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1.
Science ; 368(6493): 897-901, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32381591

RESUMO

Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes, called supramolecular attack particles (SMAPs), from CTLs to target cells. SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis, and CRISPR editing identified a carboxyl-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nanometer diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo targeted by the specificity of shell components.


Assuntos
Citotoxicidade Imunológica , Granzimas/metabolismo , Complexos Multiproteicos/metabolismo , Perforina/metabolismo , Linfócitos T Citotóxicos/metabolismo , Trombospondina 1/metabolismo , Sistemas CRISPR-Cas , Exocitose , Edição de Genes , Humanos , Células K562 , Trombospondina 1/genética , Tomografia por Raios X
2.
PLoS One ; 15(4): e0231430, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32275689

RESUMO

Extracellular vesicles (EVs) in the tumor microenvironment facilitate intercellular communication. Cancer cell-derived EVs act as an immunosuppressor by transporting cargos and presenting transmembrane proteins. By contrast, CD8+ cytotoxic T-lymphocytes (CTLs) exert anti-cancer cytotoxicity via the pore-forming protein perforin. Here, we hypothesize that although EVs are destroyed by perforin, cancer cell-derived EVs might possess mechanisms that enable them to avoid this destruction. We used a breast cancer cell line, MDA-MB-231-luc-D3H2LN (D3H2LN), to generate EVs. Destruction of the EVs by perforin was demonstrated visually using atomic force microscopy. To investigate immunosuppressive metabolites within cancer cell-derived EVs, we performed metabolomic profiling of EVs from D3H2LN cells cultured for 48 h with or without IFN-γ, which induces metabolic changes in the cells. We found that both types of EV from IFN-γ treated D3H2LN cells and non-treated D3H2LN cells contained adenosine, which has immunosuppressive effects. When we exposed cancer cell-derived EVs to CTLs, perforin secretion by CTLs fell significantly. In addition, the decreases in perforin secretion were ameliorated by treatment with adenosine deaminase, which degrades extracellular adenosine. Taken together, these results suggest that after perforin secreted by CTLs disrupts the membrane of EVs, adenosine released from the EVs acts as an immunosuppressive metabolite by binding to the adenosine receptor on the CTL membrane. This mechanism provides a novel survival strategy using cancer cell-derived EVs.


Assuntos
Adenosina/metabolismo , Vesículas Extracelulares/metabolismo , Perforina/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Vesículas Extracelulares/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Perforina/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos
3.
Nucleic Acids Res ; 48(1): e2, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31680162

RESUMO

The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using this strategy, we achieved robust and specific gene knockdown throughout the entire parasite life cycle. We also successfully silenced the endogenous gene perforin-like protein 2, phenocopying a full gene knockout. Transcriptionally restricting Ago2 expression to the liver stage further enabled us to perform a stage-specific gene knockout. The RNAi-competent Plasmodium lines reported here will be a valuable resource for loss-of-function phenotyping of the many uncharacterized genes of Plasmodium in low or high throughput, without the need to engineer the target gene locus. Thereby, our new strategy and transgenic Plasmodium lines will ultimately benefit the discovery of urgently needed antimalarial drug and vaccine candidates. Generally, the ability to render RNAi-negative organisms RNAi-competent by mere introduction of two components, Ago2 and AgoshRNA, is a unique paradigm that should find broad applicability in other species.


Assuntos
Proteínas Argonauta/genética , Engenharia Genética/métodos , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Anopheles/parasitologia , Proteínas Argonauta/metabolismo , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/antagonistas & inibidores , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Estágios do Ciclo de Vida/genética , Camundongos , Camundongos Endogâmicos C57BL , Mosquitos Vetores/parasitologia , Organismos Geneticamente Modificados , Perforina/genética , Perforina/metabolismo , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , RNA Interferente Pequeno/metabolismo , Transgenes
4.
Arthritis Rheumatol ; 72(6): 912-918, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31876120

