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1.
Scand J Immunol ; 94(5): e13102, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34755902

RESUMO

During COVID-19 infection, reduced function of natural killer (NK) cells can lead to both compromised viral clearance and dysregulation of the immune response. Such dysregulation leads to overproduction of cytokines, a raised neutrophil/lymphocyte ratio and monocytosis. This in turn increases IL-6 expression, which promotes scar and thrombus formation. Excess IL-6 also leads to a further reduction in NK function through downregulation of perforin expression, therefore forming a pathogenic auto-inflammatory feedback loop. The perforin/granzyme system of cytotoxicity is the main mechanism through which NK cells and cytotoxic T lymphocytes eliminate virally infected host cells, as well as being central to their role in regulating immune responses to microbial infection. Here, we present epidemiological evidence suggesting an association between perforin expression and resistance to COVID-19. In addition, we outline the manner in which a pathogenic auto-inflammatory feedback loop could operate and the relationship of this loop to genes associated with severe COVID-19. Such an auto-inflammatory loop may be amenable to synergistic multimodal therapy.


Assuntos
COVID-19/imunologia , Síndrome da Liberação de Citocina/imunologia , Células Matadoras Naturais/imunologia , Linfo-Histiocitose Hemofagocítica/imunologia , Neutrófilos/imunologia , Perforina/metabolismo , SARS-CoV-2/fisiologia , Animais , Autoimunidade/genética , COVID-19/epidemiologia , Síndrome da Liberação de Citocina/epidemiologia , Resistência à Doença , Humanos , Interleucina-6/metabolismo , Linfo-Histiocitose Hemofagocítica/epidemiologia , Perforina/genética
2.
Nat Commun ; 12(1): 5841, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615883

RESUMO

Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn's disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-ß strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn's disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn's disease patients and may play a role in the pathology of the disease.


Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Linfócitos/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Perforina/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Células Cultivadas , Doença de Crohn/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunidade Inata/genética , Imunidade Inata/fisiologia , Inflamação/imunologia , Inflamação/metabolismo , Linfócitos/imunologia , Perforina/genética , Reação em Cadeia da Polimerase em Tempo Real
3.
Biomedica ; 41(Sp. 2): 86-102, 2021 10 15.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-34669281

RESUMO

INTRODUCTION: Immunological markers have been described during COVID-19 and persist after recovery. These immune markers are associated with clinical features among SARSCoV-2 infected individuals. Nevertheless, studies reporting a comprehensive analysis of the immune changes occurring during SARS-CoV-2 infection are still limited. OBJECTIVE: To evaluate the production of proinflammatory cytokines, the antibody response, and the phenotype and function of NK cells and T cells in a Colombian family cluster with SARS-CoV-2 infection. MATERIALS AND METHODS: Proinflammatory cytokines were evaluated by RT-PCR and ELISA. The frequency, phenotype, and function of NK cells (cocultures with K562 cells) and T-cells (stimulated with spike/RdRp peptides) were assessed by flow cytometry. Anti-SARS-CoV-2 antibodies were determined using indirect immunofluorescence and plaque reduction neutralization assay. RESULTS: During COVID-19, we observed a high proinflammatory-cytokine production and a reduced CD56bright-NK cell and cytotoxic response. Compared with healthy controls, infected individuals had a higher frequency of dysfunctional CD8+ T cells CD38+HLA-DR-. During the acute phase, CD8+ T cells stimulated with viral peptides exhibited a monofunctional response characterized by high IL-10 production. However, during recovery, we observed a bifunctional response characterized by the co-expression of CD107a and granzyme B or perforin. CONCLUSION: Although the proinflammatory response is a hallmark of SARS-CoV-2 infection, other phenotypic and functional alterations in NK cells and CD8+ T cells could be associated with the outcome of COVID-19. However, additional studies are required to understand these alterations and to guide future immunotherapy strategies.


