RESUMO
BACKGROUND/AIM: The natural killer (NK) cell function of patients with malignant tumours may be suppressed by deficiency, and the poor prognosis of renal cell carcinoma (RCC) patients may be due to escape from NK cell cytotoxicity, especially with respect to natural cytotoxicity receptors (NCRs) on the NK cell surface. However, the specific mechanism remains unclear. Therefore, in this study, we sought to explore the role of NCR, especially NCR3 splice variants, in the process of NK cell deficiency in RCC patients. MATERIALS AND METHODS: We used flow cytometry to analyse the phenotype of NK cells from the peripheral blood and kidney tumour tissue of RCC patients. The NKp30-mediated NK cell killing function was measured by antibody-dependent cell-mediated cytotoxicity (ADCC) in NK and RCC cell coincubation. We extracted RNA from the peripheral blood mononuclear cells (PBMCs) of RCC patients and renal carcinoma tissue and carried out real-time quantitative PCR to detect the mRNA levels of NKp30a, NKp30b and NKp30c. mRNA expression levels of cytokines (IL-6, IL-8, IL-10, IL-18 and TGF-ß) based on RNA extracted from renal carcinoma tissue and adjacent normal kidney tissues were also measured by real-time quantitative PCR. RESULTS: Regarding the phenotype of NK cells in RCC patients, the proportion of NK cells in tumour tissue was significantly reduced, with changes in the NK cell proportion being most obvious in NKp30+ NK cells. Furthermore, the results of the ADCC function assay showed limited NKp30+ NK cell-mediated cytotoxicity in RCC patients. Through real-time quantitative PCR, we found lower expression of NKp30a and NKp30b, the immunostimulatory splice variants of NCR3 encoding NKp30, in RCC patients. Moreover, expression of activating cytokines (IL-6 and IL-8) in renal cancer tissue was decreased, though inhibitory cytokine (TGF-ß) expression remained unchanged, which may result in an immunosuppressive cytokine microenvironment. CONCLUSION: Decreased expression of immunostimulatory NCR3 splice variants and the inhibitory cytokine microenvironment in RCC patients may contribute to deficient NK cell cytotoxicity and renal carcinoma cell immune escape from NK cell killing, which may provide a theoretical basis for finding new immunotherapeutic targets for RCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leucócitos Mononucleares , Citocinas/genética , Citocinas/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/patologia , Células Matadoras Naturais , Fator de Crescimento Transformador beta/metabolismo , RNA Mensageiro/metabolismo , RNA/metabolismo , Microambiente Tumoral , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismoRESUMO
The NKp30 receptor is one of the three natural cytotoxic receptors reported in NK cells. This receptor is codified by the NCR3 gene, which encodes three isoforms, a consequence of the alternative splicing of exon 4. A greater expression of the three isoforms (A, B, and C), along with low levels of the NKp30 ligand B7H6, has been reported as a positive prognostic factor in different cancer types. Here, in patients with cervical cancer and precursor lesions, we report an altered immune-phenotype, characterized by non-fitness markers, that correlated with increased disease stage, from CIN 1 to FIGO IV. While overall NK cell numbers increased, loss of NKp30+ NK cells, especially in the CD56dim subpopulation, was found. Perforin levels were decreased in these cells. Decreased expression of the NKp30 C isoform and overexpression of soluble B7H6 was found in cervical cancer patients when compared against healthy subjects. PBMCs from healthy subjects downregulated NKp30 isoforms after co-culture with B7H6-expressing tumour cells. Taken together, these findings describe a unique down-modulation or non-fitness status of the immune response in cervical cancer, the understanding of which will be important for the design of novel immunotherapies against this disease.