RESUMO

OBJECTIVE: To address the independent roles of peptidylarginine deiminase type 2 (PAD2) and PAD4 in generating rheumatoid arthritis (RA) autoantigens by using a system that mimics intracellular citrullination in the RA joint. METHODS: PAD2- or PAD4-expressing 293T cells and mock-transfected cells were used as targets in cytotoxic assays using lymphokine-activated killer cells, cytotoxic YT cell granule contents, or purified human perforin. Protein citrullination and autoantigen production were determined by immunoblotting using the anti-modified citrulline-Senshu method and RA sera (n = 30), respectively. RESULTS: RA sera recognized at least 3 categories of autoantigens in PAD-expressing target cells killed by the cytotoxic lymphocyte granule-induced death pathway. These included: 1) autoantigens targeted in their native form, 2) citrullinated antigens, and 3) antigens cleaved by cytotoxic proteases (e.g., granzymes). Interestingly, although target cells expressing PAD2 or PAD4 showed prominent hypercitrullination of a broad range of proteins during cytotoxic granule-induced cell damage, autoantibodies in RA sera targeted only a very limited number of antigens in hypercitrullinated cells. Furthermore, RA sera showed distinct reactivities to autoantigens generated by PAD2 or PAD4. CONCLUSION: The cytotoxic granule-induced death pathway has the capacity to modify antigens by inducing hypercitrullination and antigen cleavage in target cells. Interestingly, among a large number of citrullinated proteins generated by PAD2 and PAD4 in cells, only a few are likely involved in the production of autoantibodies in RA.


Assuntos
Artrite Reumatoide/imunologia , Autoantígenos/imunologia , Citrulinação/imunologia , Proteína-Arginina Desiminase do Tipo 2/imunologia , Proteína-Arginina Desiminase do Tipo 4/imunologia , Artrite Reumatoide/sangue , Citrulina/metabolismo , Células HEK293 , Humanos , Perforina/metabolismo
5.
Exp Cell Res ; 386(1): 111719, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31726050

RESUMO

Gamma delta (γδ) T cell-based tumor immunotherapy has been one of the most promising cancer immunotherapeutic strategies. However, the key regulators of the Vγ9Vδ2 T cell-mediated antitumor response remain unclear. Recently, mounting reports have indicated that Tim-3 performs critical roles in the regulation of the activities of immune cells, including Vγ9Vδ2 T cells. However, the roles of Tim-3 in Vγ9Vδ2 T cell-mediated killing of colon cancer cells and the underlying mechanism remain largely unknown. Here, the proportion of Tim-3+ γδ T cells was significantly increased in both the peripheral blood and colon cancer tissue of patients and was significantly associated with TNM staging and tumor volume. Additionally, the activation of Tim-3 signaling significantly inhibited the killing efficiency of Vγ9Vδ2 T cells against colon cancer cells. In addition, Tim-3 signaling reduced the expression of perforin and granzyme B in Vγ9Vδ2 T cells. Blocking the perforin/granzyme B pathway also decreased the cytotoxicity of Vγ9Vδ2 T cells to colon cancer cells. Moreover, Tim-3 signaling reduced the perforin and granzyme B expression of Vγ9Vδ2 T cells in an ERK1/2 signaling pathway-dependent manner. This knowledge reveals that Tim-3 may be a promising therapeutic target to improve Vγ9Vδ2 T cell-based adoptive immunotherapy for colon cancer.


Assuntos
Neoplasias do Colo/imunologia , Granzimas/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Linfócitos Intraepiteliais/imunologia , Perforina/metabolismo , Idoso , Células Cultivadas , Neoplasias do Colo/terapia , Feminino , Granzimas/genética , Células HCT116 , Receptor Celular 2 do Vírus da Hepatite A/genética , Humanos , Imunoterapia/métodos , Sistema de Sinalização das MAP Quinases , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Perforina/genética
6.
Int J Nanomedicine ; 14: 9325-9336, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31819434

