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1.
Int J Mol Sci ; 21(17)2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32883007

RESUMO

When facing an acute viral infection, our immune systems need to function with finite precision to enable the elimination of the pathogen, whilst protecting our bodies from immune-related damage. In many instances however this "perfect balance" is not achieved, factors such as ageing, cancer, autoimmunity and cardiovascular disease all skew the immune response which is then further distorted by viral infection. In SARS-CoV-2, although the vast majority of COVID-19 cases are mild, as of 24 August 2020, over 800,000 people have died, many from the severe inflammatory cytokine release resulting in extreme clinical manifestations such as acute respiratory distress syndrome (ARDS) and hemophagocytic lymphohistiocytosis (HLH). Severe complications are more common in elderly patients and patients with cardiovascular diseases. Natural killer (NK) cells play a critical role in modulating the immune response and in both of these patient groups, NK cell effector functions are blunted. Preliminary studies in COVID-19 patients with severe disease suggests a reduction in NK cell number and function, resulting in decreased clearance of infected and activated cells, and unchecked elevation of tissue-damaging inflammation markers. SARS-CoV-2 infection skews the immune response towards an overwhelmingly inflammatory phenotype. Restoration of NK cell effector functions has the potential to correct the delicate immune balance required to effectively overcome SARS-CoV-2 infection.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Suscetibilidade a Doenças/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Autoimunidade , Infecções por Coronavirus/metabolismo , Humanos , Imunomodulação , Células Matadoras Naturais/metabolismo , Pandemias , Pneumonia Viral/metabolismo
2.
Front Immunol ; 11: 1692, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32754162

RESUMO

Coronavirus-induced disease-2019 (COVID-19) continues to cause significant morbidity and mortality worldwide. While studies on SARS-CoV-2 effects on immune cell function continue to progress, we know very little about the significance of depletion of key immune effectors by the virus in the mortality and morbidity of the disease. This commentary outlines what is the reported literature thus far on the effect of virus on NK cells known to kill virally infected cells. It also underscores the necessity for the future comprehensive studies of NK cells in SARS-CoV-2 infected individuals and animal models to better understand the role and significance of reported NK cell depletion and functional inactivation in disease morbidity and mortality, in hope to design effective therapeutic interventions for the disease.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/terapia , Adulto , Idoso , Animais , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/sangue , Infecções por Coronavirus/virologia , Citocinas/sangue , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neutrófilos/imunologia , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/virologia
3.
Nat Immunol ; 21(9): 1107-1118, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32788748

RESUMO

In coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the relationship between disease severity and the host immune response is not fully understood. Here we performed single-cell RNA sequencing in peripheral blood samples of 5 healthy donors and 13 patients with COVID-19, including moderate, severe and convalescent cases. Through determining the transcriptional profiles of immune cells, coupled with assembled T cell receptor and B cell receptor sequences, we analyzed the functional properties of immune cells. Most cell types in patients with COVID-19 showed a strong interferon-α response and an overall acute inflammatory response. Moreover, intensive expansion of highly cytotoxic effector T cell subsets, such as CD4+ effector-GNLY (granulysin), CD8+ effector-GNLY and NKT CD160, was associated with convalescence in moderate patients. In severe patients, the immune landscape featured a deranged interferon response, profound immune exhaustion with skewed T cell receptor repertoire and broad T cell expansion. These findings illustrate the dynamic nature of immune responses during disease progression.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Interferon Tipo I/metabolismo , Pneumonia Viral/imunologia , Receptores Imunológicos/metabolismo , Adolescente , Adulto , Idoso , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Estudos de Coortes , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , RNA-Seq , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Índice de Gravidade de Doença , Análise de Célula Única
4.
PLoS One ; 15(8): e0237034, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745117

