RESUMO
PROBLEM: Endometriosis exhibits several immune dysfunctions, including deficient natural killer (NK) cell cytotoxicity. MICA (MHC class I chain-related molecule A) is induced by biological stress and soluble MICA (sMICA) negatively modulates the expression of the activating receptor, NKG2D, reducing NK cells activities. We investigated the involvement of soluble MICA in NK cell-deficient activity in endometriosis. METHODS OF STUDY: sMICA levels (serum and peritoneal fluid-PF) were evaluated by ELISA. Circulating NK cell subsets quantification and its NKG2D receptor expression, NK cell cytotoxicity and CD107a, IFN-γ and IL-10 expressions by NK cells stimulated with K562 cells were determined by flow cytometry. RESULTS: We found higher sMICA levels (serum and PF) in endometriosis, especially in advanced and deep endometriosis. Endometriosis presented lower percentages of CD56dim CD16+ cytotoxic cells and impaired NK cell responses upon stimulation, resulting in lower CD107a and IFN-γ expressions, and deficient NK cell cytotoxicity. NK cell stimulation in the MICA-blocked condition (mimicking the effect of sMICA) showed decreased cytotoxicity in initial endometriosis stages and the emergence of a negative correlation between CD107a expression and sMICA levels. CONCLUSIONS: We suggest that soluble MICA is a potential player in endometriosis pathophysiology with involvement in disease progression and severity, contributing to NK cell impaired IFN-γ response and degranulation. NK cell compartment exhibits multiple perturbations, including quantitative deficiency and impaired cytotoxicity, contributing to inadequate elimination of ectopic endometrial tissue.
Assuntos
Endometriose , Feminino , Humanos , Degranulação Celular , Células Matadoras Naturais , Expressão Gênica , Progressão da Doença , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismoRESUMO
The involvement of NK and other cytotoxic cells is considered the first defense line against cancer. However, a significant lack of information prevails on the possible roles played by factors considered characteristic of primitive cells, such as c-kit and Sca-1, in activating these cells, particularly in melanoma models subjected to treatments with substances under investigation, such as the case of norcantharidin. In this study, B16F1 murine melanoma cells were used to induce tumors in DBA/2 mice, estimating the proportions of NK and iNKT cells; the presence of activation (CD107a+) and primitive/activation (c-kit+/Lya6A+) markers and some tumor parameters, such as the presence of mitotic bodies, nuclear factor area, NK and iNKT cell infiltration in the tumor, infiltrated tumor area, and infiltrating lymphocyte count at 10x and 40x in specimens treated with pentoxifylline, norcantharidin, and the combination of both drugs. Possible correlations were estimated with Pearson's correlation analysis. It should be noted that, despite having demonstrated multiple correlations, immaturity/activation markers were related to these cells' activation. At the tumor site, iNKT cells are the ones that exert the cytotoxic potential on tumor cells, but they are confined to specific sites in the tumor. Due to the higher number of interactions of natural killer cells with tumor cells, it is concluded that the most effective treatment was PTX at 60 mg/kg + NCTD at 0.75 mg/kg.
RESUMO
Natural killer (NK) cells can kill virus-infected cells and tumor cells without prior sensitization and secrete numerous cytokines and chemokines that modulate the activity of different cells of the immune system. The recognition of target cells is mediated by germ line-encoded receptors, and the activity of NK cells can be further regulated by soluble factors such as cytokines and Toll-like receptor ligands. Thus, NK cells display an exciting potential as a powerful immunotherapeutic tool against malignant diseases, and different strategies are being tested aiming to overcome tumor-induced NK cell suppression and restore NK-cell mediated antitumor activity. This section describes different flow cytometry-based protocols to study NK cell effector functions, which can be used to evaluate the immunomodulatory ability of different therapeutic compounds.
