RESUMO
Tumor-associated macrophages (TAM) are abundant in several tumor types and usually correlate with poor prognosis. Previously, we demonstrated that anti-inflammatory macrophages (M2) inhibit NK cell effector functions. Here, we explored the impact of TAM on NK cells in the context of clear-cell renal cell carcinoma (ccRCC). Bioinformatics analysis revealed that an exhausted NK cell signature strongly correlated with an M2 signature. Analysis of TAM from human ccRCC samples confirmed that they exhibited an M2-skewed phenotype and inhibited IFN-γ production by NK cells. Moreover, human M0 macrophages cultured with conditioned media from ccRCC cell lines generated macrophages with an M2-skewed phenotype (TAM-like), which alike TAM, displayed suppressive activity on NK cells. Moreover, TAM depletion in the mouse Renca ccRCC model resulted in delayed tumor growth and reduced volume, accompanied by an increased frequency of IFN-γ-producing tumor-infiltrating NK cells that displayed heightened expression of T-bet and NKG2D and reduced expression of the exhaustion-associated co-inhibitory molecules PD-1 and TIM-3. Therefore, in ccRCC, the tumor microenvironment polarizes TAM toward an immunosuppressive profile that promotes tumor-infiltrating NK cell dysfunction, contributing to tumor progression. In addition, immunotherapy strategies targeting TAM may result in NK cell reinvigoration, thereby counteracting tumor progression.
Assuntos
Carcinoma de Células Renais , Interferon gama , Neoplasias Renais , Células Matadoras Naturais , Macrófagos Associados a Tumor , Células Matadoras Naturais/imunologia , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Interferon gama/metabolismo , Interferon gama/imunologia , Humanos , Animais , Camundongos , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Progressão da Doença , Linhagem Celular Tumoral , Microambiente Tumoral/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Receptor de Morte Celular Programada 1/metabolismoRESUMO
NKG2D is a major natural killer (NK) cell-activating receptor that recognizes eight ligands (NKG2DLs), including MICA, and whose engagement triggers NK cell effector functions. As NKG2DLs are upregulated on tumor cells but tumors can subvert the NKG2D-NKG2DL axis, NKG2DLs constitute attractive targets for antibody (Ab)-based immuno-oncology therapies. However, such approaches require a deep characterization of NKG2DLs and NKG2D cell surface expression on primary tumor and immune cells. Here, using a bioinformatic analysis, we observed that MICA is overexpressed in renal cell carcinoma (RCC), and we also detected an association between the NKG2D-MICA axis and a diminished overall survival of RCC patients. Also, by flow cytometry (FC), we observed that MICA was the only NKG2DL over-expressed on clear cell renal cell carcinoma (ccRCC) tumor cells, including cancer stem cells (CSC) that also coexpressed NKG2D. Moreover, tumor-infiltrating leukocytes (TIL), but not peripheral blood lymphoid cells (PBL) from ccRCC patients, over-expressed MICA, ULBP3 and ULBP4. In addition, NKG2D was downregulated on peripheral blood NK cells (PBNK) from ccRCC patients but upregulated on tumor-infiltrating NK cells (TINK). These TINK exhibited impaired degranulation that negatively correlated with NKG2D expression, diminished IFN-γ production, upregulation of TIM-3, and an impaired glucose intake upon stimulation with cytokines, indicating that they are dysfunctional, display features of exhaustion and an altered metabolic fitness. We conclude that ccRCC patients exhibit a distorted MICA-NKG2D axis, and MICA emerges as the forefront NKG2DL for the development of targeted therapies in ccRCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Carcinoma de Células Renais/terapia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Neoplasias Renais/terapia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptores de Células Matadoras NaturaisRESUMO
Natural Killer (NK) cells play a key role in cancer immunosurveillance. However, NK cells from cancer patients display an altered phenotype and impaired effector functions. In addition, evidence of a regulatory role for NK cells is emerging in diverse models of viral infection, transplantation, and autoimmunity. Here, we analyzed clear cell renal cell carcinoma (ccRCC) datasets from The Cancer Genome Atlas (TCGA) and observed that a higher expression of NK cell signature genes is associated with reduced survival. Analysis of fresh tumor samples from ccRCC patients unraveled the presence of a high frequency of tumor-infiltrating PD-L1+ NK cells, suggesting that these NK cells might exhibit immunoregulatory functions. In vitro, PD-L1 expression was induced on NK cells from healthy donors (HD) upon direct tumor cell recognition through NKG2D and was further up-regulated by monocyte-derived IL-18. Moreover, in vitro generated PD-L1hi NK cells displayed an activated phenotype and enhanced effector functions compared to PD-L1- NK cells, but simultaneously, they directly inhibited CD8+ T cell proliferation in a PD-L1-dependent manner. Our results suggest that tumors might drive the development of PD-L1-expressing NK cells that acquire immunoregulatory functions in humans. Hence, rational manipulation of these regulatory cells emerges as a possibility that may lead to improved anti-tumor immunity in cancer patients.