RESUMO

Production of IFN-γ is a key innate immune mechanism that limits replication of intracellular bacteria such as Francisella tularensis (Ft) until adaptive immune responses develop. Previously, we demonstrated that the host cell types responsible for IFN-γ production in response to murine Francisella infection include not only natural killer (NK) and T cells, but also a variety of myeloid cells. However, production of IFN-γ by mouse dendritic cells (DC) is controversial. Here, we directly demonstrated substantial production of IFN-γ by DC, as well as hybrid NK-DC, from LVS-infected wild type C57BL/6 or Rag1 knockout mice. We demonstrated that the numbers of conventional DC producing IFN-γ increased progressively over the course of 8 days of LVS infection. In contrast, the numbers of conventional NK cells producing IFN-γ, which represented about 40% of non-B/T IFN-γ-producing cells, peaked at day 4 after LVS infection and declined thereafter. This pattern was similar to that of hybrid NK-DC. To further confirm IFN-γ production by infected cells, DC and neutrophils were sorted from naïve and LVS-infected mice and analyzed for gene expression. Quantification of LVS by PCR revealed the presence of Ft DNA not only in macrophages, but also in highly purified, IFN-γ producing DC and neutrophils. Finally, production of IFN-γ by infected DC was confirmed by immunohistochemistry and confocal microscopy. Notably, IFN-γ production patterns similar to those in wild type mice were observed in cells derived from LVS-infected TLR2, TLR4, and TLR2xTLR9 knockout (KO) mice, but not from MyD88 KO mice. Taken together, these studies demonstrate the pivotal roles of DC and MyD88 in IFN-γ production and in initiating innate immune responses to this intracellular bacterium.


Assuntos
Interferon gama/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Francisella tularensis/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Neutrófilos/metabolismo , Baço/metabolismo , Linfócitos T/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/imunologia , Tularemia/microbiologia
5.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(7): 930-935, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32701231

RESUMO

OBJECTIVE: To assess the effect of neutralizing CD96 on natural killer (NK) cell functions in mice with pulmonary Chlamydia muridarum infection and explore the possible mechanism. METHODS: Male BALB/c mice were randomly divided into infection group (Cm group), anti-CD96 treatment group (anti-CD96 group) and control group (n=5). In the former two groups, C. muridarum was inoculated via intranasal administration to establish mouse models of pulmonary C. muridarum infection, and the mice in the control group received intranasal administration of the inhalation buffer. In anti-CD96 group, the mice were injected with anti-CD96 antibody intraperitoneally at the dose of 250 µg every 3 days after the infection; the mice in Cm group received intraperitoneal injections of saline. The body weight of the mice was recorded daily. The mice were sacrificed 5 days after C. muridarum infection, and CD96 expression was detected by quantitative real-time PCR and Western blotting. HE staining and pathological scores were used to evaluate pneumonia of the mice. The inclusion body forming units (IFUs) were detected in the lung tissue homogenates to assess lung tissue chlamydia load. Flow cytometry and ELISA were used to assess the capacity of the lung NK cells to produce interferon-γ (IFN-γ) and regulate macrophages and Th1 cells. RESULTS: C. muridarum infection inhibited CD96 expression in NK cells of the mice. Compared with those in Cm group, the mice in antiCD96 mice showed significantly milder lung inflammation (P < 0.05) and reduced chlamydia load in the lung tissue (P < 0.05). Neutralizing CD96 with anti-CD96 significantly enhanced IFN-γ secretion by the NK cells (P < 0.05) and augmented the immunoregulatory effect of the NK cells shown by enhanced responses of the lung macrophages (P < 0.05) and Th1 cells (P < 0.05). CONCLUSIONS: Inhibition of CD96 alleviates pneumonia in C. muridarum-infected mice possibly by enhancing IFN-γ secretion by NK cells and augmenting the immunoregulatory effect of the NK cells on innate and adaptive immunity.