Assuntos
Imunomodulação/imunologia , Células Matadoras Naturais/imunologia , Degranulação Celular , Membrana Celular/metabolismo , Separação Celular , Humanos , Células Matadoras Naturais/fisiologia , Fenótipo , Coloração e RotulagemRESUMO
Classically, CD4+ T-cells have been referred as cytokine-producing cells and important players in immune responses by providing soluble factors that potentiate several effector immune functions. However, it is now evident that CD4+ T-cells can also elaborate cytotoxic responses, inducing apoptosis of target cells. Cytotoxic CD4+ T cells (CD4+ CTLs), exhibit cytolytic functions that resemble those of CD8+ T-cells; in fact, there is evidence suggesting that they may have a role in the control of viral infections. In this article, we discuss the role of CD4+ CTLs during HIV infection, where CD4+ CTLs have been associated with viral control and slow disease progression. In addition, we address the implication of CD4+ CTLs in the context of antiretroviral therapy and the partial reconstitution of CD8+ T-cells effector function.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Antivirais/imunologia , Antivirais/uso terapêutico , Apoptose , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Progressão da Doença , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Fenótipo , Subpopulações de Linfócitos T/virologia , Linfócitos T Citotóxicos/virologiaRESUMO
BACKGROUND: Natural killer (NK) cells are part of the innate immune system and provide surveillance against viruses and cancers. The ability of NK cells to kill virus-infected cells depends on the balance between the effects of inhibitory and activating NK cell receptors. This study aimed to investigate the phenotypic profile and the functional capacity of NK cells in the context of HTLV-1 infection. METHODS: This cross-sectional study sequentially recruited HTLV-1 infected individuals with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and asymptomatic HTLV-1 (AS) from the Integrated and Multidisciplinary HTLV Center in Salvador, Brazil. Blood samples from healthy blood donors served as controls. NK cell surface receptors (NKG2D, KIR2DL2/KIR2DL3, NKp30, NKG2A, NKp46, TIM-3 and PD-1), intracellular cytolytic (Granzyme B, perforin) and functional markers (CD107a for degranulation, IFN-γ) were assayed by flow cytometry in the presence or absence of standard K562 target cells. In addition, cytotoxicity assays were performed in the presence or absence of anti-NKp30. RESULTS: The frequency of NKp30+ NK cells was significantly decreased in HAM/TSP patients [58%, Interquartile Range (IQR) 30-61] compared to controls (73%, IQR 54-79, p = 0.04). The production of cytolytic (perforin, granzyme B) and functional markers (CD107a and IFN-γ) was higher in unstimulated NK cells from HAM/TSP and AS patients compared to controls. By contrast, stimulation with K562 target cells did not alter the frequency of CD107a+ NK cells in HAM/TSP subjects compared to the other groups. Blockage of the NKp30 receptor was shown to decrease cytotoxic activity (CD107a) and IFN-γ expression only in asymptomatic HTLV-1-infected individuals. CONCLUSIONS: NK cells from individuals with a diagnosis of HAM/TSP present decreased expression of the activating receptor NKp30, in addition to elevated degranulation activity that remained unaffected after blocking the NKp30 receptor.
Assuntos
Células Matadoras Naturais/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Paraparesia Espástica Tropical/imunologia , Adulto , Anticorpos Monoclonais/farmacologia , Biomarcadores/metabolismo , Estudos Transversais , Feminino , Citometria de Fluxo , Granzimas/metabolismo , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Humanos , Interferon gama/metabolismo , Células K562 , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Masculino , Pessoa de Meia-Idade , Receptor 3 Desencadeador da Citotoxicidade Natural/antagonistas & inibidores , Paraparesia Espástica Tropical/virologia , Perforina/metabolismoRESUMO
Peritoneal ascites are a distinguishable feature of patients with advanced epithelial ovarian cancer (EOC). The presence of different lymphocyte subsets has been reported in EOC-associated ascites, which also can or not contain malignant cells. The goal of this study was to analyze the functional characteristics of natural killer (NK) cells from EOC-associated ascites in terms of their expression of activating receptors and ascites' contents of lymphocyte subtypes, cytokine profile and presence of EOC cells. NK cell function was evaluated by the expression of the degranulation marker CD107a in resting and interleukin (IL)-2 stimulated NK cells from ascites and blood. Degranulation of NK cells from EOC cell-free ascites was significantly (p < 0.05) higher than all the other groups, either in their resting state or after IL-2 stimulation, suggesting a previous local stimulation. In contrast, treatment with IL-2 had no effect on NK cells from ascites with EOC cells. The amount of regulatory T cells was significantly higher in ascites with EOC cells compared to EOC cell-free ascites. Ascites with EOC cells also had higher levels of tumor necrosis factor (TNF)-α, suggesting inflammation related to the malignancy. In conclusion, the functional performance of NK cells was distinct between EOC cell-free ascites and ascites with EOC cells. The impairment of NK cell response to IL-2 in ascites with EOC cells was consistent with an immunosuppressive tumor microenvironment.