Assuntos
Antígeno B7-H1/biossíntese , Linfócitos T CD8-Positivos/citologia , Carcinoma de Células Renais/imunologia , Neoplasias Renais/imunologia , Células Matadoras Naturais/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Expressão Gênica , Humanos , Interferon gama/farmacologia , Interleucina-18/farmacologia , Células K562 , Estimativa de Kaplan-Meier , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Monitorização Imunológica , Monócitos/metabolismo , Proteínas Recombinantes/farmacologia , Regulação para CimaRESUMO
Immune checkpoint inhibitors (ICI) revolutionized the field of immuno-oncology and opened new avenues towards the development of novel assets to achieve durable immune control of cancer. Yet, the presence of tumor immune evasion mechanisms represents a challenge for the development of efficient treatment options. Therefore, combination therapies are taking the center of the stage in immuno-oncology. Such combination therapies should boost anti-tumor immune responses and/or target tumor immune escape mechanisms, especially those created by major players in the tumor microenvironment (TME) such as tumor-associated macrophages (TAM). Natural killer (NK) cells were recently positioned at the forefront of many immunotherapy strategies, and several new approaches are being designed to fully exploit NK cell antitumor potential. One of the most relevant NK cell-activating receptors is NKG2D, a receptor that recognizes 8 different NKG2D ligands (NKG2DL), including MICA and MICB. MICA and MICB are poorly expressed on normal cells but become upregulated on the surface of damaged, transformed or infected cells as a result of post-transcriptional or post-translational mechanisms and intracellular pathways. Their engagement of NKG2D triggers NK cell effector functions. Also, MICA/B are polymorphic and such polymorphism affects functional responses through regulation of their cell-surface expression, intracellular trafficking, shedding of soluble immunosuppressive isoforms, or the affinity of NKG2D interaction. Although immunotherapeutic approaches that target the NKG2D-NKG2DL axis are under investigation, several tumor immune escape mechanisms account for reduced cell surface expression of NKG2DL and contribute to tumor immune escape. Also, NKG2DL polymorphism determines functional NKG2D-dependent responses, thus representing an additional challenge for leveraging NKG2DL in immuno-oncology. In this review, we discuss strategies to boost MICA/B expression and/or inhibit their shedding and propose that combination strategies that target MICA/B with antibodies and strategies aimed at promoting their upregulation on tumor cells or at reprograming TAM into pro-inflammatory macrophages and remodeling of the TME, emerge as frontrunners in immuno-oncology because they may unleash the antitumor effector functions of NK cells and cytotoxic CD8 T cells (CTL). Pursuing several of these pipelines might lead to innovative modalities of immunotherapy for the treatment of a wide range of cancer patients.