Assuntos
Infecções por Chlamydia , Chlamydia muridarum , Interferon gama , Lesão Pulmonar , Animais , Antígenos CD/metabolismo , Infecções por Chlamydia/complicações , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/fisiopatologia , Interferon gama/genética , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Lesão Pulmonar/etiologia , Lesão Pulmonar/genética , Lesão Pulmonar/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
6.
Nat Commun ; 11(1): 3421, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647184

RESUMO

The OX40-OX40L pathway provides crucial co-stimulatory signals for CD4 T cell responses, however the precise cellular interactions critical for OX40L provision in vivo and when these occur, remains unclear. Here, we demonstrate that provision of OX40L by dendritic cells (DCs), but not T cells, B cells nor group 3 innate lymphoid cells (ILC3s), is critical specifically for the effector Th1 response to an acute systemic infection with Listeria monocytogenes (Lm). OX40L expression by DCs is regulated by cross-talk with NK cells, with IFNγ signalling to the DC to enhance OX40L in a mechanism conserved in both mouse and human DCs. Strikingly, DC expression of OX40L is redundant in a chronic intestinal Th1 response and expression by ILC3s is necessary. Collectively these data reveal tissue specific compartmentalisation of the cellular provision of OX40L and define a mechanism controlling DC expression of OX40L in vivo.


Assuntos
Microambiente Celular , Ligante OX40/metabolismo , Células Th1/imunologia , Animais , Comunicação Celular , Sinais (Psicologia) , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Humanos , Interferon gama/biossíntese , Interleucina-12/farmacologia , Intestinos/citologia , Antígeno Ki-1/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Listeria monocytogenes/fisiologia , Camundongos Endogâmicos C57BL , Receptores CXCR5/metabolismo , Receptores OX40/metabolismo , Baço/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
Transfus Med Rev ; 34(3): 165-171, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32684483

RESUMO

The urgent need for safe and effective treatments for COVID-19 has fueled the launch of many parallel complex studies of cellular therapies with small to modest enrolment projections. By pooling data from multiple studies that are similar, we can increase the ability to achieve sufficient power to determine effectiveness more quickly through meta-analysis. A scoping review of registered clinical trials using cell-based interventions for COVID-19 was conducted to identify candidate studies for meta-analysis that could support an accelerated regulatory review. ClinicalTrials.gov and WHO International Clinical Trials Registry Platform were searched April 23, 2020. Trials were included if they utilized cell or cell-derived products to treat or prevent COVID-19. Fifty-four registered cellular therapy trials were identified and included for analysis. Studies of mesenchymal stromal cells (MSCs; 41 studies; 1129 subjects projected to receive cells) and natural killer (NK) cells (5 studies; 135 projected to received cells) were observed most commonly. A subset of studies are controlled (34 studies, or 63%), including 27 studies of MSCs and 3 of NK cells. While heterogeneity in study design exists, the cumulative projected enrolment of patients from similar studies appears sufficient to allow the detection of meaningful differences in clinically important outcomes such as mortality, admission to intensive care and need for mechanical ventilation by September 2020-sooner than any individual study could determine effectiveness. MSCs are the predominant cell type in registered trials for severe or critical COVID-19 and represent the most promising candidates for future meta-analysis. Sufficient pooled sample size to detect clinically important reductions in multiple outcomes, including mortality, is anticipated by September 2020, but may require accessing supplementary data to align outcome reporting. Regulatory approval, funding and implementation by cell manufacturing partners will be accelerated by our framework for rapid meta-analysis.