RESUMO

Background: During the past few years, immune cell therapy for malignant cancer has benefited a considerable amount of patients worldwide. As one of several promising candidates for immunotherapy, Vγ9Vδ2 γδ T cells have many unique biological advantages, such as non-MHC restriction and have been noted as the earliest source of IFN-γ. However, potentiating anti-tumor functions of γδ T cells has become of particular interest to researchers studying γδ T cell applications. Purpose: In this study, we proposed a nanotechnology-based methodology for strengthening γδ T cell functions. Methods: As a type of reliable, biocompatible material, chitosan nanoparticles (CSNPs) were used to enhance anti-tumor immunity of γδ T cells. Results: First, we found that the size of prepared CSNPs distributed 50 to 100 nm, and that CSNPs had optimal immunocompatibility. Then, we observed that CSNPs could induce α-tubulin cytoskeleton polarization and rearrangement, correlating with a higher killing ability of γδ T cells. Furthermore, we revealed that CSNPs could enhance Vγ9Vδ2 T cell anti-tumor functions by upregulating killing of related receptors, including NKG2D, CD56, FasL, and perforin secretion. Conclusion: Our work provided evidence of application for CSNPs based bio-carrier in immunotherapy. More importantly, we proposed a new strategy for enhancing γδ T cell anti-tumor activity using nanobiomaterial, which could benefit future clinical applications of γδ T cells.


Assuntos
Quitosana/farmacologia , Citoesqueleto/metabolismo , Nanopartículas/química , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T Citotóxicos/imunologia , Regulação para Cima , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Ativação Linfocitária , Nanopartículas/ultraestrutura , Perforina/metabolismo , Tubulina (Proteína)/metabolismo
7.
Nat Commun ; 10(1): 5396, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776337

RESUMO

Killer T cells (cytotoxic T lymphocytes, CTLs) maintain immune homoeostasis by eliminating virus-infected and cancerous cells. CTLs achieve this by forming an immunological synapse with their targets and secreting a pore-forming protein (perforin) and pro-apoptotic serine proteases (granzymes) into the synaptic cleft. Although the CTL and the target cell are both exposed to perforin within the synapse, only the target cell membrane is disrupted, while the CTL is invariably spared. How CTLs escape unscathed remains a mystery. Here, we report that CTLs achieve this via two protective properties of their plasma membrane within the synapse: high lipid order repels perforin and, in addition, exposed phosphatidylserine sequesters and inactivates perforin. The resulting resistance of CTLs to perforin explains their ability to kill target cells in rapid succession and to survive these encounters. Furthermore, these mechanisms imply an unsuspected role for plasma membrane organization in protecting cells from immune attack.


Assuntos
Lipídeos de Membrana/química , Células T Matadoras Naturais/imunologia , Linfócitos T Citotóxicos/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Morte Celular , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/metabolismo , Lipídeos de Membrana/metabolismo , Camundongos Transgênicos , Perforina/metabolismo , Fosfatidilserinas/metabolismo , Linfócitos T Citotóxicos/química , Linfócitos T Citotóxicos/imunologia
8.
Nature ; 575(7783): 505-511, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723265

RESUMO

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.


Assuntos
Bacteriófagos/fisiologia , Enterococcus faecalis/patogenicidade , Enterococcus faecalis/virologia , Microbioma Gastrointestinal , Hepatite Alcoólica/microbiologia , Hepatite Alcoólica/terapia , Terapia por Fagos , Alcoolismo/complicações , Alcoolismo/microbiologia , Animais , Enterococcus faecalis/isolamento & purificação , Etanol/efeitos adversos , Fígado Gorduroso/complicações , Fígado Gorduroso/microbiologia , Fezes/microbiologia , Feminino , Vida Livre de Germes , Hepatite Alcoólica/complicações , Hepatite Alcoólica/mortalidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Perforina/metabolismo
9.
Immunohorizons ; 3(11): 531-546, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31732662

RESUMO

NK cell functions are tightly regulated by the balance between the inhibitory and stimulatory surface receptors. We investigated the surface expression of galectin-9 (Gal-9) and its function in NK cells from HIV-infected individuals on antiretroviral therapy, long-term nonprogressors, and progressors compared with healthy controls. We also measured the expression of TIGIT and TIM-3 on different NK cell subpopulations and compared their functionality to Gal-9 + NK cells. Our data demonstrated significant upregulation of Gal-9 on NK cells in HIV-infected groups versus healthy controls. Gal-9 expression was associated with impaired expression of cytotoxic effector molecules granzyme B, perforin, and granulysin. In contrast, Gal-9 expression significantly enhanced IFN-γ expression in NK cells of HIV-1-infected individuals. We also found an expansion of TIGIT + NK cells in HIV-infected individuals; however, dichotomous to Gal-9 + NK cells, TIGIT + NK cells expressed significantly higher amounts of cytotoxic molecules but lower IFN-γ. Moreover, lower expression of cytotoxic effector molecules in Gal-9+ NK cells was associated with higher CD107a expression, which suggests indiscriminate degranulation. Importantly, a positive correlation between the plasma viral load and Gal-9+ NK cells was observed in progressors. Finally, we found that a cytokine mixture (IL-12, IL-15, and IL-18) can improve effector functions of Gal-9+ NK cells in HIV-infected individuals, although, such an effect was observed for Gal-9- NK cells, as well. Overall, our data highlight the important role of Gal-9 in dysfunctional NK cells and, more importantly, a dichotomy for the role of Gal-9 versus TIGIT and suggest a potential new avenue for the development of therapeutic approaches.