Assuntos
Proteínas Ligadas por GPI/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Animais , Terapia Combinada , Gerenciamento Clínico , Suscetibilidade a Doenças , Proteínas Ligadas por GPI/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ligantes , Terapia de Alvo Molecular , Neoplasias/patologia , Neoplasias/terapia , Receptores de Células Matadoras Naturais/genética , Receptores de Células Matadoras Naturais/metabolismo , Transdução de Sinais , Evasão Tumoral , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Although natural killer (NK) cells infiltrate clear cell renal cell carcinomas (ccRCC), the most frequent malignancy of the kidney, tumor progression suggests that they become dysfunctional. As ccRCC-driven subversion of NK cell effector functions is usually accompanied by phenotypic changes, analysis of such alterations might lead to the identification of novel biomarkers and/or targets in immuno-oncology. Consequently, we performed a phenotypic analysis of peripheral blood NK cells (PBNK) and tumor-infiltrating NK cells (TINK) from ccRCC patients. Compared to HD, PBNK from ccRCC patients exhibited features of activated cells as shown by CD25, CD69 and CD62L expression. They also displayed increased expression of DNAM-1, CD48, CD45, MHC-I, reduced expression of NKG2D, and higher frequencies of CD85j+ and PD-1+ cells. In addition, compared to PBNK from ccRCC patients, TINK exhibited higher expression of activation markers, tissue residency features and decreased expression of the activating receptors DNAM-1, NKp30, NKp46, NKp80 and CD16, suggesting a more inhibitory phenotype. Analysis of The Cancer Genome Atlas (TCGA) revealed that CD48, CD45, CD85j and PD-1 are significantly overexpressed in ccRCC and that their expression is associated with an NK cell infiltration signature. Calculation of z-scores revealed that their expression on PBNK, alone or combined, distinguished ccRCC patients from HD. Therefore, these molecules emerge as novel potential biomarkers and our results suggest that they might constitute possible targets for immunotherapy in ccRCC patients.
Assuntos
Carcinoma de Células Renais/etiologia , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/etiologia , Neoplasias Renais/metabolismo , Células Matadoras Naturais/imunologia , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/imunologia , Idoso , Biomarcadores , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Gerenciamento Clínico , Suscetibilidade a Doenças , Feminino , Expressão Gênica , Humanos , Imunofenotipagem , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , NefrectomiaRESUMO
After recognition, NK cells can kill susceptible target cells through perforin-dependent mechanisms or by inducing death receptor-mediated apoptosis, and they can also secrete cytokines that are pivotal for immunomodulation. Despite the critical role as effector cells against tumors and virus-infected cells, NK cells have been implicated in the regulation of T cell-mediated responses in different models of autoimmunity, transplantation, and viral infections. Here, we review the mechanisms described for NK cell-mediated inhibition of adaptive immune responses, with spotlight on the emerging evidence of their regulatory role that shapes antitumor immune responses.
Assuntos
Imunidade Adaptativa/imunologia , Citotoxicidade Imunológica/imunologia , Infecções/imunologia , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Animais , HumanosRESUMO
BACKGROUND: Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity. METHODS: We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect. RESULTS: Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment. CONCLUSIONS: Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Linfoma/imunologia , Proteínas Recombinantes de Fusão/imunologia , Neoplasias da Bexiga Urinária/imunologia , Animais , Brucella/enzimologia , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Linfoma/patologia , Linfoma/terapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapiaRESUMO
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
In recent times, our understanding of the role of the immune system in different physiopathological situations has increased markedly. A new set of cells, generically known as innate lymphoid cells (ILC), has been discovered in the lymphoid compartment. Five ILC subsets can be recognized according to phenotypic and functional similarities with different subpopulations of T lymphocytes. Unlike T and B lymphocytes, ILC do not express antigen receptors nor undergo selection and clonal expansion upon activation. Instead, they respond rapidly to cytokines and danger signals in infected or inflamed tissues, producing cytokines that direct the immune response toward a type suitable for controlling the initial insult. In addition, ILC establish a crosstalk with other cells of the microenvironment that contributes to the maintenance and restoration of tissue homeostasis. Although many evidences on ILC were obtained from animal models, solid data confirm their existence in humans and their role in various inflammatory disorders. In this article, we address new knowledge on ILC, particularly on their role in the homeostasis of the immune system and in various inflammatory pathologies, in order to present new actors regulating immunity and immunopathology and affecting human health.
