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1.
Cancer Immunol Immunother ; 68(6): 961-971, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30955067

RESUMO

Hepatocellular carcinoma (HCC) is the third most lethal cancer in the world. Natural killer (NK) cell-mediated immunity is crucial for tumor surveillance and therapy. Characterization of the regulatory mechanisms of NK cell function is important for developing novel immunotherapies against HCC. In this study, we used a chemical-induced mouse HCC model to identify the upregulation of Sirtuin2 (SIRT2) in liver NK cells. In particular, SIRT2 was predominantly expressed in liver CD94+ NK cells. The HCC liver microenvironment induced SIRT2 expression in NK cells. In addition, overexpression of exogenous SIRT2 significantly upregulated the production of cytokines and cytotoxic mediators in activated NK cells. Consistently, SIRT2-overexpressing NK cells showed a stronger tumoricidal effect on hepatoma cells. Moreover, SIRT2 remarkably promoted the phosphorylation of Extracellular-signal-regulated kinase 1/2 (Erk1/2) and p38 Mitogen-activated protein kinases (MAPK) in activated NK cells. SIRT2 knockdown in liver CD94+ NK cells impaired their cytotoxic effect on hepatoma cells. Our study indicates that SIRT2 enhances the tumoricidal activity of liver NK cells in HCC.


Assuntos
Carcinoma Hepatocelular/imunologia , Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas Experimentais/imunologia , Fígado/imunologia , Sirtuína 2/imunologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Citocinas/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Imunoterapia Adotiva , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/transplante , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/terapia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Interferência de RNA , Sirtuína 2/genética , Sirtuína 2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
EBioMedicine ; 40: 106-117, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30665853

RESUMO

BACKGROUND: Adoptive T-cell transfer of therapeutic TCR holds great promise to specifically kill cancer cells, but relies on modifying the patient's own T cells ex vivo before injection. The manufacturing of T cells in a tailor-made setting is a long and expensive process which could be resolved by the use of universal cells. Currently, only the Natural Killer (NK) cell line NK-92 is FDA approved for universal use. In order to expand their recognition ability, they were equipped with Chimeric Antigen Receptors (CARs). However, unlike CARs, T-cell receptors (TCRs) can recognize all cellular proteins, which expand NK-92 recognition to the whole proteome. METHODS: We herein genetically engineered NK-92 to express the CD3 signaling complex, and showed that it rendered them able to express a functional TCR. Functional assays and in vivo efficacy were used to validate these cells. FINDINGS: This is the first demonstration that a non-T cell can exploit TCRs. This TCR-redirected cell line, termed TCR-NK-92, mimicked primary T cells phenotypically, metabolically and functionally, but retained its NK cell effector functions. Our results demonstrate a unique manner to indefinitely produce TCR-redirected lymphocytes at lower cost and with similar therapeutic efficacy as redirected T cells. INTERPRETATION: These results suggest that an NK cell line could be the basis for an off-the-shelf TCR-based cancer immunotherapy solution. FUND: This work was supported by the Research Council of Norway (#254817), South-Eastern Norway Regional Health Authority (#14/00500-79), by OUS-Radiumhospitalet (Gene Therapy program) and the department of Oncology at the University of Lausanne.


Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Respiração Celular , Citotoxicidade Imunológica/genética , Modelos Animais de Doenças , Metabolismo Energético , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Camundongos , Mitocôndrias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Yonsei Med J ; 60(1): 22-29, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30554487