Assuntos
Neoplasias do Colo do Útero , Humanos , Feminino , Perforina/genética , Células Matadoras Naturais , Isoformas de Proteínas/genética , Processamento Alternativo , Receptor 3 Desencadeador da Citotoxicidade Natural/genéticaRESUMO
Immune checkpoints are vital molecules that regulate T-cell function by activation or inhibition. Among the immune checkpoint molecules, the B7-family proteins are significantly involved in the immune escape of tumor cells. By binding to inhibitory receptors, they can suppress T-cell-mediated immunity. B7-family proteins are found at various stages of tumor microenvironment formation and promote tumorigenesis and tumor progression. B7-H6 (encoded by gene NCR3LG1) is a prominent member of the family. It has unique immunogenic properties and is involved in natural killer (NK) cell immunosurveillance by binding to the NKp30 receptor. High B7-H6 expression in certain tumor types and shortage of or low expression in healthy cells - except in cases of inflammatory or microbial stimulation - have made the protein an attractive target of research activities in recent years. The avoidance of NK-mediated B7-H6 detection is a mechanism through which tumor cells escape immune surveillance. The stimulation of tumorigenesis occurs by suppressing caspase cascade initiation and anti-apoptosis activity stimulation via the STAT3 pathway. The B7-H6-NKp30 complex on the tumor membrane activates the NK cells and releases both tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). B7-H6 is highly expressed in a wide range of tumor cells, including glioma, hematologic malignant tumors, and breast cancer cells. Clinical examination of cancer patients indicated that the expression of B7-H6 is related to distant metastasis status and permits postoperative prognosis. Because of its unique properties, B7-H6 has a high potential be utilized as a biological marker for cancer diagnosis and prognosis, as well as a target for novel treatment options.
Assuntos
Receptor 3 Desencadeador da Citotoxicidade Natural , Neoplasias , Carcinogênese , Humanos , Imunoterapia , Células Matadoras Naturais , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Neoplasias/patologia , Microambiente TumoralRESUMO
Background: B7-H6, a newly discovered member of the immunoglobulin superfamily, exerts antitumor effects by binding to NKP30 receptor on natural killer cells; it has important clinical implications. Cell surface ectodomain shedding of B7-H6 generates soluble B7-H6 (sB7-H6), which is highly expressed and serves as a valuable biomarker in multiple tumors, but the clinical significance and diagnostic value of B7-H6 in cervical squamous cell carcinoma (CSCC) remains unclear. Objective: To assess the expression and diagnostic value of B7-H6 in CSCC. Methods: In this study, 69 cervical specimens were analyzed for B7-H6 expression: 25 paired CSCC tissues were examined using quantitative real-time polymerase chain reaction, and 24 paraffin-embedded CSCC tissues and 20 normal tissues were analyzed immunohistochemically. Furthermore, plasma samples from 30 CSCC patients and 24 healthy controls were examined using ELISA. Results: B7-H6 mRNA and protein levels were significantly higher in CSCC tissues than in adjacent normal cervical tissues (p < 0.05). Immunohistochemical analysis revealed that high B7-H6 expression correlated with stromal invasion (p = 0.043), lymphovascular space involvement (p = 0.005), lymph node metastasis (p = 0.019), and International Federation of Gynecology and Obstetrics (FIGO) stage (p = 0.002). Moreover, ELISA results demonstrated that the sB7-H6 concentration in peripheral blood was higher in CSCC patients than in healthy controls (p < 0.0001). Notably, at the optimal cutoff point of 0.076 ng/mL, sB7-H6 showed 93.3% sensitivity and 62.5% specificity in the discrimination of CSCC patients from healthy controls. Conclusions: B7-H6 mRNA and protein levels are markedly increased in CSCC tissues and peripheral blood samples, and the B7-H6 level can be used as a biomarker for predicting the severity of CSCC disease and discriminating CSCC patients from healthy controls.
Assuntos
Antígenos B7/genética , Carcinoma de Células Escamosas/genética , Neoplasias do Colo do Útero/genética , Adulto , Antígenos B7/metabolismo , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/diagnóstico , China , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , RNA Mensageiro/genética , Neoplasias do Colo do Útero/diagnósticoRESUMO
B7-H6, a ligand for the NK activating receptor NKp30, has been identified as a biomarker of poor prognosis in several solid cancers. However, little is known about the role of B7-H6 and the mechanisms that control its expression in acute myeloid leukemia (AML). Epigenome modulation, including epigenomic reader dysregulation, is one of the hallmarks of AML. Bromodomain-containing protein 4 (BRD4), the best-known member of the BET family of epigenetic readers, is overexpressed in AML cells and regulates the transcription of genes involved in the pathogenesis of AML, as MYC oncogene. Here, we analyze the role of BRD4 in regulating B7-H6 in AML cells. Results demonstrated that the specific inhibition of BRD4 drastically reduces the expression of B7-H6 in AML cells. Histone acetylation mediated by CBP30/P300 facilitates the binding of BRD4 to the B7-H6 promoter, which recruits the P-TEFb elongation factor that phosphorylates RNA polymerase II, thereby activating B7-H6 transcription. BRD4 also co-bounded with JMJD6 at the distal enhancer of the B7-H6 gene. Metabolic modulation with metformin modifies the acetylation pattern in the B7-H6 promoter, impairing BRD4 binding, thereby inhibiting B7-H6 expression. B7-H6 knockdown induces the apoptosis in HEL-R cell line. Moreover, a high level of B7-H6 expression in AML patients is related to increased BRD4 levels, myelodysplastic-derived AML, and del5q, the two latter being associated with poor prognosis. Our data show that BRD4 is a positive regulator of the pro-tumorigenic molecule B7-H6 and that the blockage of the B7-H6 is a potential therapeutic target for the treatment of AML.