Assuntos
COVID-19/imunologia , Células Matadoras Naturais , SARS-CoV-2/imunologia , Linfócitos T , Adulto , Anticorpos Antivirais/análise , Antígeno CD56/imunologia , Estudos de Casos e Controles , Colômbia , Saúde da Família , Granzimas/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/sangue , Interleucina-6/sangue , Interleucina-8/sangue , Células K562 , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Perforina/metabolismo , Fenótipo , Receptores CCR7/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/sangue , Adulto Jovem
4.
Nat Commun ; 12(1): 5217, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471116

RESUMO

Lethal hit delivery by cytotoxic T lymphocytes (CTL) towards B lymphoma cells occurs as a binary, "yes/no" process. In non-hematologic solid tumors, however, CTL often fail to kill target cells during 1:1 conjugation. Here we describe a mechanism of "additive cytotoxicity" by which time-dependent integration of sublethal damage events, delivered by multiple CTL transiting between individual tumor cells, mediates effective elimination. Reversible sublethal damage includes perforin-dependent membrane pore formation, nuclear envelope rupture and DNA damage. Statistical modeling reveals that 3 serial hits delivered with decay intervals below 50 min discriminate between tumor cell death or survival after recovery. In live melanoma lesions in vivo, sublethal multi-hit delivery is most effective in interstitial tissue where high CTL densities and swarming support frequent serial CTL-tumor cell encounters. This identifies CTL-mediated cytotoxicity by multi-hit delivery as an incremental and tunable process, whereby accelerating damage magnitude and frequency may improve immune efficacy.


Assuntos
Citotoxicidade Imunológica , Melanoma/terapia , Perforina/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Apoptose/imunologia , Morte Celular , Linhagem Celular Tumoral , Dano ao DNA , Feminino , Humanos , Cinética , Células MCF-7 , Masculino , Camundongos Endogâmicos C57BL , Perforina/genética
5.
Biomolecules ; 11(8)2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34439744

RESUMO

Galectins are one of the critical players in the tumor microenvironment-tumor crosstalk and the regulation of local immunity. Galectin-9 has been in the limelight in tumor immunology. Galectin-9 possesses its multiplex biological functions both extracellularly and intracellularly, plays a pivotal role in the modulation of adaptive and innate immunity, and induces immune tolerance. NK-92MI cell lines against different malignancies were extensively studied, and recently published trials used genetically chimeric antigen receptor-transfected NK-92MI cells in tumor immunotherapy. Besides the intensive research in tumor immunotherapy, limited information is available on their immune-checkpoint expression and the impact of checkpoint ligands on their effector functions. To uncover the therapeutic potential of modulating Galectin-9-related immunological pathways in NK-cell-based therapy, we investigated the dose-dependent effect of soluble Galectin-9 on the TIM-3 checkpoint receptor and NKG2D, CD69, FasL, and perforin expression of NK-92MI cells. We also examined how their cytotoxicity and cytokine production was altered after Gal-9 treatment and in the presence of different serum supplements using flow cytometric analysis. Our study provides evidence that the Galectin-9/TIM-3 pathway plays an important role in the regulation of NK cell function, and about the modulatory role of Galectin-9 on the cytotoxicity and cytokine production of NK-92MI cells in the presence of different serum supplements. We hope that our results will aid the development of novel NK-cell-based strategies that target Galectin-9/TIM-3 checkpoint in tumors resistant to T-cell-based immunotherapy.


Assuntos
Galectinas/metabolismo , Linfoma não Hodgkin/patologia , Soro/química , Imunidade Adaptativa , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Proteína Ligante Fas/metabolismo , Expressão Gênica , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Imunidade Inata , Imunoterapia/métodos , Células K562 , Células Matadoras Naturais/metabolismo , Lectinas Tipo C/metabolismo , Linfoma não Hodgkin/metabolismo , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias/imunologia , Neoplasias/patologia , Perforina/metabolismo , Fenótipo , Proteínas Recombinantes/química , Microambiente Tumoral
6.
PLoS Biol ; 19(8): e3001328, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34343168

RESUMO

Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule-cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell-mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid "shields" against self-destruction.


Assuntos
Degranulação Celular , Células Matadoras Naturais/fisiologia , Lipídeos de Membrana/metabolismo , Perforina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Voluntários Saudáveis , Humanos , Cetocolesteróis , Cultura Primária de Células
7.
Cells ; 10(8)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34440813

