RESUMO
Natural killer group 2, member D (NKG2D) receptor is a crucial activating receptor in the immune recognition and eradication of abnormal cells by natural killer (NK) cells, and T lymphocytes. NKG2D can transmit activation signals and activate the immune system by recognizing the NKG2D ligands (NKG2D-L) on acute myeloid leukemia (AML) cells. Downregulation of NKG2D-L in AML can circumvent resistance to chemotherapy and immune recognition. Considering this effect, the exploration of targeting the NKG2D/NKG2D-L axis is considered to have tremendous potential for the discovery of novel biomacromolecule antibodies and pharmacological modulators in AML. This review was to outline the impact of NKG2D/NKG2D-L axis on intrinsic immunosurveillance and the development of AML. Furthermore, the NKG2D/NKG2D-L axis related modulators and progress in preclinical and clinical trials was also to be reviewed.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Animais , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Ligantes , Terapia de Alvo Molecular , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
Natural killer (NK) cells are potent cytotoxic effector cells of the innate immune system and play an important role in tumor immunosurveillance and control. NKG2D is an activating receptor of NK cells. The NKG2D receptor-ligand system has contributed to immune cells recognizing tumor cells and the tumor microenvironment. In order to stretch the application of NK cells on adoptive immunotherapy for B-cell malignancies, we designed and produced a novel bispecific ULBP1×CD19-scFv fusion protein, in which the extracellular domain of NKG2D ligand ULBP1 was fused to a single chain variable fragment (scFv) of anti-CD19. The vector expressing ULBP1×CD19-scFv protein was constructed and expressed in Pichia pastoris. Effects of medium composition, concentration of methanol as the inducer, induction time and broth content in shake flask on the expression of the recombinant protein were investigated. The results showed that the optimized conditions for ULBP1×CD19-scFv expression were 1% methanol induction for 96 h with 15% broth content. The secreted recombinant protein was purified using ammonium sulfate fractionation and Ni-NTA affinity chromatography and the purity is about 93%. The cytotoxicity of NK92-MI cells against CD19+ Raji cells was enhanced in the presence of purified ULBP1×CD19-scFv protein. These results indicated that ULBP1 could be used as an activating element of bispecific killer engagers (BiKEs) and Pichia pastoris yeast might be an alternative expression host for BiKEs production.
Assuntos
Anticorpos Biespecíficos , Antígenos CD19/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Anticorpos de Cadeia Única , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/isolamento & purificação , Células Hep G2 , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Proteínas Recombinantes de Fusão , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
Vitiligo is an autoimmune disease that results from the loss of melanocytes, associated with skin infiltration of CD8+ effector memory T cells with a Tc1 skewed immune response. NKG2D is an activating receptor found on immune cells, in particular natural killer and activated CD8+ T cells, that are able to produce a high amount of IFN-γ. Here we found that NKG2D expression was increased in vitiligo skin CD8+ effector memory T cells and was promoted by IL-15. Phenotypic and functional analyses showed that NKG2D+ CD8+ skin effector memory T cells displayed an activated phenotype and produced elevated levels of both IFN-γ and tumor necrosis factor-α. Additional experiments revealed that vitiligo skin dendritic cells expressed the NKG2D ligands MICA-MICB, and in vitro experiments showed that these ligands could be induced on dendritic cells by IFN-α. Cultures of IFN-α-stimulated dendritic cells with skin NKG2D+ CD8+ T cells potentiated the production of type 1 cytokines, which was next inhibited by blocking the NKG2D/MICA-MICB interaction. These data show that NKG2D is a potential marker of pathogenic skin CD8+ effector memory T cells during vitiligo. Therefore, targeting NKG2D could be an attractive strategy in vitiligo, a disease for which there is a strong need of innovative treatments.