Assuntos
Antígenos B7 , Proteínas de Ciclo Celular , Leucemia Mieloide Aguda , Fatores de Transcrição , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Epigênese Genética/genética , Humanos , Histona Desmetilases com o Domínio Jumonji , Leucemia Mieloide Aguda/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Fatores de Transcrição/genéticaRESUMO
NKp30 (Natural Cytotoxicity Receptor 1, NCR1) is a powerful cytotoxicity receptor expressed on natural killer (NK) cells which is involved in tumor cell killing and the regulation of antitumor immune responses. Ligands for NKp30, including BAG6 and B7-H6, are upregulated in virus-infected and tumor cells but rarely detectable on healthy cells. These ligands are released by tumor cells as part of the cellular secretome and interfere with NK cell activity. BAG6 is secreted via the exosomal pathway, and BAG6-positive extracellular vesicles (EV-BAG6) trigger NK cell cytotoxicity and cytokine release, whereas the soluble protein diminishes NK cell activity. However, the extracellular format and activity of B7-H6 remain elusive. Here, we used HEK293 as a model cell line to produce recombinant ligands and to study their impact on NK cell activity. Using this system, we demonstrate that soluble B7-H6 (sB7-H6), like soluble BAG6 (sBAG6), inhibits NK cell-mediated target cell killing. This was associated with a diminished cell surface expression of NKG2D and NCRs (NKp30, NKp40, and NKp46). Strikingly, a reduced NKp30 mRNA expression was observed exclusively in response to sBAG6. Of note, B7-H6 was marginally released in association with EVs, and EVs collected from B7-H6 expressing cells did not stimulate NK cell-mediated killing. The molecular analysis of EVs on a single EV level using nano flow cytometry (NanoFCM) revealed a similar distribution of vesicle-associated tetraspanins within EVs purified from wildtype, BAG6, or B7-H6 overexpressing cells. NKp30 is a promising therapeutic target to overcome NK cell immune evasion in cancer patients, and it is important to unravel how extracellular NKp30 ligands inhibit NK cell functions.
Assuntos
Antígenos B7/metabolismo , Chaperonas Moleculares/metabolismo , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Antígenos B7/genética , Vesículas Extracelulares/metabolismo , Células HEK293 , Humanos , Integrina beta1/metabolismo , Células K562 , Células Matadoras Naturais/metabolismo , Ligantes , Chaperonas Moleculares/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Evasão TumoralRESUMO
Natural killer (NK) cells play major roles in innate immunity against viruses and cancer. Natural killer receptors (NKR) expressed by NK cells recognize foreign- or self-ligands on infected and transformed cells as well as healthy cells. NKR genes are the most rapidly evolving loci in vertebrates, and it is generally difficult to detect orthologues in different taxa. The unique exception is NKp30, an activating NKR in mammals that binds to the self-ligand B7H6. The NKp30-encoding gene, NCR3, has been found in most vertebrates including sharks, the oldest vertebrates with human-type adaptive immunity. NCR3 has a special, non-rearranging VJ-type immunoglobulin superfamily (IgSF) domain that predates the emergence of the rearranging antigen receptors. Herein we show that NCR3 loci are linked to the shark major histocompatibility complex (MHC), proving NCR3's primordial association with the MHC. We identified eight subtypes of differentially expressed highly divergent shark NCR3 family genes. Using in situ hybridization, we detected one subtype, NS344823, to be expressed by predominantly single cells outside of splenic B cell zones. The expression by non-B cells was also confirmed by PCR in peripheral blood lymphocytes. Surprisingly, high expression of NS344823 was detected in the thymic cortex, demonstrating NS344823 expression in developing T cells. Finally, we show for the first time that shark T cells are found as single cells or in small clusters in the splenic red pulp, also unassociated with the large B cell follicles we previously identified.