RESUMO

Drugs targeting immune checkpoint molecules have been found effective in melanoma, lung cancer, and other malignancies treatment. Recent studies on breast cancer demonstrated the significance of inhibitory anti-CTLA-4 and anti-PD-1 in the regulation of disease progression. However, seemingly the same types of breast cancer do not always respond unambiguously to immunotherapy. Thus, here we set out to analyze the in vitro effects of inhibiting CTLA-4 and PD-1 on interactions between co-cultured lymphocytes and two selected breast adenocarcinoma cell lines. Breast cancer cells were co-cultured with lymphocytes to evaluate the effects of CTLA-4 and PD-1 inhibition. Proliferation, cell cycle, and viability assessment were measured in cancer cells. IFN-gamma, IL-10, perforin, granzyme B production, and CTLA-4 and PD-1 expression were analyzed in lymphocytes. We found that administration of anti-CTLA-4 improved the anti-cancer activity of T cells with reduced proliferation and viability of MDA-MB-231. Lack of response was observed in the context of MCF-7. In addition, differential expression of checkpoint proteins was found between studied cancer cells lines. Inhibition of molecules was followed by IL-10 and IFN-gamma decrease in lymphocytes co-cultured with MDA-MB-231, not demonstrated in reference to MCF-7. Furthermore, CTLA-4 blockage was associated with reduction of CTLA-4+ and PD-1+ lymphocytes in MDA-MB-231, with a significant increase in MCF-7, reduced by anti-PD-1. Altogether, our study revealed that anti-CTLA-4 and anti-PD-1 treatment can improve lymphocytes effects on breast cancer cells. Favorable effects seemed to be related to breast cancer cells features as differential responses were reported. Novel blocking antibodies strategies should be tested for more effective cancer inhibition.


Assuntos
Antígeno CTLA-4/imunologia , Leucócitos Mononucleares/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Adulto , Anticorpos/imunologia , Antígeno B7-1/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Antígeno CTLA-4/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Feminino , Granzimas/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Células MCF-7 , Perforina/metabolismo , Receptor de Morte Celular Programada 1/metabolismo
8.
Sci Rep ; 11(1): 14090, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34238985

RESUMO

MAIT cells have been shown to be activated upon several viral infections in a TCR-independent manner by responding to inflammatory cytokines secreted by antigen-presenting cells. Recently, a few studies have shown a similar activation of MAIT cells in response to severe acute respiratory coronavirus 2 (SARS-CoV-2) infection. In this study, we investigate the effect of SARS-CoV-2 infection on the frequency and phenotype of MAIT cells by flow cytometry, and we test in vitro stimulation conditions on the capacity to enhance or rescue the antiviral function of MAIT cells from patients with coronavirus disease 2019 (COVID-19). Our study, in agreement with recently published studies, confirmed the decline in MAIT cell frequency of hospitalized donors in comparison to healthy donors. MAIT cells of COVID-19 patients also had lower expression levels of TNF-alpha, perforin and granzyme B upon stimulation with IL-12 + IL-18. 24 h' incubation with IL-7 successfully restored perforin expression levels in COVID-19 patients. Combined, our findings support the growing evidence that SARS-CoV-2 is dysregulating MAIT cells and that IL-7 treatment might improve their function, rendering them more effective in protecting the body against the virus.


Assuntos
COVID-19/prevenção & controle , COVID-19/virologia , Interleucina-7/farmacologia , Células T Invariantes Associadas à Mucosa/fisiologia , Células T Invariantes Associadas à Mucosa/virologia , SARS-CoV-2/patogenicidade , Células Cultivadas , Feminino , Granzimas/metabolismo , Humanos , Masculino , Células T Invariantes Associadas à Mucosa/metabolismo , Perforina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
9.
J Virol ; 95(16): e0069921, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34076478

RESUMO

Functional HIV-1-specific CD8+ T cells primed from naive T cells are expected to act as effector T cells in a "shock-and-kill" therapeutic strategy for an HIV-1 cure since less functional HIV-1-specific CD8+ T cells are elicited from memory T cells in HIV-1-infected individuals on combined antiretroviral therapy (cART). CD8+ T cells specific for HIV-1 conserved and protective epitopes are candidates for such T cells. We investigated the priming with STING ligand of CD8+ T cells specific for HLA-B*52:01 or HLA-C*12:02-restricted protective epitopes from naive T cells. STING ligand 3'3'-cGAMP effectively primed CD8+ T cells specific for 3 of 4 HLA-B*52:01-restricted epitopes but failed to prime those specific for all 3 HLA-C*12:02-restricted epitopes from the naive T cells of HIV-1-uninfected individuals having an HLA-B*52:01-C*12:02 protective haplotype. These HLA-B*52:01-restricted CD8+ T cells had a strong ability to suppress HIV-1 replication and expressed a high level of cytolytic effector molecules. The viral suppression ability of these T cells was significantly correlated with the expression level of perforin and showed a trend for a positive correlation with the expression level of CD107a. The present study highlighted the priming with STING ligand of functional CD8+ T cells specific for protective epitopes, which T cells would contribute as effector T cells to a shock-and-kill therapy. IMPORTANCE The current "shock-and-kill" therapeutic strategy for HIV cure has been directed toward eliminating latent viral reservoirs by reactivation of latent reservoirs with latency-reversing agents followed by eradication of these cells by immune-mediated responses. Although HIV-1-specific T cells are expected to eradicate viral reservoirs, the function of these T cells is reduced in HIV-1-infected individuals with long-term cART. Therefore, priming of HIV-1-specific T cells with high function from naive T cells is to be expected in these individuals. In this study, we demonstrated the priming with STING ligand 3'3'-cGAMP of CD8+ T cells specific for HIV-1-protective epitopes from naive T cells. cGAMP primed CD8+ T cells specific for 3 HLA-B*52:01-restricted protective epitopes, which cells expressed a high level of cytolytic effector molecules and effectively suppressed HIV-1 replication. The present study suggested that the priming with STING ligand of functional CD8+ T cells specific for protective epitopes would be useful in a therapy for an HIV-1 cure.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , HIV-1/imunologia , Nucleotídeos Cíclicos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Granzimas/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , Soronegatividade para HIV/imunologia , Antígeno HLA-B52/imunologia , Antígenos HLA-C/imunologia , Humanos , Ligantes , Proteínas de Membrana/imunologia , Perforina/metabolismo , Replicação Viral/imunologia
10.
Theranostics ; 11(14): 6668-6681, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093846