En tiempos recientes, nuestra comprensión del rol del sistema inmune en diferentes situaciones fisiopatológicas ha aumentado notablemente. En el compartimiento linfoide se ha descubierto un conjunto de células denominadas células linfoides innatas o innate lymphoid cells (ILC). Las ILC incluyen cinco grupos, clasificados según su similitud fenotípica y funcional con diferentes subpoblaciones de linfocitos T. A diferencia de los linfocitos T y B, las ILC no expresan receptores de antígeno ni sufren selección y expansión clonal cuando se activan. En cambio, responden rápidamente frente a citoquinas y señales de peligro en tejidos infectados o inflamados produciendo citoquinas que dirigen la respuesta inmune hacia un tipo adecuado para controlar la noxa original. Además, las ILC establecen un diálogo cruzado con otras células del microambiente que contribuye al mantenimiento y la restauración de la homeostasis tisular. Si bien muchas evidencias acerca de las ILC fueron obtenidas en modelos animales, existen datos sólidos que confirman su existencia en seres humanos y su papel en diversos trastornos inflamatorios. En este artículo, abordamos los nuevos conocimientos acerca de las ILC, y su rol en la homeostasis del sistema inmune y en diversas patologías inflamatorias, con el fin de presentar nuevos actores que regulan la inmunidad y la inmunopatología, lo que repercute en la salud humana.
Assuntos
Homeostase/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Enteropatias/imunologia , Pneumopatias/imunologia , Linfócitos/imunologia , Dermatopatias/imunologia , Homeostase/fisiologia , Humanos , Inflamação/fisiopatologia , Enteropatias/fisiopatologia , Pneumopatias/fisiopatologia , Dermatopatias/fisiopatologiaRESUMO
Interleukin (IL)-23 is a member of the IL-12 family of cytokines that, as the other members of this family, is secreted by monocytes, macrophages, and dendritic cells (DC) upon recognition of bacterial, viral, and fungal components. IL-23 is critical during immunity against acute infections, and it is also involved in the development of autoimmune diseases. Although immunoregulatory effects of IL-23 on mouse natural killer (NK) cells have been described, the effect of IL-23 on human NK cells remains ill-defined. In this study, we observed that monocytes stimulated with LPS secreted IL-23 and that blockade of this cytokine during monocyte and NK cell coculture led to a diminished production of IFN-γ by NK cells. Accordingly, rIL-23-induced NK cell activation and stimulated IFN-γ production by CD56bright NK cells. This effect involved MEK1/MEK2, JNK, PI3K, mammalian target of rapamycin, and NF-κB, but not STAT-1, STAT-3, nor p38 MAPK pathways. Moreover, while NK cell-mediated cytotoxicity remained unaltered, antibody-dependent cellular cytotoxicity (ADCC) was enhanced after IL-23 stimulation. In addition, IL-23 displayed a synergistic effect with IL-18 for IFN-γ production by both CD56bright and CD56dim NK cells, and this effect was due to a priming effect of IL-23 for IL-18 responsiveness. Furthermore, NK cells pre-stimulated with IL-18 promoted an increase in CD86 expression and IL-12 secretion by DC treated with LPS, and IL-23 potentiated these effects. Moreover, IL-23-driven enhancement of NK cell "helper" function was dependent on NK cell-derived IFN-γ. Therefore, our results suggest that IL-23 may trigger NK cell-mediated "helper" effects on adaptive immunity, shaping T cell responses during different pathological situations through the regulation of DC maturation.