Assuntos
Células Matadoras Naturais/imunologia , Complexo Principal de Histocompatibilidade/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Sequência de Aminoácidos , Animais , Células Matadoras Naturais/metabolismo , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Filogenia , Homologia de Sequência , TubarõesRESUMO
Natural killer (NK) cells are innate immune effectors with potent antitumor activity. However, tumor cells can create an immunosuppressive microenvironment to escape immune surveillance. Although accumulating evidence indicates that microenvironmental hypoxia plays an important role in favoring tumor development and immune evasion, it remains unclear by what means hypoxia directly impairs NK cell antitumor activity. In this study, we confirmed that hypoxic NK cells showed significantly lower cytotoxicity against tumor cells. Consistent with this finding, we found that the reduction in NK cell cytotoxicity resulting from hypoxia correlated to the lower expression of granzyme B, IFN-γ, and degranulation marker CD107a, as well as activating receptors including NKp30, NKp46, and NKG2D expressed on the surface of NK cells. More importantly, we further demonstrated that a reduction in the phosphorylation levels of ERK and STAT3 secondary to hypoxia was strongly associated with the attenuated NK cell cytotoxicity. Focusing on the mechanism responsible for reduced phosphorylation levels of ERK and STAT3, we reveal that the activation of protein tyrosine phosphatase SHP-1 (Src homology region 2 domain-containing phosphatase-1) following hypoxia might play an essential role in this process. By knocking down SHP-1 or blocking its activity using a specific inhibitor TPI-1, we were able to partially restore NK cell cytotoxicity under hypoxia. Taken together, we demonstrate that hypoxia could impair NK cell cytotoxicity by decreasing the phosphorylation levels of ERK and STAT3 in a SHP-1-dependent manner. Therefore, targeting SHP-1 could provide an approach to enhance NK cell-based tumor immunotherapy.
Assuntos
Hipóxia/imunologia , Células Matadoras Naturais/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Linhagem Celular , Microambiente Celular , Citotoxicidade Imunológica , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Timidina/análogos & derivados , Timidina/farmacologia , Evasão TumoralRESUMO
The Natural Cytotoxicity Receptors (NCRs), NKp46, NKp44, and NKp30, were some of the first human activating Natural Killer (NK) cell receptors involved in the non-MHC-restricted recognition of tumor cells to be cloned over 20 years ago. Since this time many host- and pathogen-encoded ligands have been proposed to bind the NCRs and regulate the cytotoxic and cytokine-secreting functions of tissue NK cells. This diverse set of NCR ligands can manifest on the surface of tumor or virus-infected cells or can be secreted extracellularly, suggesting a remarkable NCR polyfunctionality that regulates the activity of NK cells in different tissue compartments during steady state or inflammation. Moreover, the NCRs can also be expressed by other innate and adaptive immune cell subsets under certain tissue conditions potentially conferring NK recognition programs to these cells. Here we review NCR biology in health and disease with particular reference to how this important class of receptors regulates the functions of tissue NK cells as well as confer NK cell recognition patterns to other innate and adaptive lymphocyte subsets. Finally, we highlight how NCR biology is being harnessed for novel therapeutic interventions particularly for enhanced tumor surveillance.
Assuntos
Células Matadoras Naturais/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Receptor 2 Desencadeador da Citotoxicidade Natural/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Neoplasias/imunologia , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Humanos , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Receptor 2 Desencadeador da Citotoxicidade Natural/genética , Receptor 2 Desencadeador da Citotoxicidade Natural/metabolismo , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismoRESUMO
INTRODUCTION: Innate immunity represents the first step of activation of the immune system and dictates the quality of adaptive immune responses. Studies have reported links between systemic inflammatory or innate immune markers and prognosis in patients with lung cancer. To our knowledge, the prospective and concomitant study of these systemic markers has never been performed. METHODS: Advanced treatment-naive non-small cell lung cancer (NSCLC) patients eligible for first-line platinum-based chemotherapy were prospectively included from December 2012 to July 2015 (N = 148). Blood samples of patients were collected before the first cycle for fresh NK cell phenotyping. Peripheral blood mononuclear cells were cryopreserved for natural cytotoxicity receptor (NCR) genotyping as well as sera for NCR's ligand quantification. Data on leukocytes, neutrophils and monocyte counts and lactate dehydrogenase (LDH) levels were extracted from electronic medical records. RESULTS: Among all studied markers, monocytosis, neutrophilia, leucocytosis, high LDH and sBAG6 levels and reduced levels of NCR3 transcripts were associated