RESUMO

PURPOSE: It is well documented that natural killer (NK) cytotoxicity against hepatocellular carcinoma (HCC) cells is impaired in HCC, which might account for the failure of anti-tumor immune response. miRNAs are considered as important regulators for the development and functions of NK cells. However, the entire role of miR-506 in NK cells remains far from being addressed. MATERIALS AND METHODS: The expressions of miR-506 and signal transducer and activator of transcription 3 (STAT3) mRNA in primary NK cells from HCC patients and healthy controls were detected by quantitative real-time PCR. NK cell cytotoxicity was assessed by CFSE/7AAD cytotoxicity assay and lactate dehydrogenase assay. Luciferase reporter assay, RNA immunoprecipitation assay, and western blot were conducted to confirm the interaction between miR-506 and STAT3. RESULTS: miR-506 expression was downregulated and STAT3 mRNA was upregulated in primary NK cells from HCC patients. Primary NK cells from HCC patients showed remarkably reduced cytotoxicity against SMMC7721 or HepG2 cells. NK cell cytotoxicity was positively correlated with miR-506 expression and negatively correlated with STAT3 mRNA expression. Additionally, miR-506 overexpression enhanced NK cell cytotoxicity against HCC cells, while miR-506 inhibitor showed the reverse effect. Moreover, miR-506 could suppress STAT3 expression by directly targeting 3'-untranslated regions of STAT3. A negative correlation between miR-506 and STAT3 mRNA expression in HCC patients was observed. Mechanistically, overexpressing STAT3 greatly reversed miR-506-mediated promotion of NK cell cytotoxicity against HCC cells. CONCLUSION: miR-506 enhanced NK cell cytotoxicity against HCC cells by targeting STAT3, suggesting that modulating miR-506 expression maybe a promising approach for enhancing NK cell-based antitumor therapies.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Citotoxicidade Imunológica/genética , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , MicroRNAs/metabolismo , Fator de Transcrição STAT3/metabolismo , Sequência de Bases , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Masculino , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Elife ; 72018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30575523

RESUMO

A recurrent theme in viral immune evasion is the sabotage of MHC-I antigen presentation, which brings virus the concomitant issue of 'missing-self' recognition by NK cells that use inhibitory receptors to detect surface MHC-I proteins. Here, we report that rodent herpesvirus Peru (RHVP) encodes a Qa-1 like protein (pQa-1) via RNA splicing to counteract NK activation. While pQa-1 surface expression is stabilized by the same canonical peptides presented by murine Qa-1, pQa-1 is GPI-anchored and resistant to the activity of RHVP pK3, a ubiquitin ligase that targets MHC-I for degradation. pQa-1 tetramer staining indicates that it recognizes CD94/NKG2A receptors. Consistently, pQa-1 selectively inhibits NKG2A+ NK cells and expression of pQa-1 can protect tumor cells from NK control in vivo. Collectively, these findings reveal an innovative NK evasion strategy wherein RHVP encodes a modified Qa-1 mimic refractory to MHC-I sabotage and capable of specifically engaging inhibitory receptors to circumvent NK activation.


Assuntos
Citotoxicidade Imunológica/imunologia , Herpesviridae/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília D de Receptores Semelhantes a Lectina de Células NK/imunologia , Sequência de Aminoácidos , Animais , Apresentação do Antígeno/imunologia , Sequência de Bases , Citotoxicidade Imunológica/genética , Células HEK293 , Herpesviridae/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mimetismo Molecular/genética , Mimetismo Molecular/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Ligação Proteica/imunologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico
5.
In Vivo ; 32(4): 771-781, 2018 Jul-Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29936458

RESUMO

BACKGROUND/AIM: Natural killer (NK) cells are one of the lymphocytes clinically used for various cancer types. Cytotoxicity of NK cells to cholangiocarcinoma (CC), however, has not yet been studied. Nor NK cell therapy against CC has been clinically applied. In this study, relevance of NK cell therapy for anti-tumor efficacy against CC was pre-clinically investigated. MATERIALS AND METHODS: Human HuCCT-1 cells, an intrahepatic CC cell line, were xenografted into nude mice. The HuCCT-1 tumor-bearing nude mice then received multiple infusions of ex vivo-expanded human NK cells (SMT01) and in vivo cytotoxic activity of the NK cells against the CC cells was evaluated. RESULTS: SMT01 infusion resulted in significant inhibition of the CC tumor growth. Body weight of the mice administrated with chemotherapy was found to be maintained at the lowest level among all treatment groups while all the SMT01 infusion groups well maintained their body weight. CONCLUSION: The present in vivo study demonstrates that NK cells contain cytolytic activity against cholangiocarcinoma and show beneficial effect of NK cell therapy in relevance to quality of life. Further investigation of the NK cell-based immunotherapy can be useful to determine cancer therapeutics for the specific tumor.