RESUMO

Background: Newly emerging cancer immunotherapy has led to significant progress in cancer treatment; however, its efficacy is limited in solid tumors since the majority of them are "cold" tumors. Oncolytic viruses, especially when properly armed, can directly target tumor cells and indirectly modulate the tumor microenvironment (TME), resulting in "hot" tumors. These viruses can be applied as a cancer immunotherapy approach either alone or in combination with other cancer immunotherapies. Cytokines are good candidates to arm oncolytic viruses. IL-23, an IL-12 cytokine family member, plays many roles in cancer immunity. Here, we used oncolytic vaccinia viruses to deliver IL-23 variants into the tumor bed and explored their activity in cancer treatment on multiple tumor models. Methods: Oncolytic vaccinia viruses expressing IL-23 variants were generated by homologue recombination. The characteristics of these viruses were in vitro evaluated by RT-qPCR, ELISA, flow cytometry and cytotoxicity assay. The antitumor effects of these viruses were evaluated on multiple tumor models in vivo and the mechanisms were investigated by RT-qPCR and flow cytometry. Results: IL-23 prolonged viral persistence, probably mediated by up-regulated IL-10. The sustainable IL-23 expression and viral oncolysis elevated the expression of Th1 chemokines and antitumor factors such as IFN-γ, TNF-α, Perforin, IL-2, Granzyme B and activated T cells in the TME, transforming the TME to be more conducive to antitumor immunity. This leads to a systemic antitumor effect which is dependent on CD8+ and CD4+ T cells and IFN-γ. Oncolytic vaccinia viruses could not deliver stable IL-23A to the tumor, attributed to the elevated tristetraprolin which can destabilize the IL-23A mRNA after the viral treatment; whereas vaccinia viruses could deliver membrane-bound IL-23 to elicit a potent antitumor effect which might avoid the possible toxicity normally associated with systemic cytokine exposure. Conclusion: Either secreted or membrane-bound IL-23-armed vaccinia virus can induce potent antitumor effects and IL-23 is a candidate cytokine to arm oncolytic viruses for cancer immunotherapy.


Assuntos
Adenocarcinoma/terapia , Neoplasias do Colo/terapia , Imunoterapia/métodos , Interleucina-23/farmacologia , Vírus Oncolíticos/genética , Microambiente Tumoral/imunologia , Vírus Vaccinia/genética , Adenocarcinoma/imunologia , Adenocarcinoma/virologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Neoplasias do Colo/imunologia , Neoplasias do Colo/virologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Granzimas/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-2/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vírus Oncolíticos/metabolismo , Perforina/metabolismo , Microambiente Tumoral/genética , Fator de Necrose Tumoral alfa/metabolismo , Vírus Vaccinia/metabolismo
11.
Front Immunol ; 12: 658562, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34113342