RESUMO
IL-27, a member of the IL-12 family of cytokines, is produced by APCs, and displays pro- and anti-inflammatory effects. How IL-27 affects human NK cells still remains unknown. In this study, we observed that mature DCs secreted IL-27 and that blockade of IL-27R (CD130) reduced the amount of IFN-γ produced by NK cells during their coculture, showing the importance of IL-27 during DC-NK-cell crosstalk. Accordingly, human rIL-27 stimulated IFN-γ secretion by NK cells in a STAT1-dependent manner, induced upregulation of CD25 and CD69 on NK cells, and displayed a synergistic effect with IL-18. Preincubation experiments demonstrated that IL-27 primed NK cells for IL-18-induced IFN-γ secretion, which was associated with an IL-27-driven upregulation of T-bet expression. Also, IL-27 triggered NKp46-dependent NK-cell-mediated cytotoxicity against Raji, T-47D, and HCT116 cells, and IL-18 enhanced this cytotoxic response. Such NK-cell-mediated cytotoxicity involved upregulation of perforin, granule exocytosis, and TRAIL-mediated cytotoxicity but not Fas-FasL interaction. Moreover, IL-27 also potentiated Ab-dependent cell-mediated cytotoxicity against mAb-coated target cells. Taken together, IL-27 stimulates NK-cell effector functions, which might be relevant in different physiological and pathological situations.
Assuntos
Células Dendríticas/imunologia , Interleucina-18/farmacologia , Interleucinas/farmacologia , Células Matadoras Naturais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/imunologia , Técnicas de Cocultura , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/imunologia , Citotoxicidade Imunológica , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Regulação da Expressão Gênica , Células HCT116 , Humanos , Interleucina-18/imunologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Interleucinas/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Cultura Primária de Células , Proteínas Recombinantes/farmacologia , Transdução de SinaisRESUMO
The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b(+)Gr-1(+), mostly CD11b(+)Ly6G(+)Ly6C(int) and CD11b(+)Ly6G(-)Ly6C(high) cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b(+)Ly6G(+)Ly6C(int) cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b(+)Ly6G(+)Ly6C(int) cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b(+)Gr-1(+) cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b(+)Gr-1(+) cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b(+)Ly6G(+)Ly6C(int) cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence.
Assuntos
Antígenos Ly/imunologia , Neoplasias da Mama/imunologia , Antígeno CD11b/imunologia , Células Matadoras Naturais/imunologia , Acetato de Medroxiprogesterona/farmacologia , Células Mieloides/imunologia , Animais , Antígenos Ly/metabolismo , Antineoplásicos Hormonais/farmacologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Antígeno CD11b/metabolismo , Diferenciação Celular , Proliferação de Células , Citotoxicidade Imunológica , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Receptores de Glucocorticoides/metabolismo , Fator de Transcrição STAT3/metabolismo , Células Tumorais CultivadasRESUMO
The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B(+) T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B(+) B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role.