Assuntos
Colangiocarcinoma/imunologia , Citotoxicidade Imunológica/imunologia , Imunoterapia Adotiva/efeitos adversos , Células Matadoras Naturais/imunologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Colangiocarcinoma/terapia , Citotoxicidade Imunológica/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Int J Mol Sci ; 19(6)2018 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-29874880

RESUMO

The innate immune system has numerous mechanisms to fight against pathogens, including the formation of neutrophil extracellular traps (NETs). By spreading out chromatin, antimicrobial peptides and enzymes, neutrophils efficiently trap pathogens like bacteria and facilitate their elimination. During this process, high concentrations of extracellular histones can be reached. Several researchers have demonstrated that the cytotoxic characteristics of these histones can trigger diseases like sepsis. Interestingly, the carbohydrate polysialic acid (polySia) can bind histones and reduce histone-mediated cytotoxicity in a chain length-dependent manner. In the present study, we examined the chain length of polySia in plasma and tested its ability to decrease the cytotoxic characteristics of extracellular histones. Remarkably, we detected polySia not only in the soluble fraction of plasma, but also on enriched extracellular vesicles (EVs). Chain length analysis revealed that polySia chains originating from human plasma can consists of more than 40 sialic acid residues and show a cytoprotective effect against extracellular histones. Intriguingly, polySia is not only present in human plasma but also in fish and other branches of vertebrates. Thus, polySia is a physiological element in plasma and may represent a natural buffer for extracellular histones.


Assuntos
Citotoxicidade Imunológica/genética , Histonas/imunologia , Sepse/metabolismo , Ácidos Siálicos/metabolismo , Carboidratos/química , Armadilhas Extracelulares/metabolismo , Histonas/efeitos adversos , Histonas/biossíntese , Humanos , Imunidade Inata/genética , Neutrófilos/imunologia , Neutrófilos/metabolismo , Sepse/etiologia , Sepse/patologia
7.
Microbiol Immunol ; 62(5): 348-356, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29577371

RESUMO

Although CD4+ T cells are generally regarded as helper T cells, some activated CD4+ T cells have cytotoxic properties. Given that CD4+ cytotoxic T lymphocytes (CTLs) often secrete IFN-γ, CTL activity among CD4+ T cells may be attributable to Th1 cells, where a T-box family molecule, T-bet serves as the "master regulator". However, although the essential contribution of T-bet to expression of IFN-γ has been well-documented, it remains unclear whether T-bet is involved in CD4+ T cell-mediated cytotoxicity. In this study, to investigate the ability of T-bet to confer cytolytic activity on CD4+ T cells, the T-bet gene (Tbx21) was introduced into non-cytocidal CD4+ T cell lines and their cytolytic function analyzed. Up-regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4+ T cells but not in untransfected parental cells. In one cell line, T-bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas- target cells. Although T-bet was found to repress up-regulation of CD40L (CD154), which controls FasL-mediated cytolysis, the extent of CD40L up-regulation on in vitro-differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T-bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Citotoxicidade Imunológica/imunologia , Proteínas com Domínio T/imunologia , Células Th1/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Ligante de CD40/metabolismo , Linhagem Celular , Citotoxicidade Imunológica/genética , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Regulação da Expressão Gênica/genética , Interferon gama/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Perforina/genética , Perforina/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Regulação para Cima
8.
Science ; 359(6377): 770-775, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29301958

RESUMO

Many human cancers are resistant to immunotherapy, for reasons that are poorly understood. We used a genome-scale CRISPR-Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T cells, the central effectors of antitumor immunity. Inactivation of >100 genes-including Pbrm1, Arid2, and Brd7, which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex-sensitized mouse B16F10 melanoma cells to killing by T cells. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T cells. Treatment-resistant tumors became responsive to immunotherapy when Pbrm1 was inactivated. In many human cancers, expression of PBRM1 and ARID2 inversely correlated with expression of T cell cytotoxicity genes, and Pbrm1-deficient murine melanomas were more strongly infiltrated by cytotoxic T cells.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Citotoxicidade Imunológica/genética , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/terapia , Linfócitos T Citotóxicos/imunologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Endonucleases , Testes Genéticos , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Imunoterapia , Interferon gama/uso terapêutico , Melanoma Experimental/genética , Camundongos , Neoplasias Cutâneas/genética , Fatores de Transcrição/genética
9.
Immunogenetics ; 70(1): 29-36, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28653095