Assuntos
Galectinas/imunologia , Infecções por HIV/imunologia , Células Matadoras Naturais/imunologia , Receptores Imunológicos/imunologia , Fármacos Anti-HIV/uso terapêutico , Estudos de Casos e Controles , Células Cultivadas , Galectinas/sangue , Granzimas/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1 , Humanos , Interferon gama/imunologia , Interleucinas/imunologia , Perforina/metabolismo , Receptores Imunológicos/sangue , Carga Viral
10.
BMC Cancer ; 19(1): 823, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31429730

RESUMO

BACKGROUND: Natural killer (NK) cell dysfunction following cancer surgery has been shown to promote metastases. Recent studies demonstrate an emerging role for lipids in the modulation of NK cell innate responses. However, the mechanisms involved in lipid modulation of NK cell postoperative anti-tumor function are unknown. This current study will determine whether the lipid accumulation via scavenger receptors on NK cells is responsible for the increase in postoperative metastasis. METHODS: Lipid content in mouse and human NK cells was evaluated by flow cytometry. NK cell scavenger receptor (SR) expression was measured by microarray analysis, validated by qRT-PCR and flow cytometry. NK cell ex vivo and in vivo tumor killing was measured by chromium-release and adoptive transfer assays, respectively. The mediating role of surgery-expanded granulocytic myeloid derived suppressor cells (gMDSC) in SR induction on NK cells was evaluated using co-culture assays. RESULTS: NK cells in surgery-treated mice demonstrated increased lipid accumulation, which occurred via up-regulation of MSR1, CD36 and CD68. NK cells with high lipid content had diminished ability to lyse tumor targets ex vivo. Adoptive transfer of lipid-laden NK cells into NK cell-deficient mice were unable to protect against a lung tumor challenge. Granulocytic MDSC from surgery-treated mice increased SR expression on NK cells. Colorectal cancer surgical patients showed increased NK cell lipid content, higher CD36 expression, decreased granzyme B and perforin production in addition to reduced cytotoxicity in the postoperative period. CONCLUSIONS: Postoperative lipid accumulation promotes the formation of metastases by impairing NK cell function in both preclinical surgical models and human surgical colorectal cancer patient samples. Understanding and targeting the mechanisms underlying lipid accumulation in innate immune NK cells can improve prognosis in cancer surgical patients.


Assuntos
Neoplasias Colorretais/metabolismo , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ácidos Palmíticos/metabolismo , Transferência Adotiva , Animais , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos CD36/genética , Neoplasias Colorretais/cirurgia , Modelos Animais de Doenças , Feminino , Granzimas/metabolismo , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Perforina/metabolismo , Período Pós-Operatório , Receptores Depuradores/genética , Receptores Depuradores Classe A/genética
11.
Mediators Inflamm ; 2019: 6519427, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31316301