Assuntos
Subpopulações de Linfócitos/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Linfócitos T Citotóxicos/imunologia , Vitiligo/imunologia , Adulto , Idoso , Biomarcadores/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interleucina-15/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/citologia , Pele/imunologia , Pele/patologia , Linfócitos T Citotóxicos/metabolismo , Vitiligo/tratamento farmacológico , Vitiligo/patologiaRESUMO
Mice deficient in IFN-γ (IFN-γ knockout [KO] mice) develop a systemic inflammatory syndrome in response to CFA, in contrast to CFA-challenged wild-type (WT) mice who only develop a mild inflammation. Symptoms in CFA-challenged IFN-γ KO resemble systemic juvenile idiopathic arthritis (sJIA), a childhood immune disorder of unknown cause. Dysregulation of innate immune cells is considered to be important in the disease pathogenesis. In this study, we used this murine model to investigate the role of NK cells in the pathogenesis of sJIA. NK cells of CFA-challenged IFN-γ KO mice displayed an aberrant balance of activating and inhibitory NK cell receptors, lower expression of cytotoxic proteins, and a defective NK cell cytotoxicity. Depletion of NK cells (via anti-IL-2Rß and anti-Asialo-GM1 Abs) or blockade of the NK cell activating receptor NKG2D in CFA-challenged WT mice resulted in increased severity of systemic inflammation and appearance of sJIA-like symptoms. NK cells of CFA-challenged IFN-γ KO mice and from anti-NKG2D-treated mice showed defective degranulation capacities toward autologous activated immune cells, predominantly monocytes. This is in line with the increased numbers of activated inflammatory monocytes in these mice which was particularly reflected in the expression of CCR2, a chemokine receptor, and in the expression of Rae-1, a ligand for NKG2D. In conclusion, NK cells are defective in a mouse model of sJIA and impede disease development in CFA-challenged WT mice. Our findings point toward a regulatory role for NK cells in CFA-induced systemic inflammation via a NKG2D-dependent control of activated immune cells.
Assuntos
Artrite Juvenil/imunologia , Artrite Juvenil/metabolismo , Suscetibilidade a Doenças , Imunomodulação , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Animais , Artrite Juvenil/patologia , Biomarcadores , Citotoxicidade Imunológica , Modelos Animais de Doenças , Imunofenotipagem , Interferon gama/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Osteoclastos/imunologia , Osteoclastos/metabolismoRESUMO
Cellular senescence is a tumor suppressive mechanism that can paradoxically contribute to aging pathologies. Despite evidence of immune clearance in mouse models, it is not known how senescent cells (SnCs) persist and accumulate with age or in tumors in individuals. Here, we identify cooperative mechanisms that orchestrate the immunoevasion and persistence of normal and cancer human SnCs through extracellular targeting of natural killer receptor signaling. Damaged SnCs avoid immune recognition through MMPs-dependent shedding of NKG2D-ligands reinforced via paracrine suppression of NKG2D receptor-mediated immunosurveillance. These coordinated immunoediting processes are evident in residual, drug-resistant tumors from cohorts of >700 prostate and breast cancer patients treated with senescence-inducing genotoxic chemotherapies. Unlike in mice, these reversible senescence-subversion mechanisms are independent of p53/p16 and exacerbated in oncogenic RAS-induced senescence. Critically, the p16INK4A tumor suppressor can disengage the senescence growth arrest from the damage-associated immune senescence program, which is manifest in benign nevi lesions where indolent SnCs accumulate over time and preserve a non-pro-inflammatory tissue microenvironment maintaining NKG2D-mediated immunosurveillance. Our study shows how subpopulations of SnCs elude immunosurveillance, and reveals secretome-targeted therapeutic strategies to selectively eliminate -and restore the clearance of- the detrimental SnCs that actively persist after chemotherapy and accumulate at sites of aging pathologies.