Assuntos
Ascite/imunologia , Ascite/patologia , Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Idoso , Biomarcadores , Carcinoma Epitelial do Ovário , Estudos de Casos e Controles , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Feminino , Expressão Gênica , Humanos , Imunofenotipagem , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologiaRESUMO
BACKGROUND: Cutaneous leishmaniasis (CL) is caused by Leishmania (Viannia) braziliensis, which infects dermal macrophages and dendritic cells, causing an intense immune-mediated-tissue inflammation and a skin ulcer with elevated borders that can heal spontaneously or after antimonial therapy. The resolution of lesions depends on an adaptive immune response, and cytotoxic cells seem to have a fundamental role in this process. The aim of this study is to better understand the role of cytotoxicity mediated mechanisms that occur during the immune response in the CL lesion milieu, considering distinct cytotoxic-related CD107a+ cells, such as CD8+, CD4+, CD4neg CD8neg (double-negative, DN) and CD4+CD8+ (double-positive, DP) T lymphocytes, as well as NK and NKT cells. METHODS: Lesion derived cells were assessed for T cell subpopulations and NK cells, as well as CD107a expression by flow cytometry. In addition, cytometric bead array (CBA) was used to quantify cytokines and granzyme B concentrations in supernatants from macerated lesions. RESULTS: Flow cytometry analyses revealed that NKT cells are the major CD107a-expressing cell population committed to cytotoxicity in CL lesion, although we also observed high frequencies of CD4+ and DN T cells expressing CD107a. Analysing the pool of CD107a+-cell populations, we found a higher distribution of DN T cells (44%), followed by approximately 25% of NKT cells. Interestingly, NK and CD8+ T cells represented only 3 and 4% of the total-CD107a+-cell pool, respectively. CONCLUSIONS: The cytotoxicity activity that occurs in the lesion milieu of CL patients seems to be dominated by DN T and NKT cells. These findings suggest the need for a reevaluation of the role of classical-cytotoxic NK and CD8+ T cells in the pathogenesis of CL, implicating an important role for other T cell subpopulations.
Assuntos
Citotoxicidade Imunológica , Leishmaniose Cutânea/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Células T Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Antígenos de Protozoários/imunologia , Biópsia , Brasil/epidemiologia , Citocinas/biossíntese , Citocinas/genética , Feminino , Citometria de Fluxo , Granzimas/análise , Humanos , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/epidemiologia , Proteína 1 de Membrana Associada ao Lisossomo/genética , Masculino , Pessoa de Meia-Idade , Pele/imunologia , Pele/parasitologia , Pele/patologiaRESUMO
Cutaneous leishmaniasis (CL) is an important public health issue worldwide. The control of Leishmania infection depends on cellular immune mechanisms, and the inflammatory response may contribute to pathogenesis. A beneficial role of CD8(+) T lymphocytes has been proposed; nevertheless, other studies suggest a cytotoxic role of CD8(+) T lymphocytes involved in tissue damage, showing controversial role of these cells. The goal of the current study was to understand the immunopathology of CL and determine the profile of cytotoxic cells--such as CD4(+) T, natural killer and natural killer T cells--that might be involved in triggering immunological mechanisms, and may lead to cure or disease progression. The frequencies of cytotoxic cell populations in peripheral blood, obtained from patients with active disease, during treatment and after clinical healing, were assessed by flow cytometry. Cytotoxicity could not be related to a deleterious role in Leishmania braziliensis infection, as patients with active CL showed similar percentages of degranulation to healthy individuals (HI). Cured patients exhibited a lower percentage of degranulating cells, which may be due to a downregulation of the immune response. The understanding of the immunopathological mechanisms involved in CL and the commitment of cytotoxic cells enables improvements in therapeutic strategies.