RESUMO

This study is to investigate the role of regulatory B (Breg) cells in cervical cancer. In total, 70 cases of cervical cancer, 52 cases of cervical intraepithelial neoplasia (CIN), and 40 normal controls were enrolled. The percentage of Breg cells was detected by flow cytometry. Serum levels of IL-10 were measured by ELISA. The correlation between Breg cells and the clinical characterizations of cervical cancer was analyzed. The inhibition effect of Breg cells on CD8+ T cells was tested by blocking IL-10 in vitro. The percentage of CD19+CD5+CD1d+ Breg cells and the level of IL-10 of patients with cervical cancer or CIN were significantly higher than those in the control group (P < 0.05). And the postoperative levels of Breg cells and IL-10 were significantly lower than the preoperative levels (P < 0.05). Breg cells and the IL-10 level were positively correlated in cervical cancer patients (r = 0.516). In addition, the Breg cell percentage was closely related to the FIGO stages, lymph node metastasis, tumor differentiation, HPV infection, and the tumor metastasis of cervical cancer (P < 0.05). The Breg cell percentage was negatively correlated with CD8+ T cells of cervical cancer patients (r = -0.669). The level of IL-10 in the culture supernatant of Bregs treated with CpG was significantly higher than that of non-Bregs (P < 0.05). After coculture with Bregs, the quantity of CD8+ T cells to secrete perforin and Granzyme B was significantly decreased, and this effect was reversed after blocking IL-10 by a specific antibody. Breg cells are elevated in cervical cancer and associated with disease progression and metastasis. Moreover, they can inhibit the cytotoxicity of CD8+ T cells.


Assuntos
Linfócitos B Reguladores/imunologia , Neoplasia Intraepitelial Cervical/imunologia , Neoplasias do Colo do Útero/imunologia , Adulto , Idoso , Antígenos CD19/sangue , Antígenos CD1d/sangue , Antígenos CD5/sangue , Linfócitos T CD8-Positivos/citologia , Estudos de Casos e Controles , Ilhas de CpG , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Granzimas/metabolismo , Humanos , Interleucina-10/sangue , Metástase Linfática , Pessoa de Meia-Idade , Perforina/metabolismo
12.
Ann Clin Transl Neurol ; 6(7): 1151-1164, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31353869

RESUMO

OBJECTIVE: CD8+ T cells are the most prevailing lymphocyte population in inflammatory lesions of patients with multiple sclerosis (MS) but it is not even known whether they are merely passive bystanders or actively communicate with other cells in the brain. To identify their potential interaction partners, we analyzed CD8+ T cells that contained vectorially oriented cytotoxic granules and analyzed the areas to which the granules pointed. METHODS: We stained cryo-sections of active MS lesions of an index patient with antibodies to CD8 and perforin, searched for vectorially oriented perforin granules, and isolated target areas opposing the granules and control areas by laser-microdissection. From both areas, we analyzed cell-type specific transcripts by next-generation sequencing. In parallel, we stained samples from the index-patient and other patients by four-color immunohistochemistry (IHC). RESULTS: We found transcripts of the mononuclear phagocyte (MP) specific markers CD163 and CD11b only in the microdissected target areas but not in control areas. We validated the finding that MPs are communication partners of CD8+ T cells in MS lesions by classical IHC in samples from the index-patient and other patients with acute and progressive MS and other inflammatory neurological diseases. INTERPRETATION: Because CD163 and CD11b are specifically expressed in MPs, our findings suggest that CD8+ T cells communicate with local MPs. Although it is still unclear if these interactions lead to killing of the communication partners by CD8+ T cells, our data underline that CD8+ T cells play an active role in the pathogenesis of MS.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/imunologia , Sistema Fagocitário Mononuclear/imunologia , Esclerose Múltipla/imunologia , Fagócitos/imunologia , Adulto , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Encéfalo/patologia , Antígeno CD11b/imunologia , Linfócitos T CD8-Positivos/patologia , Humanos , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Bainha de Mielina/patologia , Perforina/metabolismo , Fagócitos/patologia , Receptores de Superfície Celular/imunologia
13.
Hum Immunol ; 80(10): 871-877, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31326139

RESUMO

A subset of natural killer (NK) cells with CD56+/brightCD16dim/- expression is recently shown to present critical regulatory functions. Functional characteristics of CD56+/bright NK cells in osteoarthritis (OA) patients remains unknown. Here, we remedied this problem by comparing the NK cells from healthy controls and OA patients. Data showed that the CD56brightCD16- NK subset was significantly enriched in OA patients. These CD56brightCD16- NK cells from OA patients presented significantly higher IFNG transcription and IFN-γ protein secretion than those from healthy controls, both directly ex vivo and after activation via various stimulating reagents, including IL-2/IL-15, K562, and PMA/ionomycin. On the other hand, the transcription and secretion of granzyme A (Gzm-A), Gzm-B, and perforin were significantly lower in CD56brightCD16- NK cells from OA patients than in CD56brightCD16- NK cells from healthy controls. Also, the CD56brightCD16- NK cells from OA patients were less capable of suppressing the proliferation of autologous CD4+ T cells, in a manner that was dependent on the expression of Gzm-B and perforin. Interestingly, CD4+ T cells co-incubated with CD56brightCD16- NK cells were prone to express a higher level of IFNG, and the CD56brightCD16- NK cells from OA patients were more potent at stimulating IFNG than the CD56brightCD16- NK cells from healthy controls. Overall, our investigation demonstrated that CD56brightCD16- NK cells from osteoarthritis patients were shifted toward an IFN-γ-promoting phenotype and with reduced regulatory functions.