with poor overall survival (OS) in univariate analysis. The levels of NCR3 transcripts was linked to age, number of metastatic sites, monocyte counts, LDH and sBAG6 levels. Neutrophilia was associated to high sBAG6 levels. NCR3 was the unique innate immune parameter that remained as an independent factor associated with both OS (P = 0.003) and progression-free survival (P = 0.009) in the multivariate analysis. CONCLUSION: This study brought evidence that these biomarkers are entangled; parameters associated with an inflammatory process were related to reduced levels of NCR3 transcripts. Finally, the level of NCR3 transcripts was independently associated with outcomes in treatment-naive patients with advanced NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Imunidade Inata/imunologia , Neoplasias Pulmonares/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Células Matadoras Naturais/imunologia , L-Lactato Desidrogenase/metabolismo , Contagem de Leucócitos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/imunologia , Monócitos/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Neutrófilos/imunologia , Prognóstico , Intervalo Livre de Progressão , Modelos de Riscos Proporcionais , RNA Mensageiro/metabolismo , Taxa de SobrevidaRESUMO
The anti-leukemia activity of NK cells helps prevent relapse during hematopoietic stem cell transplantation (HSCT) in leukemia patients. However, the factors that determine the sensitivity or resistance of leukemia cells in the context of NK-mediated cytotoxicity are not well-established. Here, we performed a genome-wide CRISPR screen in the human chronic-myelogenous-leukemia (CML) cell line K562 to identify genes that regulate the vulnerability of leukemia cells to killing by primary human NK cells. The distribution of guide RNAs (gRNAs) in K562 cells that survived co-incubation with NK cells showed that loss of NCR3LG1, which encodes the ligand of the natural cytotoxicity receptor NKp30, protected K562 cells from killing. In contrast, loss of genes that regulate the antigen-presentation and interferon-γ-signaling pathways increased the vulnerability of K562 cells. The addition of IFN-γ neutralizing antibody increased the susceptibility of K562 cells to NK-mediated killing. Upregulation of MHC class I on K562 cells after co-incubation with NK cells was dependent on IFNGR2. Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low IFNGR2 expression in cancer tissues was associated with improved overall survival in acute myeloid leukemia (AML) and Kidney Renal Clear Cell Carcinoma (KIRC) patients. Our results, showing that the upregulation of MHC class I by NK-derived IFN-γ leads to resistance to NK cytotoxicity, suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.
Assuntos
Carcinoma de Células Renais , Interferon gama , Células Matadoras Naturais/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva , Evasão Tumoral , Antígenos B7/genética , Antígenos B7/imunologia , Sistemas CRISPR-Cas , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Estudo de Associação Genômica Ampla , Humanos , Interferon gama/genética , Interferon gama/imunologia , Células K562 , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Células Matadoras Naturais/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Receptores de Interferon/genética , Receptores de Interferon/imunologiaRESUMO
Natural killer (NK) cells play a pivotal role in the immune response against infections and malignant transformation, and adopted transfer of NK cells is thought to be a promising therapeutic approach for cancer patients. Previous reports describing the phenotypic features of canine NK cells have produced inconsistent results. Canine NK cells are still defined as non-B and non-T (CD3-CD21-) large granular lymphocytes. However, a few reports have demonstrated that canine NK cells share the phenotypic characteristics of T lymphocytes, and that CD3+CD5dimCD21- lymphocytes are putative canine NK cells. Based on our previous reports, we hypothesized that phenotypic modulation could occur between these two populations during activation. In this study, we investigated the phenotypic and functional differences between CD3+CD5dimCD21- (cytotoxic large granular lymphocytes) and CD3-CD5-CD21- NK lymphocytes before and after culture of peripheral blood mononuclear cells isolated from normal dogs. The results of this study show that CD3+CD5dimCD21- lymphocytes can be differentiated into non-B, non-T NK (CD3-CD5-CD21-TCRαß-TCRγδ-GranzymeB+) lymphocytes through phenotypic modulation in response to cytokine stimulation. In vitro studies of purified CD3+CD5dimCD21- cells showed that CD3-CD5-CD21- cells are derived from CD3+CD5dimCD21- cells through phenotypic modulation. CD3+CD5dimCD21- cells share more NK cell functional characteristics compared with CD3-CD5-CD21- cells, including the expression of T-box transcription factors (Eomes, T-bet), the production of granzyme B and interferon-γ, and the expression of NK cell-related molecular receptors such as NKG2D and NKp30. In conclusion, the results of this study suggest that CD3+CD5dimCD21- and CD3-CD5-CD21- cells both contain a subset of putative NK cells, and the difference between the two populations may be due to the degree of maturation.