RESUMO

Natural killer (NK) cells are becoming valuable tools for cancer therapy because of their cytotoxicity against tumor cells without prior sensitization and their involvement in graft-versus-host disease; however, it is difficult to obtain highly cytotoxic NK cells without adding extra feeder cells. In this study, we developed a new method for obtaining highly cytotoxic NK cells from peripheral blood mononuclear cells (PBMCs) independently of extra feeder cell addition using rituximab not coated on a flask (non-coated rituximab). We found that rituximab could promote both the activation and expansion of NK cells from PBMCs, irrespective of being coated on a flask or not. However, NK cells activated by non-coated rituximab had much greater antitumor activity against cancer cells, and these effects were dependent on autologous living B cells. The antibody-dependent cellular cytotoxicity effect of NK cells activated by non-coated rituximab was also more substantial. Furthermore, these cells expressed higher levels of CD107a, perforin, granzyme B, and IFN-γ. However, there was no difference in the percentage, apoptosis, and cell-cycle progression of NK cells induced by coated and non-coated rituximab. Non-coated rituximab activated NK cells by increasing AKT phosphorylation, further enhancing the abundance of XBP1s. In conclusion, we developed a new method for amplifying NK cells with higher antitumor functions with non-coated rituximab via autologous B cells from PBMCs, and this method more efficiently stimulated NK cell activation than by using coated rituximab.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos Imunológicos/farmacologia , Linfócitos B/imunologia , Fatores Imunológicos/farmacologia , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Rituximab/farmacologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos Imunológicos/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Citocinas/biossíntese , Granzimas/metabolismo , Humanos , Fatores Imunológicos/imunologia , Imunofenotipagem , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Perforina/metabolismo , Rituximab/imunologia
12.
Int Immunopharmacol ; 97: 107685, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33951560

RESUMO

BACKGROUND: The 2019 Coronavirus (COVID-19) pandemic poses a huge threat internationally; however, the role of the host immune system in the pathogenesis of COVID-19 is not well understood. METHODS: Cytokine and chemokine levels and characterisation of immune cell subsets from 20 COVID-19 cases after hospital admission (17 critically ill and 3 severe patients) and 16 convalescent patients were determined using a multiplex immunoassay and flow cytometry, respectively. RESULTS: IP-10, MCP-1, MIG, IL-6, and IL-10 levels were significantly higher in acute severe/critically ill patients with COVID-19, whereas were normal in patients who had reached convalescence. CD8 T cells in severe and critically ill COVID-19 patients expressed high levels of cytotoxic granules (granzyme B and perforin)and was hyperactivated as evidenced by the high proportions of CD38. Furthermore, the cytotoxic potential of natural killer (NK) cells, and the frequencies of myeloid dendritic cells and plasmacytoid dendritic cells was reduced in patients with severe and critical COVID-19; however, these dysregulations were found to be restored in convalescent phases. CONCLUSION: Thus, elicitation of the hyperactive cytokine-mediated inflammatory response, dysregulation of CD8 T and NK cells, and deficiency of host myeloid and plasmacytoid DCs, may contribute to COVID-19 pathogenesis and provide insights into potential therapeutic targets and strategies.


Assuntos
COVID-19/sangue , COVID-19/imunologia , Convalescença , Inflamação/etiologia , ADP-Ribosil Ciclase 1/sangue , Doença Aguda , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Quimiocina CCL2/sangue , Quimiocina CXCL10/sangue , Quimiocina CXCL9/sangue , Estado Terminal , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/imunologia , Células Dendríticas/imunologia , Feminino , Granzimas/metabolismo , Humanos , Interleucina-10/sangue , Interleucina-6/sangue , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , Masculino , Glicoproteínas de Membrana/sangue , Pessoa de Meia-Idade , Perforina/metabolismo
13.
Methods Mol Biol ; 2325: 65-77, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34053051

RESUMO

Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T-cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T-cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T-cell (CTL) studies have taken advantage with this high-throughput technology by providing insights of quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T-cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T-cells is of relevance for immune diagnostic. The most utilized Elispot assay is the Interferon-gamma (IFN-γ) test, a marker for CD8+ CTL activation, but Elispot can be also used to distinguish different subsets of activated T-cells by using other cytokines such as T-helper (Th) 1 type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21 and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10 and IL-13), and Th17 (IL-17) cells.The reliability of Elispot generated data, by the evaluation of T-cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot Assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Milteny cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T-cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot Assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and Perforin Elispot assays will be described.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , ELISPOT/métodos , Humanos , Interferon gama/metabolismo , Perforina/metabolismo
14.
Scand J Immunol ; 94(1): e13047, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33914954

RESUMO

Perforin, a pore-forming glycoprotein, has been demonstrated to play key roles in clearing virus-infected cells and tumour cells due to its ability of forming 'pores' on the cell membranes. Additionally, perforin is also found to be associated with human diseases such as tumours, virus infections, immune rejection and some autoimmune diseases. Until now, plenty of perforin genes have been identified in vertebrates, especially the mammals and teleost fish. Conversely, vertebrate homologue of perforin gene was not identified in the invertebrates. Although recently there have been several reviews focusing on perforin and granzymes in mammals, no one highlighted the current advances of perforin in the other vertebrates. Here, in addition to mammalian perforin, the structure, evolution, tissue distribution and function of perforin in bony fish are summarized, respectively, which will allow us to gain more insights into the perforin in lower animals and the evolution of this important pore-forming protein across vertebrates.