RESUMO

Granzyme B (GzmB) is a component of cytolytic granules within NK cells and is involved in several pathologies. It has previously been reported that there are three non-synonymous coding SNPs (rs8192917; Q48R, rs11539752; P88A, and rs2236338; Y245H) in the GZMB gene and that the QPY/RAH allele was clustered together close to the C-terminal α-helix. However, it is unknown whether the function of GzmB produced from NK cells is influenced by QPY/RAH polymorphism. The authors investigated the distribution of QPY/RAH polymorphism of the GZMB gene in a Japanese population (n = 106), and the involvement of Q48R polymorphism in NK cell cytotoxicity, degranulation, and production of GzmB. A strong linkage disequilibrium was observed among these SNPs, and NK cell cytotoxicity was influenced by rs8192917 (Q48R). Moreover, it found that R48-GzmB is a stable protein that accumulates to similar levels in activated NK cells as Q48-GzmB. rs8192917 polymorphism may influence antitumor activity and the effect of antitumor cellular immunotherapy. The authors expect that these new informations about QPY/RAH polymorphism of the GZMB gene could help to assess the impact of NK cell cytotoxicity in several pathologies and aid their treatment.


Assuntos
Granzimas/genética , Células Matadoras Naturais/imunologia , Adolescente , Adulto , Alelos , Grupo com Ancestrais do Continente Asiático/genética , Citotoxicidade Imunológica/genética , Feminino , Frequência do Gene , Genótipo , Granzimas/imunologia , Granzimas/metabolismo , Haplótipos , Humanos , Células Matadoras Naturais/citologia , Desequilíbrio de Ligação , Masculino , Polimorfismo de Nucleotídeo Único
10.
Surg Today ; 48(1): 119-126, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28573328

RESUMO

PURPOSE: Various strategies, such as the generation of alpha-1,3-galactosyltransferase knocked-out pigs and CD55 transgenic pigs, have been investigated to inhibit pig to human xenogeneic rejection. Our aim is to develop strategies to overcome the hurdle of not only hyper acute rejection, but also that of cellular xenogeneic rejection (CXR). Although macrophages have been well known to play a critical role in CXR, monocyte/macrophage-mediated xenogeneic rejection has not been well studied. In this study, we evaluated the effect of CD200 in xenogeneic rejection by macrophages. METHODS: Naïve swine endothelial cells (SEC) and SEC/CD200 were co-cultured with M0 macrophages and the cytotoxicity was measured by a WST-8 assay. The phagocytosis of SEC and SEC/CD200 by macrophages was analyzed by flow cytometry. RESULTS: While CD200 failed to suppress a significant amount of cytotoxicity against SEC by monocytes, M0 macrophage-mediated cytotoxicity was significantly suppressed by human CD200. The phagocytosis by M0 macrophages was also tested. The phagocytosis assay revealed that human CD200 suppresses M0 macrophage-mediated phagocytosis. CONCLUSIONS: Our findings indicate that human CD200 suppresses the xenogeneic rejection by CD200R+ macrophages and that the generation of hCD200 transgenic pigs for use in xenografts is very attractive for preventing the macrophage-mediated rejection.


Assuntos
Antígenos CD/fisiologia , Citotoxicidade Imunológica/genética , Células Endoteliais/imunologia , Macrófagos/imunologia , Fagocitose/genética , Animais , Células Cultivadas , Citometria de Fluxo , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Humanos , Suínos
11.
Proc Natl Acad Sci U S A ; 114(50): 13236-13241, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29180433