Assuntos
Células Matadoras Naturais/classificação , Células Matadoras Naturais/imunologia , Animais , Complexo CD3/genética , Antígenos CD5/genética , Diferenciação Celular , Citotoxicidade Imunológica , Cães , Granzimas/imunologia , Interferon gama/imunologia , Leucócitos Mononucleares/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Fenótipo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Complemento 3d/genética , Proteínas com Domínio T/genética , Linfócitos T/imunologiaRESUMO
Immunomodulatory drugs (IMiDs) are anticancer drugs with immunomodulatory, anti-angiogenesis, anti-proliferative, and pro-apoptotic properties. IMiDs are currently used for the treatment of multiple myeloma, myelodysplastic syndrome, and B-cell lymphoma; however, little is known about efficacy in acute myeloid leukemia (AML). We proposed in this study to investigate the relevance of IMiDs therapy for AML treatment. We evaluated the effect of IMiDs on primary AML blasts (n = 24), and the impact in natural killer (NK) cell-mediated immunosurveillance of AML. Using primary AML cells and an immunodeficient mouse leukemia xenograft model, we showed that IMiDs induce AML cell death in vitro and impair leukemia progression in vivo. In addition, treatment of AML blasts with IMiDs resulted in enhanced allogeneic NK cell anti-leukemia reactivity. Treatment by pomalidomide of AML blasts enhanced lysis, degranulation, and cytokine production by primary allogeneic NK cells. Furthermore, the treatment with lenalidomide of patients with myeloid malignancies resulted in NK cell phenotypic changes similar to those observed in vitro. IMiDs increased CD56 and decreased NKp30, NKp46, and KIR2D expression on NK cells. Finally, AML blasts treatment with IMiDs induced phenotypic alterations including downregulation of HLA-class I. The effect of pomalidomide was not correlated with cereblon expression and A/G polymorphism in AML cells. Our data revealed, a yet unobserved, dual effects on AML affecting both AML survival and their sensitivity to NK immunotherapy using IMiDs. Our study encourages continuing investigation for the use of IMiDs in AML, especially in combination with conventional therapy or immunotherapy strategies.
Assuntos
Antineoplásicos/uso terapêutico , Fatores Imunológicos/uso terapêutico , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno CD56/genética , Antígeno CD56/imunologia , Células Cultivadas , Citotoxicidade Imunológica , Genes MHC Classe I , Humanos , Células K562 , Lenalidomida/uso terapêutico , Masculino , Camundongos , Pessoa de Meia-Idade , Monitorização Imunológica , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Talidomida/análogos & derivados , Talidomida/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Granulocyte colony-stimulating factor (G-CSF) has been widely used in the field of allogeneic haematopoietic stem cell transplantation (allo-HSCT) for priming donor stem cells from the bone marrow (BM) to peripheral blood (PB) to collect stem cells more conveniently. Donor-derived natural killer (NK) cells have important antitumour functions and immune regulatory roles post-allo-HSCT. The aim of this study was to evaluate the effect of G-CSF on donors' NK cells in BM and PB. The percentage of NK cells among nuclear cells and lymphocyte was significantly decreased and led to increased ratio of T and NK cells in BM and PB post-G-CSF in vivo application. Relative expansion of CD56bri NK cells led to a decreased ratio of CD56dim and CD56bri NK subsets in BM and PB post-G-CSF in vivo application. The expression of CD62L, CD54, CD94, NKP30 and CXCR4 on NK cells was significantly increased in PB after G-CSF treatment. G-CSF treatment decreased the IFN-γ-secreting NK population (NK1) dramatically in BM and PB, but increased the IL-13-secreting NK (NK2), TGF-ß-secreting NK (NK3) and IL-10-secreting NK (NKr) populations significantly in BM. Clinical data demonstrated that higher doses of NK1 infused into the allograft correlated with an increased incidence of chronic graft-vs-host disease post-transplantation. Taken together, our results show that the in vivo application of G-CSF can modulate NK subpopulations, leading to an increased ratio of T and NK cells and decreased ratio of CD56dim and CD56bri NK cells as well as decreased NK1 populations in both PB and BM.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Doença Enxerto-Hospedeiro/genética , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Adolescente , Adulto , Transplante de Medula Óssea/métodos , Antígeno CD56/genética , Criança , Feminino , Doença Enxerto-Hospedeiro/sangue , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/terapia , Fator Estimulador de Colônias de Granulócitos/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Molécula 1 de Adesão Intercelular/genética , Interferon gama/genética , Interleucina-13/genética , Células Matadoras Naturais/transplante , Selectina L/genética , Masculino , Pessoa de Meia-Idade , Subfamília D de Receptores Semelhantes a Lectina de Células NK/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptores CXCR4/genética , Transplante Homólogo/métodos , Adulto JovemRESUMO