Assuntos
Infecções por Coronavirus/terapia , Pneumonia Viral/terapia , Betacoronavirus , Ensaios Clínicos como Assunto , Cuidados Críticos , Saúde Global , Humanos , Imunização Passiva , Células Matadoras Naturais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Pandemias , Sistema de Registros , Reprodutibilidade dos Testes , Projetos de Pesquisa , Respiração Artificial , Tamanho da Amostra , Resultado do Tratamento
8.
Cancer Sci ; 111(9): 3132-3141, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32579769

RESUMO

Esophageal squamous cell carcinoma (ESCC) is the main prevalent histological type of esophageal cancer, predominantly constituting 90% of cases worldwide. Despite the development of multidisciplinary therapeutic approaches, its prognosis remains unfavorable. Recently, the development of monoclonal antibodies inhibiting programmed death 1 (PD-1) or programmed death-ligand 1 (PD-L1) has led to marked therapeutic responses among multiple malignancies including ESCC. However, only a few patients achieved clinical benefits due to resistance. Therefore, precise and accurate predictive biomarkers should be identified for personalized immunotherapy in clinical settings. Because the tumor immune microenvironment can potentially influence the patient's response to immune checkpoint inhibitors, tumor immunity, such as PD-L1 expression on tumors, tumor-infiltrating lymphocytes, tumor-associated macrophages, and myeloid-derived suppressor cells, in ESCC should be further investigated. In this review, accumulated evidence regarding the tumor immune microenvironment and immune checkpoint inhibitors in ESCC are summarized.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias Esofágicas/etiologia , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/etiologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Animais , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/patologia , Humanos , Imunomodulação/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Mutação , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo
9.
Nat Med ; 26(7): 1070-1076, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32514174

RESUMO

There is an urgent need to better understand the pathophysiology of Coronavirus disease 2019 (COVID-19), the global pandemic caused by SARS-CoV-2, which has infected more than three million people worldwide1. Approximately 20% of patients with COVID-19 develop severe disease and 5% of patients require intensive care2. Severe disease has been associated with changes in peripheral immune activity, including increased levels of pro-inflammatory cytokines3,4 that may be produced by a subset of inflammatory monocytes5,6, lymphopenia7,8 and T cell exhaustion9,10. To elucidate pathways in peripheral immune cells that might lead to immunopathology or protective immunity in severe COVID-19, we applied single-cell RNA sequencing (scRNA-seq) to profile peripheral blood mononuclear cells (PBMCs) from seven patients hospitalized for COVID-19, four of whom had acute respiratory distress syndrome, and six healthy controls. We identify reconfiguration of peripheral immune cell phenotype in COVID-19, including a heterogeneous interferon-stimulated gene signature, HLA class II downregulation and a developing neutrophil population that appears closely related to plasmablasts appearing in patients with acute respiratory failure requiring mechanical ventilation. Importantly, we found that peripheral monocytes and lymphocytes do not express substantial amounts of pro-inflammatory cytokines. Collectively, we provide a cell atlas of the peripheral immune response to severe COVID-19.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus , Imunidade Celular , Leucócitos Mononucleares , Pandemias , Pneumonia Viral , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/genética , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , RNA-Seq/métodos , Índice de Gravidade de Doença , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto Jovem
10.
Anticancer Res ; 40(6): 3231-3237, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487617

RESUMO

BACKGROUND/AIM: We previously established a novel type of epidermal growth factor receptor variant III (EGFRvIII)-specific chimeric antigen receptor (CAR)-expressing natural killer (NK) cell line, designated EvCAR-KHYG-1, which inhibited the growth of glioblastoma (GBM) cells in vitro via apoptosis. MATERIALS AND METHODS: We investigated the cytokine-producing effect of EvCAR-KHYG-1 cells on GBM-like cell lines and their antitumour effect using in vivo xenograft assays. RESULTS: EvCAR-KHYG-1 cells produced interleukin-2, interferon-γ, and tumour necrosis factor-α on EGFRvIII-expressing U87MG cells. In vivo xenograft assays showed that EvCAR-KHYG-1 cells did not reduce the volume of subcutaneous tumours derived from EGFRvIII-expressing U87MG cells but did reduce tumour cell occupancy. CONCLUSION: EvCAR-KHYG-1 cells led to expression of cellular immunity-related cytokines on EGFRvIII-expressing U87MG in vitro but did not inhibit tumour progression due to the induction of a pseudo progression-like pathological feature. Future studies investigating the effect of different conditions in vivo are required to study the inhibition of tumour progression in GBM.