RESUMO

CD8+ T cells are preprogrammed for cytotoxic differentiation in the thymus as they acquire expression of the transcription factor Runx3. However, a subset of effector CD8+ T cells (Tc17) produce IL-17 and fail to express cytotoxic genes. Here, we show that the transcription factors directing IL-17 production, STAT3 and RORγt, inhibit cytotoxicity despite persistent Runx3 expression. Cytotoxic gene repression did not require the transcription factor Thpok, which in CD4+ T cells restrains Runx3 functions and cytotoxicity; and STAT3 restrained cytotoxic gene expression in CD8+ T cells responding to viral infection in vivo. STAT3-induced RORγt represses cytotoxic genes by inhibiting the functions but not the expression of the "cytotoxic" transcription factors T-bet and Eomesodermin. Thus, the transcriptional circuitry directing IL-17 expression inhibits cytotoxic functions. However, by allowing expression of activators of the cytotoxic program, this inhibitory mechanism contributes to the instability of IL-17-producing T cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/genética , Fator de Transcrição STAT3/metabolismo , Animais , Células Cultivadas , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo
12.
Cancer Res ; 77(24): 7049-7058, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29055013

RESUMO

T cell-based immunotherapies are a promising approach for patients with advanced cancers. However, various obstacles limit T-cell efficacy, including suboptimal T-cell receptor (TCR) activation and an immunosuppressive tumor environment. Here, we developed a fusion protein by linking CD8α and MyD88 (CD8α:MyD88) to enhance CD8+ T-cell responses to weakly immunogenic and poorly expressed tumor antigens. CD8α:MyD88-engineered T cells exhibited increased proliferation and expression of effector and costimulatory molecules in a tumor antigen-dependent manner. These effects were accompanied by elevated activation of TCR and Toll-like receptor signaling-related proteins. CD8α:MyD88-expressing T cells improved antitumor responses in mice. Enhanced antitumor activity was associated with a unique tumor cytokine/chemokine signature, improved T-cell infiltration, reduced markers of T-cell exhaustion, elevated levels of proteins associated with antigen presentation, and fewer macrophages with an immunosuppressive phenotype in tumors. Given these observations, CD8α:MyD88 represents a unique and versatile approach to help overcome immunosuppression and enhance T-cell responses to tumor antigens. Cancer Res; 77(24); 7049-58. ©2017 AACR.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Apresentação do Antígeno/efeitos dos fármacos , Apresentação do Antígeno/imunologia , Antígenos CD8/genética , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/genética , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Proteínas Recombinantes de Fusão/genética
13.
Exp Oncol ; 39(3): 212-218, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28967637

RESUMO

AIM: The aim of this study is to investigate the effects of miR150 transfection on NK-like cells differentiated from adipose tissue derived mesenchymal stem cells (AD-MSCs). METHODS: NK-like cells were differentiated from AD-MSCs and activated by miR150 transfection. Transfected/non-transfected NK-like cells were characterized by immunohistochemical and RT-PCR analyzes. Apoptotic efficiency of the transfected/non-transfected NK-like cells on pancreatic cancer cells PANC1 were determined by TUNEL and RT-PCR. RESULTS: In miR150-transfected cells, the increased expression of NK cell-specific genes such as GKMB, KIR2DL2, CD16, CD56, NKG2D, NKp46 and increased immunoreactivity of NK cell-specific surface marker CD314 (NKG2D) were evident. TUNEL assays showed that NK-like cells with/without transfection induced apoptosis in PANC1 cells in the same manner. The decrease in oncogene expression and the increase in the tumor suppressor gene expression in PANC1 cells upon co-culture with NK-like cells differentiated from AD-MSCs were more prominent following miRNA150 transfection. CONCLUSION: It was shown in vitro that NK-like cells could be obtained by differentiation from AD-MSCs and their efficiency could be increased via miR150 transfection. The results are encouraging for further clinical studies in improvement of immunotherapeutic approaches for cancer therapy.


Assuntos
Tecido Adiposo/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Apoptose/genética , Apoptose/imunologia , Biomarcadores , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Expressão Gênica , Humanos , Células Matadoras Naturais/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Transfecção
14.
Cancer Res ; 77(24): 7059-7071, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29042417

RESUMO

Immune checkpoint therapies target tumor antigen-specific T cells, but less is known about their effects on natural killer (NK) cells, which help control metastasis. In studying the development of lung metastases, we found that NK cells lose their cytotoxic capacity and acquire a molecular signature defined by the expression of coinhibitory receptors. In an effort to overcome this suppressive mechanism, we evaluated NK cell responses to the immunostimulatory cytokine IL12. Exposure to IL12 rescued the cytotoxicity of NK cells but also led to the emergence of an immature NK cell population that expressed high levels of the coinhibitory molecules PD-1, Lag-3, and TIGIT, thereby limiting NK cell-mediated control of pulmonary metastases. Notably, checkpoint blockade therapy synergized with IL12 to fully enable tumor control by NK cells, demonstrating that checkpoint blockers are not only applicable to enhance T cell-mediated immunotherapy, but also to restore the tumor-suppressive capacity of NK cells. Cancer Res; 77(24); 7059-71. ©2017 AACR.