Assuntos
Antígeno CD56/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Osteoartrite/imunologia , Fenótipo , Receptores de IgG/metabolismo , Idoso , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Técnicas de Cocultura/métodos , Feminino , Proteínas Ligadas por GPI/metabolismo , Granzimas/genética , Granzimas/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Perforina/genética , Perforina/metabolismo
14.
Aquat Toxicol ; 213: 105223, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31207538

RESUMO

Methylmercury (MeHg), cadmium (Cd) and arsenic (As(III)) are among the most toxic metals in aquatic systems that have been associated with multiple animal and human health problems. This study investigated cytotoxic, oxidative stress, and apoptosis effects on fish leukocytes following their exposure to metals. A preliminary study indicated that leukocytes exposed to MeHg at a concentration of 0.01 mM, Cd at 0.05 mM, and As(III) at 2 mM showed a time-dependent cell viability reduction (around 40%), so they were selected for further experiments. To evaluate the effect of MeHg, Cd and As(III) on Pacific red snapper Lutjanus peru, we measured cytotoxicity, reactive oxygen species, antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT)), nitric oxide production, apoptosis-related and immune-related genes on head-kidney and spleen leukocytes following exposure to MeHg (0.01 mM), Cd (0.05 mM) and As(III) (2 mM) for 30 min and 2 h. Reactive oxygen species (ROS) generation highly increased in time-dependent doses in head-kidney leukocytes compared with the control group. Regarding antioxidant activity, SOD increased significantly in leukocytes exposed to any heavy metals after two h. Expressly, CAT activity decreased in those leukocytes exposed to Cd and As(III). Apoptotic function genes (Casp-2, Casp-3, and Casp-7) strongly up-regulated after heavy metal exposure, but Cd was more toxic. Finally, granzyme A and perforin 1 strongly up-regulated in leukocytes exposed to MeHg and As(III) compared with the control group. Our data showed that MeHg, Cd, and As(III) might have been cytotoxic and induced oxidative stress and apoptosis with possible biological consequences in fish.


Assuntos
Apoptose/efeitos dos fármacos , Arsênico/toxicidade , Cádmio/toxicidade , Leucócitos/metabolismo , Compostos de Metilmercúrio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Perciformes/metabolismo , Testes de Toxicidade , Animais , Antioxidantes/metabolismo , Catalase/metabolismo , Morte Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Granzimas/metabolismo , Leucócitos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Óxido Nítrico/biossíntese , Perforina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Poluentes Químicos da Água/toxicidade
15.
Transpl Int ; 32(11): 1203-1215, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31225919

RESUMO

We have previously reported that ICOS-Ig expressed locally by a PIEC xenograft induces a perigraft cellular accumulation of CD4+ CD25+ Foxp3+ T cells and specific xenograft prolongation. In the present study we isolated and purified CD4+ CD25+ T cells from ICOS-Ig secreting PIEC grafts to examine their phenotype and mechanism of xenograft survival using knockout and mutant mice. CD4+ CD25+ T cells isolated from xenografts secreting ICOS-Ig were analysed by flow cytometry and gene expression by real-time PCR. Regulatory function was examined by suppression of xenogeneic or allogeneic primed CD4 T cells in vivo. Graft prolongation was shown to be dependent on a pre-existing Foxp3+ Treg, IL-10, perforin and granzyme B. CD4+ CD25+ Foxp3+ T cells isolated from xenografts secreting ICOS-Ig demonstrated a phenotype consistent with nTreg but with a higher expression of CD275 (ICOSL), expression of CD278 (ICOS) and MHC II and loss of CD73. Moreover, these cells were functional and specifically suppressed xenogeinic but not allogeneic primed T cells in vivo.