Assuntos
Neoplasias Encefálicas/terapia , Receptores ErbB/imunologia , Glioblastoma/terapia , Células Matadoras Naturais/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Citocinas/biossíntese , Citocinas/imunologia , Progressão da Doença , Feminino , Glioblastoma/genética , Glioblastoma/imunologia , Glioblastoma/patologia , Células HEK293 , Humanos , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Distribuição Aleatória , Receptores de Antígenos Quiméricos/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
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
12.
Medicine (Baltimore) ; 99(18): e20073, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32358389

RESUMO

Individuals infected with hepatitis B virus (HBV) are often coinfected with human immunodeficiency virus (HIV). However, individuals with chronic HBV infection living with acute HIV infection have a significantly lower HBV viral load, along with higher HBeAg and HBsAg loss than HBV-infected individuals alone. Here, we investigated the possible role of natural killer cells (NK cell) function in this progressive course to explore the relationship between phenotypic/functional changes in NK cells during acute HIV infection and HBV clearance in patients with HIV/HBV coinfection.Peripheral blood NK cells from 38 patients with primary HIV infection, including 20 with untreated HIV infection and 18 treatment-naïve patients with HIV/HBV coinfection and 16 patients with chronic HBV infection, were enrolled in this study.We found that the HIV/HBV-coinfected individuals had higher levels of NK cells than the HBV-infected individuals, due to expansion of the CD56 NK cell population. The proportion of NK cells in CD56 and CD56 NK subsets was not found significant difference between HIV/HBV-coinfected and HBV-infected individuals. However, NKG2C levels on NK cells and subsets were significantly higher in HIV/HBV-coinfected individuals than in HBV-infected individuals, whereas NKG2A levels were unaffected or decreased. In addition, the levels of degranulation CD107a, cytotoxicity and IFN-γ production of NK cells were increased in HIV/HBV-coinfected individuals than in HBV-infected individuals. The level of IL-10 production of NK cells was decreased in HIV/HBV-coinfected individuals than in HBV-infected individuals. Furthermore, the level of HBV-DNA was inversely correlated with the proportion of NKG2C and NKG2CNKG2A NK cells, while positively correlated with the proportion of NKG2A and NKG2CNKG2A NK cells. IFN-γ production was inversely correlated with levels of HBV-DNA, but the CD107a expression and IL-10 production of NK cells were not correlated with HBV-DNA levels.These results demonstrate that the upregulation of NKG2C expression, but not of NKG2A expression on the surface of NK cells increases cytolytic capacity and the amounts of cytokines produced and may play a crucial role in HBV clearance during HIV/HBV-coinfection.


Assuntos
Infecções por HIV/epidemiologia , Hepatite B Crônica/epidemiologia , Células Matadoras Naturais/metabolismo , Adulto , Pequim , Estudos Transversais , Citometria de Fluxo , Antígenos de Superfície da Hepatite B/biossíntese , Antígenos E da Hepatite B/biossíntese , Humanos , Masculino , Carga Viral
13.
Cancer Sci ; 111(7): 2234-2247, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32333709