Assuntos
Pontos de Checagem do Ciclo Celular , Citotoxicidade Imunológica , Interleucina-12/farmacologia , Células Matadoras Naturais/imunologia , Neoplasias Mamárias Experimentais/terapia , Terapia de Alvo Molecular/métodos , Metástase Neoplásica/imunologia , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/imunologia , Citotoxicidade Imunológica/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imunoterapia/métodos , Interleucina-12/genética , Interleucina-12/uso terapêutico , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Metástase Neoplásica/genética , Células Tumorais Cultivadas
15.
Cancer Biomark ; 20(4): 609-615, 2017 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-28869441

RESUMO

OBJECTIVE: To investigate the improvement of cytotoxicity of autologous CIKs from patients with breast cancer to MCF-7 cells by suppressed PD-1 expression. METHODS: The Lentiviral Vector/PD-1 carrying the gene that can suppressed PD-1 was transferred to CIK cells from patients with breast cancer to inhibit PD-1 expression. The PD-1 protein were detected by RT-PCR and Western blot. The positive PD-1 of CIKs and PD-L1 of MCF-7 cells were detected by FCM, and cytotoxicity of CIKs to MCF-7 was assayed by CCK-8. RESULTS: The PD-1 positive CIKs with Lentiviral Vector/PD-1 transferred from patients with breast cancer were 16.02%, 14.36% and 14.64% at 14th, 21st and 28th day, obviously inhibited as compared to 50.54%, 74.50% and 73.36% in CIKs without transinfection (P< 0.05); the Lentiviral Vector/PD-1 decreased the PD-1 mRNA levels in CIK cells, and Lentiviral Vector/PD-1-transferred CIKs had lower PD-1 expression; CCK-8 detection showed that at 14th day, the cytotoxicity rates of CIKs with blank plasmids and those with PD-1 transfection to MCF-7 cells were 58.78% and 68.14%, respectively. CONCLUSION: MCF-7 cells have a strong PD-L1 expression at its surface, and inhibition of PD-1 expression can improve the cytotoxicity of CIK cells.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Células Matadoras Induzidas por Citocinas/imunologia , Células Matadoras Induzidas por Citocinas/metabolismo , Citotoxicidade Imunológica/genética , Regulação da Expressão Gênica , Receptor de Morte Celular Programada 1/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Células MCF-7 , Receptor de Morte Celular Programada 1/metabolismo , Transfecção
16.
J Immunol ; 199(6): 2030-2042, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28784847

RESUMO

Alternative splicing occurs frequently in many genes, especially those involved in immunity. Unfortunately, the functions of many alternatively spliced molecules from immunologically relevant genes remain unknown. Classical HLA-I molecules are expressed on almost all nucleated cells and play a pivotal role in both innate and adaptive immunity. Although splice variants of HLA-I genes have been reported, the details of their functions have not been reported. In the current study, we determined the characteristics, expression, and function of a novel splice variant of HLA-A11 named HLA-A11svE4 HLA-A11svE4 is located on the cell surface without ß2-microglobulin (ß2m). Additionally, HLA-A11svE4 forms homodimers as well as heterodimers with HLA-A open conformers, instead of combining with ß2m. Moreover, HLA-A11svE4 inhibits the activation of NK cells to protect target cells. Compared with ß2m and HLA-A11, the heterodimer of HLA-A11svE4 and HLA-A11 protected target cells from lysis by NK cells more effectively. Furthermore, HLA-AsvE4 expression was upregulated by HIV-1 in vivo and by HSV, CMV, and hepatitis B virus in vitro. In addition, our findings indicated that HLA-A11svE4 molecules were functional in activating CD8+ T cells through Ag presentation. Taken together, these results suggested that HLA-A11svE4 can homodimerize and form a novel heterodimeric complex with HLA-A11 open conformers. Furthermore, the data are consistent with HLA-A11svE4 playing a role in the immune escape of HIV-1.


Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Antígeno HLA-A11/metabolismo , Células Matadoras Naturais/fisiologia , Isoformas de Proteínas/metabolismo , Processamento Alternativo , Apresentação do Antígeno/genética , Células Cultivadas , Citotoxicidade Imunológica/genética , Regulação da Expressão Gênica , Antígeno HLA-A11/genética , Humanos , Evasão da Resposta Imune , Domínios Proteicos/genética , Isoformas de Proteínas/genética , Multimerização Proteica , Deleção de Sequência/genética
17.
Int Immunol ; 29(6): 277-289, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28814066

RESUMO

Specific variants of endoplasmic reticulum-associated aminopeptidase 1 (ERAP1) identified by genome-wide association study modify the risk for developing ankylosing spondylitis. We previously confirmed that disease-associated ERAP1 variants have altered enzymatic abilities that can impact upon the production of pro-inflammatory cytokines from cells expressing the same ERAP1 variants. To determine if these ERAP1 variants also impacted immune responses in vivo, we generated two strains of transgenic mice expressing human ERAP1 genes containing non-synonymous single-nucleotide polymorphisms associated with an increased (ERAP1-High) or decreased (ERAP1-Low) risk for developing autoimmune disease. After vaccination with foreign antigens, ERAP1-High mice generated unique populations of antigen-specific T-cell clones. The expression of ERAP1-High also reduced MHC-I expression on the surface of multiple cell types, demonstrating a global impact on the MHC-I peptidome. ERAP1 variants also affected the innate immune system, because NK cells from murine ERAP1 (mERAP1) knockout mice and ERAP1-High/mERAP1-/- mice had decreased surface expression of the activating receptor NKG2D on their NK and T cells, and NK cells derived from mERAP1-/- mice or ERAP1-Low mice demonstrated more active NK cell killing than NK cells derived from wild-type or ERAP1-High mice. Finally, these studies were conducted in female mice, as all male ERAP1-High mice died in utero or shortly after birth, making ERAP1-High one of the only dominant lethal autosomal genes known in mammals. Together, these results present the first direct evidence that human disease-associated ERAP1 variants can greatly alter survival, as well as antigen presentation, T-cell repertoire and NK cell responses in vivo.


Assuntos
Aminopeptidases/genética , Citotoxicidade Imunológica/genética , Células Matadoras Naturais/fisiologia , Antígenos de Histocompatibilidade Menor/genética , Espondilite Anquilosante/genética , Linfócitos T/fisiologia , Imunidade Adaptativa/genética , Animais , Apresentação do Antígeno , Células Clonais , Feminino , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunidade Inata/genética , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Polimorfismo de Nucleotídeo Único , Receptores de Antígenos de Linfócitos T/genética , Risco , Transgenes/genética
18.
Cell Immunol ; 319: 35-42, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28735814

RESUMO

BACKGROUND: Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcÆ´R+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. METHODS: HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. RESULTS: HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. CONCLUSIONS: In the presence of effector cells with high cytotoxic capacity, TKIs have the ability to augment the passive immunotherapeutic potential of trastuzumab in SKBR3, a model of HER2+ breast cancer. ADCC levels detected by LDH release assays are extremely low in MCF-7; the flow cytometry-based CFSE/7-AAD method is more sensitive and consistent for the determination of ADCC in HER2-low models.