Assuntos
Linfócitos T CD4-Positivos/citologia , Sobrevivência de Enxerto , Xenoenxertos/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Animais , Apoptose , Linhagem Celular , Fatores de Transcrição Forkhead/metabolismo , Granzimas/metabolismo , Interleucina-10/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Perforina/metabolismo , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo
16.
Cancer Immunol Immunother ; 68(8): 1287-1301, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31253998

RESUMO

Patchy infiltration of tumors by cytotoxic T cells (CTLs) predicts poorer prognosis for cancer patients. The factors limiting intratumoral CTL dissemination, though, are poorly understood. To study CTL dissemination in tumors, we histologically examined human melanoma samples and used mice to image B16-OVA tumors infiltrated by OT-I CTLs using intravital two-photon microscopy. In patients, most CTLs concentrated around peripheral blood vessels, especially in poorly infiltrated tumors. In mice, OT-I CTLs had to cluster around tumor cells to efficiently kill them in a contact-and perforin-dependent manner and cytotoxicity was strictly antigen-specific. OT-I CTLs as well as non-specific CTLs concentrated around peripheral vessels, and cleared the tumor cells around them. This was also the case when CTLs were injected directly into the tumors. CTLs crawled rapidly only in areas within 50 µm of flowing blood vessels and transient occlusion of vessels immediately, though reversibly, stopped their migration. In vitro, oxygen depletion and blockade of oxidative phosphorylation also reduced CTL motility. Taken together, these results suggest that hypoxia limits CTL migration away from blood vessels, providing immune-privileged niches for tumor cells to survive. Normalizing intratumoral vasculature may thus synergize with tumor immunotherapy.


Assuntos
Vasos Sanguíneos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Neoplasias Cutâneas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/imunologia , Movimento Celular , Citotoxicidade Imunológica , Humanos , Melanoma/irrigação sanguínea , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Neovascularização Patológica , Fosforilação Oxidativa , Perforina/metabolismo , Neoplasias Cutâneas/irrigação sanguínea
17.
PLoS Pathog ; 15(6): e1007825, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31220184

RESUMO

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.


Assuntos
Toxinas Bacterianas/metabolismo , Células Epiteliais/metabolismo , Epitélio Anterior/metabolismo , Infecções por Proteus/metabolismo , Proteus/metabolismo , Infecções por Serratia/metabolismo , Serratia marcescens/metabolismo , Animais , Toxinas Bacterianas/genética , Morte Celular , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Epitélio Anterior/microbiologia , Epitélio Anterior/patologia , Humanos , Camundongos , Perforina/genética , Perforina/metabolismo , Proteus/genética , Infecções por Proteus/genética , Infecções por Proteus/microbiologia , Infecções por Proteus/patologia , Células RAW 264.7 , Infecções por Serratia/genética , Infecções por Serratia/microbiologia , Infecções por Serratia/patologia , Serratia marcescens/genética , Suínos , Sistemas de Secreção Tipo V/genética , Sistemas de Secreção Tipo V/metabolismo
18.
J Pediatr Hematol Oncol ; 41(5): e277-e283, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31107368

RESUMO

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease associated with immune system hyperactivation and the appearance of serious systemic disturbances. The purpose of this study was an assessment of natural killer (NK) cell disturbances in a group of children with clinical signs of HLH. A total of 43 children with HLH and 17 healthy children were enrolled in the study. NK phenotyping, intracellular perforin staining, and cytotoxicity tests were performed by using the flow cytometry method. HLH patients were divided into 6 HLH types: 9% infection-related HLH; 7% malignancy-related HLH; 21% macrophage activating syndrome; 12% familial hemophagocytic lymphohistiocytosis; 2% X-linked lymphoproliferative syndrome; and 49% as HLH of unknown background. A positive correlation was observed between cytotoxicity and NK cells in children with HLH (P=0.01). In all HLH groups, the percentage of NK cells was significantly lower than in the control population. The spontaneous cytotoxicity was significantly lower in HLH patients. The results presented in this study indicate the importance of impaired function and the number of NK cells in the pathogenesis of HLH. Nonetheless, the background of disturbances seems to be different in various cases.