RESUMO

Natural killer group 2 member D (NKG2D) ligands (NKG2DLs) on tumor cells engage NKG2D and mediate killing by NKG2D+ immune cells. However, tumor cells with high levels of NKG2DLs are still malignant and proliferate rapidly. We investigated the reason for NKG2DL-expressing cell progression. Tumor cells in mice were assessed for their NKG2DL expression, ability to attract immune cells, tumorigenicity, mTOR, and signal transducer and activator of transcription 3 (STAT3) signaling activation. Antibody blockade was used to determine the effect of NKG2DL-NKG2D interaction on signaling activation in vitro. Retinoic acid early inducible gene 1 (Rae1) was related to the expression of other NKG2DLs, the promotion of tumorigenicity, Mmp2 expression, mTOR and STAT3 phosphorylation in GL261 cells, and the recruitment of NKG2D+ cells in mice. Rae1 also induced NKG2DL expression, mTOR, and STAT3 phosphorylation in GL261 cells and LLC cells, but not in B16 and Pan02 cells, which did not express NKG2DLs, when cocultured with PBMCs; the induced phosphorylation was eliminated by Rae1-NKG2D blockade. Inhibition of mTOR and/or STAT3 decreased PBMC-induced migration and proliferation of GL261 cells in vitro. Rae1, a NKG2DL on tumor cells, plays a driving role in the expression of other NKG2DLs and in tumor development in mice by activating mTOR and STAT3 pathways, relying on its interaction with NKG2D on immune cells.


Assuntos
Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Testes Imunológicos de Citotoxicidade , Suscetibilidade a Doenças , Feminino , Expressão Gênica , Imuno-Histoquímica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Neoplasias/etiologia , Neoplasias/patologia , Proteínas Associadas à Matriz Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Ligação Proteica
14.
Nat Immunol ; 21(6): 684-694, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32231301

RESUMO

Aging is associated with remodeling of the immune system to enable the maintenance of life-long immunity. In the CD8+ T cell compartment, aging results in the expansion of highly differentiated cells that exhibit characteristics of cellular senescence. Here we found that CD27-CD28-CD8+ T cells lost the signaling activity of the T cell antigen receptor (TCR) and expressed a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promoted cytotoxicity against cells that expressed NKG2D ligands. Immunoprecipitation and imaging cytometry indicated that the NKG2D-DAP12 complex was associated with sestrin 2. The genetic inhibition of sestrin 2 resulted in decreased expression of NKG2D and DAP12 and restored TCR signaling in senescent-like CD27-CD28-CD8+ T cells. Therefore, during aging, sestrins induce the reprogramming of non-proliferative senescent-like CD27-CD28-CD8+ T cells to acquire a broad-spectrum, innate-like killing activity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Senescência Celular/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citotoxicidade Imunológica , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Células Matadoras Naturais/metabolismo , Transdução de Sinais , Febre Amarela/genética , Febre Amarela/imunologia , Febre Amarela/metabolismo , Febre Amarela/virologia , Vírus da Febre Amarela/imunologia
15.
PLoS One ; 15(4): e0231664, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302338

RESUMO

Natural killer (NK) cells are innate lymphocytes with functions that include target cell killing, inflammation and regulation. NK cells integrate incoming activating and inhibitory signals through an array of germline-encoded receptors to gauge the health of neighbouring cells. The reactive potential of NK cells is influenced by microRNA (miRNA), small non-coding sequences that interfere with mRNA expression. miRNAs are highly conserved between species, and a single miRNA can have hundreds to thousands of targets and influence entire cellular programs. Two miRNA species, miR-155-5p and miR-146a-5p are known to be important in controlling NK cell function, but research to best understand the impacts of miRNA species within NK cells has been bottlenecked by a lack of techniques for altering miRNA concentrations efficiently and without off-target effects. Here, we describe a non-viral and straightforward approach for increasing or decreasing expression of miRNA in primary human NK cells. We achieve >90% transfection efficiency without off-target impacts on NK cell viability, education, phenotype or function. This opens the opportunity to study and manipulate NK cell miRNA profiles and their impacts on NK cellular programs which may influence outcomes of cancer, inflammation and autoimmunity.