Assuntos
Doença Celíaca/genética , Duodeno/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Mucosa Intestinal/metabolismo , Estresse Fisiológico/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Pré-Escolar , Duodeno/patologia , Enterócitos/metabolismo , Enterócitos/patologia , Feminino , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Mucosa Intestinal/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Plasmócitos/metabolismo , Plasmócitos/patologia , Índice de Gravidade de Doença , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Two populations of human natural killer (NK) cells can be identified in peripheral blood. The majority are CD3(-)CD56(dim) cells while the minority exhibits a CD3(-)CD56(bright) phenotype. In vitro evidence indicates that CD56(bright) cells are precursors of CD56(dim) cells, but in vivo evidence is lacking. Here, we studied NK cells from a patient that suffered from a melanoma and opportunistic fungal infection during childhood. The patient exhibited a stable phenotype characterized by a reduction in the frequency of peripheral blood CD3(-)CD56(dim) NK cells, accompanied by an overt increase in the frequency and absolute number of CD3(-)CD56(bright) cells. These NK cells exhibited similar expression of perforin, CD57 and CD158, the major activating receptors CD16, NKp46, NKG2D, DNAM-1, and 2B4, as well as the inhibitory receptor CD94/NKG2A, on both CD56(bright) and CD56(dim) NK cells as healthy controls. Also, both NK cell subpopulations produced IFN-γ upon stimulation with cytokines, and CD3(-)CD56(dim) NK cells degranulated in response to cytokines or K562 cells. However, upon stimulation with cytokines, a substantial fraction of CD56(dim) cells failed to up-regulate CD57 and CD158, showed a reduction in the percentage of CD16(+) cells, and CD56(bright) cells did not down-regulate CD62L, suggesting that CD56(dim) cells could not acquire a terminally differentiated phenotype and that CD56(bright) cells exhibit a maturation defect that might result in a potential altered migration pattern. These observations, support the notion that NK cells of this patient display a maturation/activation defect that precludes the generation of mature NK cells at a normal rate accompanied by CD56(dim) NK cells that cannot completely acquire a terminally differentiated phenotype. Thus, our results provide evidence that support the concept that in vivo CD56(bright) NK cells differentiate into CD56(dim) NK cells, and contribute to further understand human NK cell ontogeny.
Assuntos
Antígeno CD56 , Diferenciação Celular , Linhagem da Célula , Células Matadoras Naturais , Antígeno CD56/sangue , Antígeno CD56/genética , Antígeno CD56/imunologia , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citometria de Fluxo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Células K562 , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologiaRESUMO
HDACi are being used as a novel, therapeutic approach for leukemias and other hematological malignancies. However, their effect on immune cells remains ill-defined, as HDACi may impair immune surveillance. In this work, we demonstrate that TSA, VPA, and NaB inhibited IFN-γ production by CD56(dim) and CD56(bright) NK cells and NK cell-mediated cytotoxicity against K562 target cells. HDACi promoted minor NK cell apoptosis but inhibited nuclear mobilization of NF-κB p50, which was accompanied by a robust down-regulation of NKG2D and NKp46 on resting NK cells and of NKG2D, NKp44, NKp46, and CD25 on cytokine-activated NK cells. Decreased CD25 expression promoted a weakened IFN-γ secretion upon restimulation of NK cells with IL-2, whereas reduced expression of NKG2D and NKp46 was accompanied by an impaired NKG2D- and NKp46-dependent cytotoxicity. Moreover, NK cells from normal mice treated in vivo with TSA displayed a diminished expression of NK1.1, NKG2D, and NKp46 and secreted reduced amounts of IFN-γ upon ex vivo stimulation with cytokines. Thus, our preclinical results indicate that HDACi exert deleterious effects on NK cell function, which may weaken immune surveillance and facilitate relapse of the malignant disease in HDACi-treated patients.