Assuntos
Peixes/metabolismo , Mamíferos/metabolismo , Perforina/metabolismo , Animais , Membrana Celular/metabolismo , Humanos
15.
Front Immunol ; 12: 578548, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815355

RESUMO

Objectives: To explore the potential role of CD3+CD8+CD161high TCRVα7.2+ mucosal-associated invariant T (MAIT) cells in the pathogenesis of primary biliary cholangitis (PBC). Methods: We enrolled 55 patients with PBC, 69 healthy controls (HCs), and 8 patients with hepatic hemangioma. Circulating MAIT cells and their chemokine receptor profiles and cytokine production were quantified using flow cytometry. Liver-resident MAIT cells were examined by immunofluorescence staining. CXCL12-mediated chemotaxis of MAIT cells was measured using a transwell migration assay. Plasma interleukin (IL)-18 was measured using ELISA, and cytokine production in IL-18-stimulated MAIT cells was detected using flow cytometry. Result: Peripheral MAIT cells were found to be significantly lower in patients with PBC (3.0 ± 3.2% vs. 9.4 ± 8.0%, p < 0.01) and negatively correlated with alkaline phosphatase (ALP) levels (r = -0.3209, p < 0.05). Liver immunofluorescence staining suggested that MAIT cells might accumulate in PBC liver. MAIT cells from patients with PBC expressed higher levels of CXCR4 (84.8 ± 18.0% vs. 58.7 ± 11.4%, p < 0.01), and the expression of CXCL12 was higher in PBC liver. CXCL12 promoted MAIT cell chemotaxis (70.4 ± 6.8% vs. 52.2 ± 3.5%, p < 0.01), which was attenuated by CXCR4 antagonist. MAIT cells from PBC produced significantly more interferon-γ (IFN-γ) (88.3 ± 4.2% vs. 64.2 ± 10.1%, p < 0.01), tumor necrosis factor-α (TNF-α) (93.0 ± 1.1% vs. 80.1 ± 5.3%, p < 0.01), Granzyme B (89.3 ± 3.3% vs. 72.1 ± 7.0%, p < 0.01), and perforin (46.8 ± 6.6% vs. 34.8 ± 7.7%, p < 0.05). MAIT cells from PBC expressed higher levels of IL18-Rα (83.8 ± 10.2% vs. 58.3 ± 8.7%, p < 0.01). Plasma IL-18 was more abundant in patients with PBC (286.8 ± 75.7 pg/ml vs. 132.9 ± 78.1 pg/ml, p < 0.01). IL-18 promoted IFN-γ production in MAIT cells (74.9 ± 6.6% vs. 54.7 ± 6.7%, p < 0.01), which was partially attenuated by blocking IL-18R (68.6 ± 8.3% vs. 43.5 ± 4.2%, p < 0.01). Conclusion: Mucosal-associated invariant T cells from patients with PBC accumulated in the liver via CXCL12-CXCR4-mediated chemotaxis, produced pro-inflammatory cytokines, and contributed to portal inflammation, which was potentially mediated by elevated IL-18. Targeting MAIT cells might be a therapeutic approach for PBC.


Assuntos
Quimiocina CXCL12/imunologia , Cirrose Hepática Biliar/imunologia , Fígado/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Receptores CXCR4/imunologia , Adulto , Fosfatase Alcalina/imunologia , Fosfatase Alcalina/metabolismo , Quimiocina CXCL12/metabolismo , Quimiotaxia/imunologia , Feminino , Granzimas/imunologia , Granzimas/metabolismo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-18/imunologia , Interleucina-18/metabolismo , Fígado/metabolismo , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Células T Invariantes Associadas à Mucosa/metabolismo , Perforina/imunologia , Perforina/metabolismo , Receptores CXCR4/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
16.
Genes Cells ; 26(5): 313-327, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33662167