Assuntos
Envelhecimento/imunologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Senescência Celular/imunologia , Resistencia a Medicamentos Antineoplásicos/imunologia , Neoplasias da Próstata/tratamento farmacológico , Evasão Tumoral/imunologia , Envelhecimento/patologia , Animais , Antineoplásicos/uso terapêutico , Biópsia , Mama/patologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Dano ao DNA/efeitos dos fármacos , Conjuntos de Dados como Assunto , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Vigilância Imunológica/efeitos dos fármacos , Vigilância Imunológica/imunologia , Masculino , Metaloendopeptidases/metabolismo , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Próstata/patologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Análise Serial de Tecidos , Evasão Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Multiple myeloma (MM) is an incurable hematologic malignancy of plasma cells, with an estimated 30,000 new cases diagnosed each year in the United States, signifying the need for new therapeutic approaches. We hypothesized that targeting MM using a bispecific antibody (biAb) to simultaneously engage both innate and adaptive cytolytic immune cells could present potent antitumor activity. We engineered a biAb by fusing an anti-CS1 single-chain variable fragment (scFv) and an anti-NKG2D scFv (CS1-NKG2D biAb). Although NKG2D is a potent activation receptor ubiquitously expressed on mostly cytolytic immune cells including NK cells, CD8+ T cells, γδ T cells, and NKT cells, the CS1 tumor-associated antigen on MM represents a promising target. CS1-NKG2D biAb engaged human MM cell lines and NKG2D+ immune cells, forming immune synapses. In effector cells, CS1-NKG2D biAb triggered the phosphorylation of AKT, a downstream protein kinase of the activated NKG2D-DAP10 complex. The EC50 values of CS1-NKG2D biAb for CS1high and for CS1low MM cell lines with effector PBMCs were 10-12 and 10-9 mol/L, respectively. CS1-NKG2D biAb acted through multiple types of immune cells, and this induced cytotoxicity was both CS1- and NKG2D-specific. In vivo, survival was significantly prolonged using CS1-NKG2D biAb in a xenograft NOD-SCIDIL2γc-/- (NSG) mouse model engrafted with both human PBMCs and MM cell lines. Collectively, we demonstrated that the CS1-NKG2D biAb facilitated an enhanced immune synapse between CS1+ MM cells and NKG2D+ cytolytic innate and antigen-specific effector cells, which, in turn, activated these immune cells for improved clearance of MM. Cancer Immunol Res; 6(7); 776-87. ©2018 AACR.
Assuntos
Anticorpos Biespecíficos/farmacologia , Citotoxicidade Imunológica , Mieloma Múltiplo/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Família de Moléculas de Sinalização da Ativação Linfocitária/antagonistas & inibidores , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Animais , Biomarcadores , Linhagem Celular Tumoral , Feminino , Humanos , Sinapses Imunológicas , Imunofenotipagem , Interferon gama/metabolismo , Camundongos , Mieloma Múltiplo/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Subpopulações de Linfócitos T/metabolismoRESUMO
Chimeric Antigen Receptor (CAR) T cells expressing the fusion of the NKG2D protein with CD3ζ (NKG2D-CAR T Cells) acquire a specificity for stress-induced ligands expressed on hematological and solid cancers. However, these stress ligands are also transiently expressed by activated T cells implying that NKG2D-based T cells may undergo self-killing (fratricide) during cell manufacturing or during the freeze thaw cycle prior to infusion in patients. To avoid target-driven fratricide and enable the production of NKG2D-CAR T cells for clinical application, two distinct approaches were investigated. The first focused upon the inclusion of a Phosphoinositol-3-Kinase inhibitor (LY294002) into the production process. A second strategy involved the inclusion of antibody blockade of NKG2D itself. Both processes impacted T cell fratricide, albeit at different levels with the antibody process being the most effective in terms of cell yield. While both approaches generated comparable NKG2D-CAR T cells, there were subtle differences, for example in differentiation status, that were fine-tuned through the phasing of the inhibitor and antibody during culture in order to generate a highly potent NKG2D-CAR T cell product. By means of targeted inhibition of NKG2D expression or generic inhibition of enzyme function, target-driven CAR T fratricide can be overcome. These strategies have been incorporated into on-going clinical trials to enable a highly efficient and reproducible manufacturing process for NKG2D-CAR T cells.
Assuntos
Citotoxicidade Imunológica/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Imunoterapia Adotiva/métodos , Células K562 , Ligantes , Morfolinas/farmacologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
Natural killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNγ, MIP-1α, and TNFα) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as IL12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a Transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA-NKG2D interactions resulted in reductions in IFNγ production. Depletion of monocytes in vivo resulted in decreased IFNγ production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock-depleted controls (P < 0.05). These data suggest that NK cell-monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer. Cancer Immunol Res; 5(9); 778-89. ©2017 AACR.