Assuntos
Antineoplásicos/farmacologia , Butiratos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Vigilância Imunológica/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Animais , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/imunologia , Células Cultivadas/metabolismo , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Citotoxicidade Imunológica , Inibidores de Histona Desacetilases/efeitos adversos , Humanos , Interferon gama/metabolismo , Interleucinas/farmacologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/biossíntese , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , VorinostatRESUMO
The MHC class I chain-related protein A (MICA) is an inducible molecule almost not expressed by normal cells but strongly up-regulated in tumor cells. MICA-expressing cells are recognized by natural killer (NK) cells, CD8+ abTCR and gdTCR T lymphocytes through the NKG2D receptor. Engagement of NKG2D by MICA triggers IFN-g secretion and cytotoxicity against malignant cells. Although most solid tumors express MICA and this molecule is a target during immune surveillance against tumors, it has been observed that high grade tumors from different histotypes express low amounts of cell surface MICA due to a metalloprotease-induced shedding. Also, melanomas develop after a complex process of neotransformation of normal melanocytes. However, the expression of MICA in premalignant stages (primary human quiescent melanocytic nevi) remains unknown. Here, we assessed expression of MICA by flow cytometry using cell suspensions from 15 primary nevi isolated from 11 patients. When collected material was abundant, cell lysates were prepared and MICA expression was also analyzed by Western blot. We observed that MICA was undetectable in the 15 primary nevi (intradermic, junction, mixed, lentigo and congenital samples) as well as in normal skin, benign lesions (seborrheic keratosis), premalignant lesions (actinic keratosis) and benign basocellular cancer. Conversely, a primary recently diagnosed melanoma showed intense cell surface MICA. We conclude that the onset of MICA expression is a tightly regulated process that occurs after melanocytes trespass the stage of malignant transformation. Thus, analysis of MICA expression in tissue sections of skin samples may constitute a useful marker to differentiate between benign and malignant nevi.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Nevo/metabolismo , Lesões Pré-Cancerosas/metabolismo , Neoplasias Cutâneas/metabolismo , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Nevo/imunologia , Nevo/patologia , Lesões Pré-Cancerosas/imunologia , Lesões Pré-Cancerosas/patologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologiaRESUMO
The inflammatory response is a self-limiting process which involves the sequential activation of signaling pathways leading to the production of both pro- and anti-inflammatory mediators. Galectin-1 (Gal-1), an endogenous lectin found in peripheral lymphoid organs and inflammatory sites, elicits a broad spectrum of biological functions predominantly by acting as a potent anti-inflammatory factor and as a suppressive agent for T-cell responses. However, the molecular pathways underlying Gal-1 expression and function remain poorly understood. Here we identified a regulatory loop linking Gal-1 expression and function to NF-κB activation. NF-κB-activating stimuli increased Gal-1 expression on T cells, an effect which could be selectively prevented by inhibitors of NF-κB signaling. Accordingly, transient transfection of the p65 subunit of NF-κB was sufficient to induce high Gal-1 expression. Using in silico studies and chromatin immunoprecipitation analysis we have identified a functional NF-κB binding site within the first intron of the LGALS1 gene. In addition, our results show that exogenous Gal-1 can attenuate NF-κB activation, as shown by inhibition of IκB-α degradation induced by pro-inflammatory stimuli, higher cytoplasmic retention of p65, lower NF-κB DNA binding activity and impaired transcriptional activation of target genes. The present study suggest a novel regulatory loop by which NF-κB induces expression of Gal-1, which in turn may lead to negative control of NF-κB signaling.
Assuntos
Galectina 1/biossíntese , Regulação da Expressão Gênica/imunologia , NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Sítios de Ligação , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Retroalimentação Fisiológica/fisiologia , Galectina 1/genética , Galectina 1/imunologia , Expressão Gênica , Humanos , Microscopia Confocal , NF-kappa B/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Linfócitos T/metabolismo , TransfecçãoRESUMO
Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)-gamma secretion on engagement of the natural-killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family, such as NKp46, by ligands expressed on tumour cells. However, it remains unknown whether T cells can regulate NK cell-mediated anti-tumour responses. Here, we investigated the early events occurring during T cell-tumour cell interactions, and their impact on NK cell functions. We observed that on co-culture with some melanomas, activated CD4(+) T cells promoted degranulation, and NKG2D- and NKp46-dependent IFN-gamma secretion by NK cells, probably owing to the capture of NKG2D and NKp46 ligands from the tumour-cell surface (trogocytosis). This effect was observed in CD4(+), CD8(+) and resting T cells, which showed substantial amounts of cell surface major histocompatibility complex class I chain-related protein A on co-culture with tumour cells. Our findings identify a new, so far, unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity.
Assuntos
Células Matadoras Naturais/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Neoplasias/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/fisiologia , Microscopia Confocal , Mitógenos/farmacologia , Fito-Hemaglutininas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/fisiologiaRESUMO
Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.