Assuntos
Células Matadoras Naturais/patologia , Linfo-Histiocitose Hemofagocítica/imunologia , Estudos de Casos e Controles , Criança , Testes Imunológicos de Citotoxicidade , Citotoxicidade Imunológica , Feminino , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Contagem de Linfócitos , Linfo-Histiocitose Hemofagocítica/epidemiologia , Linfo-Histiocitose Hemofagocítica/etiologia , Masculino , Perforina/metabolismo , Polônia
19.
BMC Infect Dis ; 19(1): 433, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101076

RESUMO

BACKGROUND: Natural killer (NK) cells are part of the innate immune system and provide surveillance against viruses and cancers. The ability of NK cells to kill virus-infected cells depends on the balance between the effects of inhibitory and activating NK cell receptors. This study aimed to investigate the phenotypic profile and the functional capacity of NK cells in the context of HTLV-1 infection. METHODS: This cross-sectional study sequentially recruited HTLV-1 infected individuals with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and asymptomatic HTLV-1 (AS) from the Integrated and Multidisciplinary HTLV Center in Salvador, Brazil. Blood samples from healthy blood donors served as controls. NK cell surface receptors (NKG2D, KIR2DL2/KIR2DL3, NKp30, NKG2A, NKp46, TIM-3 and PD-1), intracellular cytolytic (Granzyme B, perforin) and functional markers (CD107a for degranulation, IFN-γ) were assayed by flow cytometry in the presence or absence of standard K562 target cells. In addition, cytotoxicity assays were performed in the presence or absence of anti-NKp30. RESULTS: The frequency of NKp30+ NK cells was significantly decreased in HAM/TSP patients [58%, Interquartile Range (IQR) 30-61] compared to controls (73%, IQR 54-79, p = 0.04). The production of cytolytic (perforin, granzyme B) and functional markers (CD107a and IFN-γ) was higher in unstimulated NK cells from HAM/TSP and AS patients compared to controls. By contrast, stimulation with K562 target cells did not alter the frequency of CD107a+ NK cells in HAM/TSP subjects compared to the other groups. Blockage of the NKp30 receptor was shown to decrease cytotoxic activity (CD107a) and IFN-γ expression only in asymptomatic HTLV-1-infected individuals. CONCLUSIONS: NK cells from individuals with a diagnosis of HAM/TSP present decreased expression of the activating receptor NKp30, in addition to elevated degranulation activity that remained unaffected after blocking the NKp30 receptor.


Assuntos
Células Matadoras Naturais/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Paraparesia Espástica Tropical/imunologia , Adulto , Anticorpos Monoclonais/farmacologia , Biomarcadores/metabolismo , Estudos Transversais , Feminino , Citometria de Fluxo , Granzimas/metabolismo , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Humanos , Interferon gama/metabolismo , Células K562 , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Masculino , Pessoa de Meia-Idade , Receptor 3 Desencadeador da Citotoxicidade Natural/antagonistas & inibidores , Paraparesia Espástica Tropical/virologia , Perforina/metabolismo
20.
Int Immunol ; 31(6): 385-396, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31051036

RESUMO

Burkholderia cepacia complex (Bcc), which includes B. cenocepacia and B. multivorans, pose a life-threatening risk to patients with cystic fibrosis. Eradication of Bcc is difficult due to the high level of intrinsic resistance to antibiotics, and failure of many innate immune cells to control the infection. Because of the pathogenesis of Bcc infections, we wondered if a novel mechanism of microbial host defense involving direct antibacterial activity by natural killer (NK) cells might play a role in the control of Bcc. We demonstrate that NK cells bound Burkholderia, resulting in Src family kinase activation as measured by protein tyrosine phosphorylation, granule release of effector proteins such as perforin and contact-dependent killing of the bacteria. These studies provide a means by which NK cells could play a role in host defense against Bcc infection.


Assuntos
Infecções por Burkholderia/imunologia , Burkholderia cepacia/fisiologia , Burkholderia/fisiologia , Fibrose Cística/imunologia , Células Matadoras Naturais/imunologia , Adesão Celular , Degranulação Celular , Linhagem Celular , Citotoxicidade Imunológica , Humanos , Imunidade Celular , Perforina/metabolismo , Fosforilação , Transdução de Sinais , Quinases da Família src/metabolismo
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