RESUMO

As the sentinels of innate and adaptive immune system, dendritic cells (DCs) have been considered to hold a great promise for medical application. Among the diverse types of DCs, monocyte-derived DCs (mo-DCs) generated in vitro have been most commonly employed. We have been improving the culture protocol and devised a protocol to produce mature interferon-α-induced DCs (IFN-DCs), hereinafter called (mat)IFN-DCs. While exploring the relationship between the expression of CD56 and the cytotoxic activity of (mat)IFN-DCs, we unexpectedly found that sorting of (mat)IFN-DCs with CD56 antibody-coated microbeads (MB) resulted in fractionating cells with tumoricidal activity into the flow-through (FT) but not MB-bound fraction. We uncovered that the FT fraction contains cells expressing low but substantial level of CD56. Moreover, those cells express granzyme B (GrB), perforin (PFN), and serpin B9 at high levels. By employing a specific inhibitor of PFN, we confirmed that direct tumoricidal activity relies on the GrB/PFN pathway. We designated subpopulation in FT fraction as CD56dim and that in CD56 positively sorted fraction as CD56bright , respectively. This is the first time, to our knowledge, to identify subpopulations of CD56-positive IFN-DCs with distinct tumoricidal activity which is ascribed to high expression of the components of GrB/PFN pathway.


Assuntos
Antígeno CD56/metabolismo , Células Dendríticas/metabolismo , Granzimas/metabolismo , Interferon-alfa/farmacologia , Perforina/metabolismo , Serpinas/metabolismo , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Macrolídeos/farmacologia , Monócitos/metabolismo
17.
Cancer Med ; 10(9): 3129-3138, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33769705

RESUMO

BACKGROUND: A simple measure of immune cytolytic activity (CYT) base on mRNA expression levels of two genes, GZMA and PRF1, was recently reported. Here, we aimed to evaluate the CYT score's potential as a measure of antitumor immunity and predictor of clinical outcome in gastric cancer (GC) patients. MATERIALS AND METHODS: We evaluated the correlations between tumor-infiltrating immune cells and the CYT score in 238 GC samples from The Cancer Genome Atlas (TCGA). Next, we investigated CYT score associations with molecular subtypes, somatic mutation load, and immune checkpoint molecules in GC samples from TCGA and Asian Cancer Research Group (ACRG). Moreover, we evaluated the clinical significance of the CYT score calculated by reverse transcription (RT)-quantitative PCR (qPCR) data in 123 GC samples and the association of the CYT score with the response to anti-PD-1 therapy in 7 GC samples from Kyushu University Hospital. RESULTS: The CYT score positively correlated with the proportions of tumor-infiltrating CD8+ T cells and macrophages and negatively correlated with the proportion of regulatory T cells in GC tissues. A high CYT score was associated with common immune checkpoint molecules, a high mutation, the Epstein-Barr virus subtype, and the microsatellite instability subtype in GC. Moreover, a low CYT score was a poor prognosis factor in patients with GC. Finally, the CYT score was higher in a responder to anti-PD-1 therapy compared to nonresponders. CONCLUSION: The CYT score reflects antitumor immunity and predicts clinical outcome in GC patients.


Assuntos
Biomarcadores Tumorais/imunologia , Linfócitos T CD8-Positivos/imunologia , Granzimas/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Perforina/metabolismo , Neoplasias Gástricas/imunologia , Idoso , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Estudos de Coortes , Bases de Dados Genéticas , Feminino , Granzimas/genética , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Proteínas de Checkpoint Imunológico/metabolismo , Imunidade Celular , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/citologia , Masculino , Mutação/imunologia , Perforina/genética , Prognóstico , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/terapia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Resultado do Tratamento , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia
18.
Front Immunol ; 12: 612583, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33746956

RESUMO

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of immune dysregulation characterized by hyperactivation of the immune system, excessive cytokine secretion and severe systemic inflammation. HLH is classified as familial (FHL) when associated with mutations in PRF1, UNC13D, STX11, and STXBP2 genes. There is limited information available about the clinical and mutational spectrum of FHL patients in Indian population. This study is a retrospective analysis of 101 molecularly characterized FHL patients over the last 10 years from 20 different referral centers in India. FHL2 and FHL3 together accounted for 84% of cases of FHL in our cohort. Patients belonging to different FHL subtypes were indistinguishable based on clinical and biochemical parameters. However, flow cytometry-based assays viz. perforin expression and degranulation assay were found to be specific and sensitive in diagnosis and classification of FHL patients. Molecular characterization of respective genes revealed 76 different disease-causing mutations including 39 (51%) novel mutations in PRF1, UNC13D, STX11, and STXBP2 genes. Overall, survival was poor (28%) irrespective of the age of onset or the type of mutation in our cohort. Altogether, this article sheds light on the current scenario of FHL in India. Our data reveal a wide genetic heterogeneity of FHL in the Indian population and confirms the poor prognosis of FHL. This study also emphasizes that though mutational analysis is important for diagnostic confirmation of FHL, flow cytometry based assays help significantly in rapid diagnosis and functional validation of novel variants identified.