Assuntos
Neoplasias da Mama/terapia , Antígenos de Histocompatibilidade Classe I/imunologia , Interferon gama/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Antígenos de Histocompatibilidade Classe I/efeitos dos fármacos , Humanos , Interleucina-12/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos , Monócitos/imunologia , Monócitos/patologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Receptores Fc/administração & dosagem , Receptores Fc/imunologia , Trastuzumab/administração & dosagem , Trastuzumab/imunologiaRESUMO
Cancer cells may co-opt the NKG2D lymphocyte receptor to complement the presence of its ligands for autonomous stimulation of oncogenic signaling. Previous studies raise the possibility that cancer cell NKG2D may induce high malignancy traits, but its full oncogenic impact is unknown. Using epithelial ovarian cancer as model setting, we show here that ex vivo NKG2D+ cancer cells have stem-like capacities, and provide formal in vivo evidence linking NKG2D stimulation with the development and maintenance of these functional states. NKG2D+ ovarian cancer cell populations harbor substantially greater capacities for self-renewing in vitro sphere formation and in vivo tumor initiation in immunodeficient (NOD scid gamma) mice than NKG2D- controls. Sphere formation and tumor initiation are impaired by NKG2D silencing or ligand blockade using antibodies or a newly designed pan ligand-masking NKG2D multimer. In further support of pathophysiological significance, a prospective study of 47 high-grade serous ovarian cancer cases revealed that the odds of disease recurrence were significantly greater and median progression-free survival rates higher among patients with above and below median NKG2D+ cancer cell frequencies, respectively. Collectively, our results define cancer cell NKG2D as an important regulator of tumor initiation in ovarian cancer and presumably other malignancies and thus challenge current efforts in immunotherapy aimed at enhancing NKG2D function.
Assuntos
Biomarcadores Tumorais/genética , Proliferação de Células/efeitos dos fármacos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos/administração & dosagem , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Alopecia em Áreas/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Alopecia em Áreas/metabolismo , Alopecia em Áreas/patologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Interferon gama/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C3H , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Projetos Piloto , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Human papillomavirus (HPV) is the most common sexually transmitted agent worldwide and is etiologically linked to several cancers, including cervical and genital cancers. NKG2D, an activating receptor expressed by NK cells, plays an important role in cancer immune-surveillance. We analyzed the impact of a NKG2D gene variant, rs1049174, on the incidence of HPV-related cancers in Vietnamese patients and utilized various molecular approaches to elucidate the mechanisms of NKG2D receptor regulation by rs1049174. In a group of 123 patients with HPV+ anogenital cancers, the low cytotoxicity allele LNK was significantly associated with increased cancer susceptibility (p = 0.016). Similar results were also observed in a group of 153 women with cervical cancer (p = 0.05). In functional studies, NK cells from individuals with LNK genotype showed a lower NKG2D expression and displayed less efficient NKG2D-mediated functions than NK cells with HNK genotype. Notably, the rs1049174 variant occurs within a targeting site for miR-1245, a negative regulator of NKG2D expression. Compared with the higher cytotoxicity allele HNK, the LNK allele was more efficiently targeted by miR-1245 and thus determined lower NKG2D expression in NK cells with the LNK genotype. The NKG2D variants may influence cancer immunosurveillance and thus determine susceptibility to various malignancies, including HPV-induced cancers.
Assuntos
Células Matadoras Naturais/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Papillomaviridae/isolamento & purificação , Neoplasias Urogenitais/patologia , Regiões 3' não Traduzidas , Adulto , Idoso , Alelos , Sequência de Bases , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Suscetibilidade a Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Genótipo , Células HeLa , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Masculino , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência , Fator de Crescimento Transformador beta1/farmacologia , Neoplasias Urogenitais/virologiaRESUMO
Tetramethypyrazine (TMP), one of the active compounds extracted from the traditional Chinese medicinal herb (Chuanxiong), has been verified as an anticancer compound against several types of cancer. However, understanding of the molecular mechanisms have not been fully elucidated. In the present study, the anticancer efficacy of TMP was investigated in human clear cell renal cell carcinoma (ccRCC) cells. We showed that TMP significantly inhibited ccRCC cell viability, proliferation, apoptosis, invasion and migration through the methods of MTT, flow cytometry, wound healing and transwell assays. Furthermore, reverse transcription polymerase chain reaction (RT-PCR), western blotting and immunofluorescence results demonstrated TMP upregulation of the expression of NKG2D ligands (NKG2DLs) MHC class I chain-related molecules A and B (MICA/B) and epithelial cell expression marker of E-cadherin, and downregulation of mesenchymal cell expression markers of vimentin and fibronectin. Taken together, the inhibition of TMP on ccRCC cells might be mediated via inhibition of NKG2D related signaling pathway to further suppress epithelial-mesenchymal transition (EMT) progression. The binding of NKG2D to its ligands activates NK cells, giving the rationale for studies on the utilization of TMP as a potential cancer therapeutic compound to increase NK cells-mediated cytotoxicity against high MICA/B expression in cancer cells.