Assuntos
Leishmaniose Cutânea/imunologia , Adulto , Antiprotozoários/uso terapêutico , Contagem de Linfócito CD4 , Degranulação Celular , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/parasitologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/tratamento farmacológico , Masculino , Meglumina/uso terapêutico , Antimoniato de Meglumina , Pessoa de Meia-Idade , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/parasitologia , Compostos Organometálicos/uso terapêutico , Adulto JovemRESUMO
This study aimed at evaluating the functional activation and activating receptors expression on resting, short- and long-term NK and NK-like T cells from blood of ovarian neoplasia patients. Blood from patients with adnexal benign alterations (n = 10) and ovarian cancer (grade I-IV n = 14) were collected after signed consent. Effector cells activation was evaluated by the expression of the CD107a molecule. Short-term culture was conducted overnight with IL-2 and long-term culture for 21 days, by a method designed to expand CD56(+) lymphocytes. Short-term culture significantly increased NK cells activation compared to resting NK cells (p<0.05), however, the long-term procedure supported an even higher increase (p<0.001). Resting NK-like T cells showed poor activation, which was not altered by the culture procedures. The long-term culture effectively increased the expression of the activating receptors on NK and NK-like T cells, either by increasing the number of cells expressing a given receptor and/or by up-regulating their expression intensity. As a conclusion, the long-term culture system employed, resulted in a high number of functional NK cells. The culture system was particularly efficient on the up-regulation of NKp30 and DNAM-1 receptors on NK cells.
Assuntos
Adenocarcinoma Mucinoso/genética , Antígenos de Diferenciação de Linfócitos T/genética , Cistadenocarcinoma Seroso/genética , Regulação Neoplásica da Expressão Gênica , Células Matadoras Naturais/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Neoplasias Ovarianas/genética , Adenocarcinoma Mucinoso/imunologia , Adenocarcinoma Mucinoso/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Diferenciação de Linfócitos T/imunologia , Estudos de Casos e Controles , Cistadenocarcinoma Seroso/imunologia , Cistadenocarcinoma Seroso/patologia , Feminino , Humanos , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/patologia , Ativação Linfocitária , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Pessoa de Meia-Idade , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Estadiamento de Neoplasias , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Ovário/imunologia , Ovário/patologia , Cultura Primária de Células , Transdução de Sinais , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Tuberculous pleural effusion is characterized by a T helper type 1 (Th1) profile, but an excessive Th1 response may also cause tissue damage that might be controlled by regulatory mechanisms. In the current study we investigated the role of regulatory T cells (Treg ) in the modulation of Th1 responses in patients with tuberculous (TB) pleurisy. Using flow cytometry we evaluated the proportion of Treg (CD4(+) CD25(high) forkhead box protein 3(+) ), interferon (IFN)-γ and interleukin (IL)-10 expression and CD107 degranulation in peripheral blood (PB) and pleural fluid (PF) from patients with TB pleurisy. We demonstrated that the proportion of CD4(+) CD25(+) , CD4(+) CD25(high) FoxP3(+) and CD8(+) CD25(+) cells were increased in PF compared to PB samples. Mycobacterium tuberculosis stimulation increased the proportion of CD4(+) CD25(low/neg) IL-10(+) in PB and CD4(+) CD25(low/neg) IFN-γ(+) in PF; meanwhile, CD25(high) mainly expressed IL-10 in both compartments. A high proportion of CD4(+) CD107(+) and CD8(+) CD107(+) cells was observed in PF. Treg depletion enhanced the in-vitro M. tuberculosis-induced IFN-γ and CD4(+) and CD8(+) degranulation responses and decreased CD4(+) IL-10(+) cells in PF. Our results demonstrated that in TB pleurisy Treg cells effectively inhibit not only IFN-γ expression but also the ability of CD4(+) and CD8(+) cells to degranulate in response to M. tuberculosis.