Assuntos
Biomarcadores , Suscetibilidade a Doenças , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/etiologia , Fenótipo , Alelos , Criança , Pré-Escolar , Terapia Combinada , Biologia Computacional/métodos , Bases de Dados Genéticas , Gerenciamento Clínico , Suscetibilidade a Doenças/imunologia , Feminino , Predisposição Genética para Doença , Humanos , Índia , Lactente , Linfo-Histiocitose Hemofagocítica/metabolismo , Linfo-Histiocitose Hemofagocítica/terapia , Masculino , Mutação , Perforina/genética , Perforina/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Resultado do Tratamento
19.
Am J Pathol ; 191(6): 1064-1076, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33713685

RESUMO

In neuron-specific ovalbumin-transgenic CKTAC mice, antigen-specific OT-I CD8 T cells home to the enteric nervous system, where they attack and destroy neurons of the myenteric and submucosal plexus. Clinically, experimental autoimmune enteric ganglionitis (EAEG) manifests with gastrointestinal dysmotility and rapidly progresses to lethal ileus. Although interferon-γ has been identified as capable of damaging neurons in EAEG, the role of perforin, Fas/FasL, and tumor necrosis factor-α (TNF-α) in this disease is still a matter of debate. Thus, CKTAC mice were adoptively transferred with either perforin-/- or wild-type OT-I CD8 T cells. In addition, CKTAC mice that had received wild-type OT-I CD8 T cells were treated by either anti-TNF-α or anti-FasL. Furthermore, wild-type OT-I CD8 T cells were adoptively transferred into CKTAC mice with neuron-specific deletion of Fas. Although neither inactivation of enteric neuronal Fas nor anti-FasL treatment improved the disease, the absence of perforin from OT-I CD8 T cells and anti-TNF-α treatment significantly ameliorated EAEG and prevented lethal ileus by rescue of enteric neurons. Thus, these experiments identify perforin and TNF-α as important in the pathogenesis of EAEG.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Sistema Nervoso Entérico/patologia , Doenças Inflamatórias Intestinais/patologia , Neurônios/patologia , Perforina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Camundongos
20.
Cancer Res ; 81(8): 1965-1976, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589517

RESUMO

Inactivation of tumor-infiltrating lymphocytes (TIL) is one of the mechanisms mitigating antitumor immunity during tumor onset and progression. Epigenetic abnormalities are regarded as a major culprit contributing to the dysfunction of TILs within tumor microenvironments. In this study, we used a murine model of melanoma to discover that Tet2 inactivation significantly enhances the antitumor activity of TILs with an efficacy comparable to immune checkpoint inhibition imposed by anti-PD-L1 treatment. Single-cell RNA-sequencing analysis suggested that Tet2-deficient TILs exhibit effector-like features. Transcriptomic and ATAC-sequencing analysis showed that Tet2 ablation reshapes chromatin accessibility and favors binding of transcription factors geared toward CD8+ T-cell activation. Furthermore, the ETS family of transcription factors contributed to augmented CD8+ T-cell function following Tet2 depletion. Overall, our study establishes that Tet2 constitutes one of the epigenetic barriers that account for dysfunction of TILs and that Tet2 inactivation could promote antitumor immunity to suppress tumor growth. SIGNIFICANCE: This study suggests that ablation of TET2+ from TILs could promote their antitumor function by reshaping chromatin accessibility for key transcription factors and enhancing the transcription of genes essential for antitumor activity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Ligação a DNA/deficiência , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Proteínas Proto-Oncogênicas/deficiência , Transferência Adotiva/métodos , Animais , Cromatina/metabolismo , Desmetilação do DNA , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Epigênese Genética , Deleção de Genes , Inativação Gênica , Inibidores de Checkpoint Imunológico/uso terapêutico , MAP Quinase Quinase Quinases , Melanoma Experimental/metabolismo , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Perforina/metabolismo , Proteínas Proto-Oncogênicas/genética , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/metabolismo
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