Assuntos
Carcinoma de Células Renais/patologia , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Renais/patologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Pirazinas/farmacologia , Western Blotting , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Vasodilatadores/farmacologiaRESUMO
It is now clear that recognition of nascent tumors by the immune system is critical for survival of the host against cancer. During cancer immunoediting, the ability of the tumor to escape immune recognition is important for tumor development. The immune system recognizes tumors via the presence of classical Ags and also by conserved innate mechanisms. One of these mechanisms is the NKG2D receptor that recognizes ligands whose expression is induced by cell transformation. In this study, we show that in NKG2D receptor-deficient mice, increasing numbers of B cells begin to express NKG2D ligands as they age. Their absence in wild-type mice suggests that these cells are normally cleared by NKG2D-expressing cells. NKG2D-deficient mice and mice constitutively expressing NKG2D ligands had increased incidence of B cell tumors, confirming that the inability to clear NKG2D ligand-expressing cells was important in tumor suppression and that NKG2D ligand expression is a marker of nascent tumors. Supporting a role for NKG2D ligand expression in controlling the progression of early-stage B cell lymphomas in humans, we found higher expression of a microRNA that inhibits human NKG2D ligand expression in tumor cells from high-grade compared with low-grade follicular lymphoma patients.
Assuntos
Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Animais , Progressão da Doença , Humanos , Ligantes , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/deficiência , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genéticaRESUMO
Cytokine-induced killer (CIK) cells represent a realistic approach in cancer immunotherapy with confirmed survival benefits in the context of metastatic solid tumors. However, therapeutic effects are limited to a fraction of patients. In this study, immune-resistance elements and ideal combination therapies were explored. Initially, phenotypic analysis was performed to document CD3, CD56, NKG2D, DNAM-1, PD-L1, PD-1, CTLA-4, TIM-3, 2B4, and LAG-3 on CIK cells. Upon engagement of CIK cells with the tumor cells, expression of PD-1 on CIK cells and PD-L1 on both cells were up-regulated. Over-expression of PD-L1 levels on tumor cells via lentiviral transduction inhibited tumoricidal activity of CIK cells, and neutralizing of PD-L1/PD-1 signaling axis could enhance their tumor-killing effect. Conversely, blockade of NKG2D, a major activating receptor of CIK cells, largely caused dysfunction of CIK cells. Functional study showed an increase of NKG2D levels along with PD-L1/PD-1 blockade in the presence of other immune effector molecule secretion. Additionally, combined therapy of CIK infusion and PD-L1/PD-1 blockade caused a delay of in vivo tumor growth and exhibited a survival advantage over untreated mice. These results provide a preclinical proof-of-concept for simultaneous PD-L1/PD-1 pathways blockade along with CIK infusion as a novel immunotherapy for unresectable cancers.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Citocinas/metabolismo , Neoplasias Gastrointestinais/terapia , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Children with advanced stages (relapsed/refractory and stage IV) of rhabdomyosarcoma (RMS) have a poor prognosis despite intensive chemotherapy and autologous stem cell rescue, with 5-year survival rates ranging from 5 to 35 %. Development of new, additional treatment modalities is necessary to improve the survival rate. In this preclinical study, we investigated the potential of resting and cytokine-activated natural killer (NK) cells to lyse RMS cell lines, as well as the pathways involved, to explore the eventual clinical application of (activated) NK cell immunotherapy. RMS cell lines (n = 3 derived from embryonal RMS and n = 2 derived from alveolar RMS) were susceptible to cytolysis mediated by resting NK cells, and this susceptibility was significantly increased using IL-15-activated NK cells. Flow cytometry and cytolytic assays were used to define the activating and inhibitory pathways of NK cells involved in recognizing and lysing RMS cells. NKG2D and DNAM-1 receptor-ligand interactions were essential in cytolysis by resting NK cells, as simultaneous blocking of both pathways resulted in almost complete abrogation of the cytotoxicity. In contrast, combined blocking of DNAM-1 and NKG2D only led to partial reduction of the lytic activity of IL-15-activated NK cells. In this respect, residual lysis was, at least partly, mediated by pathways involving the natural cytotoxicity receptors NKp30 and NKp46. These findings support further exploration of NK cell-based immunotherapy as adjuvant modality in current treatment strategies of RMS.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Citotoxicidade Imunológica , Interleucina-15/imunologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Rabdomiossarcoma/terapia , Linhagem Celular Tumoral , Citocinas/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Ativação Linfocitária/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Rabdomiossarcoma/imunologiaRESUMO
