6335, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286456

RESUMO

Numerous cell types modulate hematopoiesis through soluble and membrane bound molecules. Whether developing hematopoietic progenitors of a particular lineage modulate the differentiation of other hematopoietic lineages is largely unknown. Here we aimed to investigate the influence of myeloid progenitors on CD34+ cell differentiation into CD56+ innate lymphocytes. Sorted CD34+ cells cultured in the presence of stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (FLT3L) give rise to numerous cell types, including progenitors that expressed the prolactin receptor (PRLR). These CD34+PRLR+ myeloid-lineage progenitors were derived from granulocyte monocyte precursors (GMPs) and could develop into granulocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Moreover, CD34+PRLR+ myeloid progenitors lacked lymphoid developmental potential, but when stimulated with prolactin (PRL) they increased the differentiation of other CD34+ cell populations into the NK lineage in a non-contact dependent manner. Both mRNA and protein analyses show that PRL increased mothers against decapentaplegic homolog 7 (SMAD7) in CD34+PRLR+ myeloid cells, which reduced the production of transforming growth factor beta 1 (TGF-ß1), a cytokine known to inhibit CD56+ cell development. Thus, we uncover an axis whereby CD34+PRLR+ GMPs inhibit CD56+ lineage development through TGF-ß1 production and PRL stimulation leads to SMAD7 activation, repression of TGF-ß1, resulting in CD56+ cell development.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Linfopoese/genética , Prolactina/genética , Receptores da Prolactina/genética , Proteína Smad7/genética , Fator de Crescimento Transformador beta1/genética , Antígenos CD34/genética , Antígenos CD34/imunologia , Antígeno CD56/genética , Antígeno CD56/imunologia , Diferenciação Celular/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Linfócitos/citologia , Linfócitos/imunologia , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética
19.
Exerc Immunol Rev ; 26: 116-131, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32139354

RESUMO

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease that targets and destroys insulin-secreting pancreatic beta cells. Although T cell mediated, a number of other immune cells are also critically involved in coordinating the events leading to T1D. Specifically, innate subsets play an important role in the pathogenesis of T1D. NK cells are one of the first cell types to infiltrate the pancreas, causing damage and release of beta cell antigens. Previous work in our group has shown differential mobilisation of highly differentiated CD8+ T cells during vigorous intensity exercise in T1D compared to a control cohort. Here, we aimed to explore exercise-induced mobilisation of other cell types involved in T1D pathogenesis. In this study, we investigated the effects of a single bout of vigorous (80% predicted VO2max) intensity exercise on innate cell mobilisation in T1D and control participants. T1D (N=12, mean age 33.2yrs, predicted VO2max 32.2 ml.kg.min⁻¹, BMI 25.3 kg.m⁻²) and control (N=12, mean age 29.4yrs, predicted VO2 max 38.5 ml.kg.min⁻¹, BMI 23.7 kg.m⁻² male participants completed a 30-minute bout of cycling at 80% predicted VO2 max in a fasted state. Peripheral blood was collected at baseline, immediately post-exercise, and 1 hour post-exercise. NK cell subsets mobilised during vigorous intensity exercise in both control and T1D participants. However, mature NK cells, defined as the CD56dimCD16bright subset, displayed a lower percentage increase following vigorous intensity exercise in T1D participants (Control: 185.12%, T1D: 97.06%). This blunted mobilisation was specific to early mature NK cells (KIR+) but not later differentiated NK cells (KIR+CD57+). Myeloid lineage subsets mobilised to a similar extent in both control and T1D participants. In conclusion, vigorous exercise mobilises innate immune cells in people with T1D albeit to a different extent to those without T1D. This mobilisation of innate immune cells provides a mechanistic argument to support exercise in people with T1D where it has the potential to improve surveillance for infection and to modulate the autoimmune response to the beta cell.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Exercício Físico , Células Matadoras Naturais/citologia , Ativação Linfocitária , Adulto , Antígeno CD56 , Proteínas Ligadas por GPI , Humanos , Masculino , Receptores de IgG
20.
PLoS Pathog ; 16(3): e1008339, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163523

RESUMO

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.


Assuntos
Antirretrovirais/administração & dosagem , Linfócitos B/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Interleucina-15/antagonistas & inibidores , Células Matadoras Naturais/efeitos dos fármacos , Proteínas/administração & dosagem , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/fisiopatologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Interleucina-15/genética , Interleucina-15/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Macaca mulatta , Proteínas Recombinantes de Fusão , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/fisiologia , Latência Viral/efeitos dos fármacos
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