The activating natural cytotoxicity receptors on natural killer (NK) cells play a fundamental role in immunosurveillance of infections and cancer. Phylogenetic analyses showed that NKp30 is highly conserved in almost all jawed vertebrates and thus, represents one of the most ancient NK cell receptors. However, in contrast to other higher vertebrates, NKp30 is only a pseudogene in mouse, which contains two premature stop codons. To decipher the evolutionary role and biological function of NKp30 in mouse, we removed these premature stop codons and expressed the putative mouse NKp30 (mNKp30) protein as soluble Fc fusion construct and as full-length receptor on A5-GFP reporter cells. Interestingly, even though both NKp30 variants were expressed, maturation and targeting to the plasma membrane were impaired. Previous studies implicated that N-linked glycosylation is crucial for plasma membrane targeting and ligand binding of human NKp30. However, even though present in all other jawed vertebrates analyzed so far, these three N-linked glycosylation sites are missing in mouse NKp30. Interestingly, reconstitution of N-linked glycosylation enabled secretion of a mNKp30-Fc fusion protein which recognized a yet unknown ligand on the plasma membrane of mastocytoma cells. Based on these data, our study is the first to show expression and functional analysis of a mNKp30 protein suggesting that the mouse NKp30 pseudogene is the result of a species-specific loss of function.
Assuntos
Códon de Terminação/genética , Evolução Molecular , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Pseudogenes/genética , Animais , Glicosilação , Humanos , Células Matadoras Naturais/imunologia , Ligantes , Camundongos , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Filogenia , Ligação Proteica , Pseudogenes/imunologia , Especificidade da EspécieRESUMO
Linkage studies have revealed a linkage of mild malaria to chromosome 6p21 that contains the NCR3 gene encoding a natural killer cell receptor, whereas NCR3-412G>C (rs2736191) located in its promoter region was found to be associated with malaria in Burkina Faso. Here we confirmed the association of rs2736191 with mild malaria in a Congolese cohort and investigated its potential cis-regulatory effect. Luciferase assay results indicated that rs2736191-G allele had a significantly increased promoter activity compared to rs2736191-C allele. Furthermore, EMSAs demonstrated an altered binding of two nuclear protein complexes to the rs2736191-C allele in comparison to rs2736191-G allele. Finally, after in silico identification of transcription factor candidates, pull-down western blot experiments confirmed that both STAT4 and RUNX3 bind the region encompassing rs2736191 with a higher affinity for the G allele. To our knowledge, this is the first report that explored the functional role of rs2736191. These results support the hypothesis that genetic variation within natural killer cell receptors alters malaria resistance in humans.
Assuntos
Malária Falciparum/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Sítios de Ligação , Congo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Células K562 , Masculino , Fator de Transcrição STAT4/metabolismoRESUMO
As a stress-inducible natural killer (NK) cell ligand, B7H6 plays a role in innate tumor immunosurveillance and is a fairly tumor selective marker expressed on a variety of solid and hematologic cancer cells. Here, we describe the isolation and characterization of a new family of single chain fragment variable (scFv) molecules targeting the human B7H6 ligand. Through directed evolution of a yeast surface displayed non-immune human-derived scFv library, eight candidates comprising a single family of clones differing by up to four amino acid mutations and exhibiting nM avidities for soluble B7H6-Ig were isolated. A representative clone re-formatted as an scFv-CH1-Fc molecule demonstrated specific binding to both B7H6-Ig and native membrane-bound B7H6 on tumor cell lines with a binding avidity comparable to the previously characterized B7H6-targeting antibody, TZ47. Furthermore, these clones recognized an epitope distinct from that of TZ47 and the natural NK cell ligand NKp30, and demonstrated specific activity against B7H6-expressing tumor cells when expressed as a chimeric antigen receptor (CAR) in T cells.