Assuntos
Engenharia Celular/métodos , Células Matadoras Naturais/metabolismo , MicroRNAs/genética , Transfecção/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Meios de Cultura Livres de Soro/farmacologia , Voluntários Saudáveis , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Cultura Primária de Células
16.
Nat Med ; 26(4): 511-518, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251406

RESUMO

Cellular immunity is critical for controlling intracellular pathogens, but individual cellular dynamics and cell-cell cooperativity in evolving human immune responses remain poorly understood. Single-cell RNA-sequencing (scRNA-seq) represents a powerful tool for dissecting complex multicellular behaviors in health and disease1,2 and nominating testable therapeutic targets3. Its application to longitudinal samples could afford an opportunity to uncover cellular factors associated with the evolution of disease progression without potentially confounding inter-individual variability4. Here, we present an experimental and computational methodology that uses scRNA-seq to characterize dynamic cellular programs and their molecular drivers, and apply it to HIV infection. By performing scRNA-seq on peripheral blood mononuclear cells from four untreated individuals before and longitudinally during acute infection5, we were powered within each to discover gene response modules that vary by time and cell subset. Beyond previously unappreciated individual- and cell-type-specific interferon-stimulated gene upregulation, we describe temporally aligned gene expression responses obscured in bulk analyses, including those involved in proinflammatory T cell differentiation, prolonged monocyte major histocompatibility complex II upregulation and persistent natural killer (NK) cell cytolytic killing. We further identify response features arising in the first weeks of infection, for example proliferating natural killer cells, which potentially may associate with future viral control. Overall, our approach provides a unified framework for characterizing multiple dynamic cellular responses and their coordination.


Assuntos
Comunicação Celular , Infecções por HIV/genética , Infecções por HIV/imunologia , Imunidade Celular/fisiologia , Análise de Célula Única/métodos , Doença Aguda , Reação de Fase Aguda/genética , Reação de Fase Aguda/imunologia , Reação de Fase Aguda/patologia , Adolescente , Adulto , Comunicação Celular/genética , Comunicação Celular/imunologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/imunologia , Infecções por HIV/patologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Estudos Longitudinais , Análise de Sequência de RNA/métodos , Integração de Sistemas , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Carga Viral/genética , Carga Viral/imunologia , Adulto Jovem
17.
Nat Commun ; 11(1): 1773, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286313

RESUMO

In amyotrophic lateral sclerosis (ALS), immune cells and glia contribute to motor neuron (MN) degeneration. We report the presence of NK cells in post-mortem ALS motor cortex and spinal cord tissues, and the expression of NKG2D ligands on MNs. Using a mouse model of familial-ALS, hSOD1G93A, we demonstrate NK cell accumulation in the motor cortex and spinal cord, with an early CCL2-dependent peak. NK cell depletion reduces the pace of MN degeneration, delays motor impairment and increases survival. This is confirmed in another ALS mouse model, TDP43A315T. NK cells are neurotoxic to hSOD1G93A MNs which express NKG2D ligands, while IFNγ produced by NK cells instructs microglia toward an inflammatory phenotype, and impairs FOXP3+/Treg cell infiltration in the spinal cord of hSOD1G93A mice. Together, these data suggest a role of NK cells in determining the onset and progression of MN degeneration in ALS, and in modulating Treg recruitment and microglia phenotype.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Células Matadoras Naturais/metabolismo , Neurônios Motores/metabolismo , Adulto , Idoso , Esclerose Amiotrófica Lateral/imunologia , Esclerose Amiotrófica Lateral/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Córtex Motor/imunologia , Córtex Motor/metabolismo , Córtex Motor/patologia , Neurônios Motores/imunologia , Neurônios Motores/patologia , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia
18.
PLoS One ; 15(4): e0226444, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240177