Assuntos
Antineoplásicos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/genética , Trastuzumab/farmacologia , Afatinib , Linhagem Celular Tumoral , Citotoxicidade Imunológica/genética , Interações de Medicamentos , Humanos , Células K562 , L-Lactato Desidrogenase/metabolismo , Lapatinib , Células MCF-7 , Quinazolinas/farmacologia , Quinolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Transdução de Sinais
19.
Oncol Rep ; 38(2): 693-702, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28677817

RESUMO

Immunotherapy is expected to be promising as a next generation cancer therapy. Immunoreceptors are often activated constitutively in cancer cells, however, such levels of ligand expression are not effectively recognized by the native immune system due to tumor microenvironmental adaptation. Studies have demonstrated that natural-killer group 2, member D (NKG2D), a major activating immunoreceptor, responds to DNA damage. The upregulation of major histocompatibility complex class I-related chain A and B (MICA/B) (members of NKG2D ligands) expression after DNA damage is associated with NK cell-mediated killing of cancer cells. However, the regulation of DNA damage-induced MICA/B expression has not been fully elucidated in the context of the types of cancer cell lines. In the present study, we found that MICA/B expression varied between cancer cell lines after DNA damage. Screening in terms of chromatin remodeling identified that inhibitors related to chromatin relaxation via post-translational modification on histone H3K9, i.e. HDAC, Suv39 or G9a inhibition, restored DNA damage-dependent MICA/B expression in insensitive cells. In addition, we revealed that the restored MICA/B expression was dependent on ATR as well as E2F1, a transcription factor. We further revealed that low­dose treatment of an HDAC inhibitor was sufficient to restore MICA/B expression in insensitive cells. Finally, we demonstrated that HDAC inhibition restored DNA damage­dependent cytotoxic NK activity against insensitive cells. Thus, the present study revealed that DNA damage­dependent MICA/B expression in insensitive cancer cells can be restored by chromatin relaxation via the HDAC/Suv39/G9a pathway. Collectively, manipulation of chromatin status by therapeutic cancer drugs may potentiate the antitumor effect by enhancing immune activation following radiotherapy and DNA damage-associated chemotherapy.


Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Histona Desacetilases/genética , Neoplasias/imunologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Montagem e Desmontagem da Cromatina/genética , Citotoxicidade Imunológica/genética , Dano ao DNA/genética , Fator de Transcrição E2F1/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Histona Desacetilases/imunologia , Histona-Lisina N-Metiltransferase/genética , Humanos , Células Matadoras Naturais/imunologia , Metiltransferases/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas Repressoras/genética , Microambiente Tumoral/imunologia
20.
Am J Hematol ; 92(10): 981-988, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28646491

RESUMO

Sickle erythrocytes' (SSRBCs) unique physical adaptation to hypoxic conditions renders them able to home to hypoxic tumor niches in vivo, shut down tumor blood flow and induce tumoricidal responses. SSRBCs are also useful vehicles for transport of encapsulated drugs and oncolytic virus into hypoxic tumors with enhanced anti-tumor effects. In search of additional modes for arming sickle cells with cytotoxics, we turned to a lentiviral ß-globin vector with optimized Locus Control Region/ß-globin coding region/promoter/enhancers. We partially replaced the ß-globin coding region of this vector with genes encoding T cell cytolytics, perforin and granzyme or immune modulating superantigens SEG and SEI. These modified vectors efficiently transduced Sca+ ckit- Lin- hematopoietic stem cells (HSCs) from humanized sickle cell knockin mice. Irradiated mice reconstituted with these HSCs displayed robust expression of transgenic RNAs and proteins in host sickle cells that was sustained for more than 10 months. SSRBCs from reconstituted mice harboring SEG/SEI transgenes induced robust proliferation and a prototypical superantigen-induced cytokine reaction when exposed to human CD4+/CD8+ cells. The ß-globin lentiviral vector therefore produces a high level of functional, erythroid-specific immune modulators and cytotoxics that circulate without toxicity. Coupled with their unique ability to target and occlude hypoxic tumor vessels these armed SSRBCs constitute a potentially useful tool for treatment of solid tumors.


Assuntos
Anemia Falciforme , Citotoxicidade Imunológica , Eritrócitos Anormais/imunologia , Neoplasias Experimentais/imunologia , Neovascularização Patológica/imunologia , Globinas beta/genética , Anemia Falciforme/sangue , Animais , Citotoxicidade Imunológica/genética , Sistemas de Liberação de Medicamentos , Eritrócitos Anormais/metabolismo , Eritrócitos Anormais/transplante , Técnicas de Introdução de Genes , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Hipóxia , Lentivirus/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/terapia , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia
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