Assuntos
Anticorpos Antineoplásicos/química , Antígenos B7/química , Biomarcadores Tumorais/química , Proteínas Mutantes Quiméricas/química , Receptores de Antígenos de Linfócitos T/química , Anticorpos de Cadeia Única/química , Substituição de Aminoácidos , Animais , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/genética , Antígenos B7/genética , Antígenos B7/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Linhagem Celular Tumoral , Técnicas de Visualização da Superfície Celular , Citotoxicidade Imunológica , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Expressão Gênica , Células HEK293 , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Camundongos , Modelos Moleculares , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/imunologia , Mutação , Receptor 3 Desencadeador da Citotoxicidade Natural/química , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genéticaRESUMO
PROBLEM: In molecular analysis of tissue biopsy specimens, one crucial aspect is characterization of immune cell populations. This is especially important for evaluation of uterine receptivity by assessing levels of lymphocyte populations including CD56bright CD16- uterine NK cells and CD56dim CD16+ conventional NK cells. Our objective was to investigate whether measuring total RNA transcripts from a tissue specimen would accurately reflect immune cell levels and be a new technique to assess immune cell subsets. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMCs) and endometrial tissues were used. Flow cytometry was utilized for the analysis of lymphocyte subsets in PBMCs, and RT-qPCR was applied to quantify RNA transcripts indicative of lymphocyte and granulocyte populations. RESULTS: In PBMC specimens, there were significant correlations between gene expression levels and cell subsets. NK cells correlated with CD16A, NKp46, and CD56 transcripts, B cells correlated with EBF1, and CD8+ T cells correlated with CD8ß. Finally, endometrial tissues displayed high CD56 expression and very low CD3ε, CD16A, and NKp30, reflecting the characteristic endometrial NK cell subsets. CONCLUSION: Strong correlations between RT-qPCR data and levels of lymphocyte subsets indicate that gene expression analysis will be a useful technique for characterizing levels of CD56+ cells in endometrial tissues.
Assuntos
Células Sanguíneas/imunologia , Endométrio/imunologia , Imunofenotipagem/métodos , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Biópsia , Antígeno CD56/genética , Antígeno CD56/metabolismo , Separação Celular , Feminino , Citometria de Fluxo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica , Humanos , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Reação em Cadeia da Polimerase , Receptores de IgG/genética , Receptores de IgG/metabolismoRESUMO
Human cytomegalovirus (HCMV) is a major human pathogen, causing serious diseases in immunocompromised populations and congenially infected neonates. One of the main immune cells acting against the virus are Natural Killer (NK) cells. Killing by NK cells is mediated by a small family of activating receptors such as NKp30 that interact with the cellular ligand B7-H6. The outcome of B7-H6-NKp30 interaction was, so far, mainly studied with regard to NK recognition and killing of tumors. Here, we demonstrated that the expression of B7-H6 is upregulated following HCMV infection and that HCMV uses two of its genes: US18 and US20, to interfere with B7-H6 surface expression, in a mechanism involving endosomal degradation, in order to evade NK cell recognition.
Assuntos
Antígenos B7/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Interações Hospedeiro-Patógeno , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Proteínas Virais/genética , Antígenos B7/metabolismo , Linhagem Celular , Infecções por Citomegalovirus/metabolismo , Citotoxicidade Imunológica , Regulação da Expressão Gênica , Ordem dos Genes , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Lisossomos/metabolismo , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Virulência/imunologiaRESUMO
Cytogenetics and European Leukemia Net (ELN) genetic classification predict patients at increased risk of relapse in acute myeloid leukemia (AML) except in the intermediate risk group for which further prognostic determinants are required. We have previously shown that Natural Killer (NK) cell defects in AML are predictors of poor overall survival (OS). This study aimins at validating NKp30, a receptor that mediates NK activation, as a prognostic biomarker for AML patients with intermediate prognosis.NKp30 expression was prospectively assessed at diagnosis on NK cells from peripheral blood by flow cytometry (N = 201 patients). Clinical outcome was evaluated with regard to NKp30 status.In patients with intermediate cytogenetic (N = 162), NKp30high phenotype at diagnosis was predictive of better OS (HR = 0.26; 95%CI = [0.14-0.50]; P < 0.0001) and relapse-free survival (RFS) (HR = 0.21; 95%CI = [0.08-0.52]; P = 0.0007). In patients with intermediate ELN (N = 116), NKp30high phenotype at diagnosis was predictive of better OS (HR = 0.33; 95%CI = [0.16-0.67]; P = 0.0019) and RFS (HR = 0.24; 95%CI = [0.08-0.67]; P = 0.0058). In multivariate analysis, high NKp30 expression independently predicted improved OS (HR = 0.56, P = 0.046) and RFS (HR = 0.37, P = 0.048). Consistently, cumulative incidence of relapse (CIR) was lower in patients with high NKp30 expression (HR = 0.37, P = 0.026).In conclusion, we propose NKp30 status as a simple and early prognostic biomarker that identifies intermediate-risk patients with poor prognosis who otherwise may not be identified with existing risk stratification systems.