RESUMO

Glioblastoma, the most aggressive form of glioma, has a 5-year survival rate of <5%. While radiation and immunotherapies are routinely studied in the murine Gl261 glioma model, little is known about its inherent immune response. This study quantifies the temporal and spatial localization of immune cell populations and mediators during glioma development. Eight-week old male C57Bl/6 mice were orthotopically inoculated with 1x106 Gl261 cells and tumor morphology, local and systemic immune cell populations, and plasma cytokines/chemokines assessed at day 0, 1, 3, 7, 14, and 21 post-inoculation by magnetic resonance imaging, chromogenic immunohistochemistry, multiplex immunofluorescent immunohistochemistry, flow cytometry and multiplex immunoassay respectively. From day 3 tumors were distinguishable with >30% Ki67 and increased tissue vascularization (p<0.05). Increasing tumor proliferation/malignancy and vascularization were associated with significant temporal changes in immune cell populations within the tumor (p<0.05) and systemic compartments (p = 0.02 to p<0.0001). Of note, at day 14 16/24 plasma cytokine/chemokines levels decreased coinciding with an increase in tumor cytotoxic T cells, natural killer and natural killer/T cells. Data derived provide baseline characterization of the local and systemic immune response during glioma development. They reveal that type II macrophages and myeloid-derived suppressor cells are more prevalent in tumors than regulatory T cells, highlighting these cell types for further therapeutic exploration.


Assuntos
Glioma/imunologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Linhagem da Célula/imunologia , Proliferação de Células/genética , Quimiocinas/sangue , Quimiocinas/imunologia , Citocinas/sangue , Citocinas/imunologia , Progressão da Doença , Citometria de Fluxo , Glioma/sangue , Glioma/patologia , Humanos , Células Matadoras Naturais/metabolismo , Camundongos , Linfócitos T Citotóxicos/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(15): 8563-8572, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32220963

RESUMO

The small GTPase RABL3 is an oncogene of unknown physiological function. Homozygous knockout alleles of mouse Rabl3 were embryonic lethal, but a viable hypomorphic allele (xiamen [xm]) causing in-frame deletion of four amino acids from the interswitch region resulted in profound defects in lymphopoiesis. Impaired lymphoid progenitor development led to deficiencies of B cells, T cells, and natural killer (NK) cells in Rabl3 xm/xm mice. T cells and NK cells exhibited impaired cytolytic activity, and mice infected with mouse cytomegalovirus (MCMV) displayed elevated titers in the spleen. Myeloid cells were normal in number and function. Biophysical and crystallographic studies demonstrated that RABL3 formed a homodimer in solution via interactions between the effector binding surfaces on each subunit; monomers adopted a typical small G protein fold. RABL3xm displayed a large compensatory alteration in switch I, which adopted a ß-strand configuration normally provided by the deleted interswitch residues, thereby permitting homodimer formation. Dysregulated effector binding due to conformational changes in the switch I-interswitch-switch II module likely underlies the xm phenotype. One such effector may be GPR89, putatively an ion channel or G protein-coupled receptor (GPCR). RABL3, but not RABL3xm, strongly associated with and stabilized GPR89, and an N-ethyl-N-nitrosourea (ENU)-induced mutation (explorer) in Gpr89 phenocopied Rabl3 xm.


Assuntos
Linfócitos B/imunologia , Linfopoese , Proteínas Mutantes/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Linfócitos T/imunologia , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Cristalografia por Raios X , Feminino , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muromegalovirus/imunologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , Conformação Proteica , Linfócitos T/metabolismo , Linfócitos T/patologia
20.
Nat Med ; 26(2): 259-269, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32042191

RESUMO

Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9-dependent resistance to natural killer cells. Loss of developmental stage-specific constraint in macrometastases triggered by natural killer cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis.


Assuntos
Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Sistema Imunitário/fisiologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Metástase Neoplásica , Animais , Brônquios/metabolismo , Diferenciação Celular , Linhagem da Célula , Análise por Conglomerados , Bases de Dados Genéticas , Progressão da Doença , Endoderma/metabolismo , Feminino , Humanos , Hidrogéis/química , Células Matadoras Naturais/metabolismo , Pulmão/patologia , Camundongos , Fenótipo , Alvéolos Pulmonares/metabolismo , Regeneração , Transdução de Sinais
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