In allogeneic hematopoietic stem cell transplantation (HSCT), controlling graft-versus-host disease (GVHD) while maintaining graft-versus-tumor (GVT) responses is of critical importance. Using a mouse model of allogeneic HSCT, we hereby demonstrate that NKG2D expression by CD8(+) T cells plays a major role in mediating GVHD and GVT effects by promoting the survival and cytotoxic function of CD8(+) T cells. The expression of NKG2D ligands was not induced persistently on normal tissues of allogeneic HSCT-recipient mice treated with anti-NKG2D antibody, suggesting that transient NKG2D blockade might be sufficient to attenuate GVHD and allow CD8(+) T cells to regain their GVT function. Indeed, short-term treatment with anti-NKG2D antibody restored GVT effects while maintaining an attenuated GVHD state. NKG2D expression was also detected on CD8(+) T cells from allogeneic HSCT patients and trended to be higher in those with active GVHD. Together, these data support a novel role for NKG2D expression by CD8(+) T cells during allogeneic HSCT, which could be potentially therapeutically exploited to separate GVHD from GVT effects.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Regulação da Expressão Gênica/imunologia , Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Tumor/imunologia , Transplante de Células-Tronco Hematopoéticas , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Aloenxertos , Animais , Anticorpos Neutralizantes/farmacologia , Linfócitos T CD8-Positivos/patologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Efeito Enxerto vs Tumor/efeitos dos fármacos , Efeito Enxerto vs Tumor/genética , Camundongos , Camundongos Knockout , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genéticaRESUMO
The interaction between keratinocytes (KCs) and skin-resident immune cells has an important role in induction of contact hypersensitivity. A specific subset of γδ T cells termed dendritic epidermal T cells (DETCs) are located in mouse epidermis, and we have recently shown that DETCs become activated and produce IL-17 in an IL-1ß-dependent manner during contact hypersensitivity. Various receptors on DETCs, including NKG2D, are involved in DETC responses against tumors and during wound healing. The ligands for NKG2D (NKG2DL) are stress-induced proteins such as mouse UL16-binding protein-like transcript 1 (Mult-1), histocompatibility 60 (H60), and retinoic acid early inducible-1 (Rae-1) in mice and major histocompatibility complex (MHC) class I-chain-related A (MICA), MHC class I-chain-related B, and UL16-binding protein in humans. Here, we show that allergens upregulate expression of the NKG2DL Mult-1, H60, and Rae-1 in cultured mouse KCs and of MICA in primary human KCs. We demonstrate that Mult-1 is expressed in mouse skin exposed to allergen. Furthermore, we find that the vast majority of DETCs in murine epidermis and skin-homing cutaneous lymphocyte-associated antigen positive γδ T cells in humans express NKG2D. Finally, we demonstrate that blocking of NKG2D partially inhibits allergen-induced DETC activation. These findings demonstrate that NKG2D and NKG2DL are involved in allergen-induced activation of DETCs and indicate that the NKG2D/NKG2DL pathway might be a potential target for treatment of contact hypersensitivity.
Assuntos
Dermatite de Contato/metabolismo , Dermatite de Contato/patologia , Células de Langerhans/metabolismo , Células de Langerhans/patologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Alérgenos/efeitos adversos , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cultivadas , Dermatite de Contato/etiologia , Modelos Animais de Doenças , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/efeitos dos fármacos , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismoRESUMO
PURPOSE: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive forms of cancer with no curative therapies available. To date, strategies to target ATC by immunotherapy have not been evaluated. We investigated whether ATC would be a suitable target for natural killer (NK) cell-based immunotherapy. EXPERIMENTAL DESIGN: We first established seven new cell lines from ATC tumors, three from papillary thyroid carcinoma tumors and analyzed them together with eight additional ATC cell lines. Cells were analyzed for sensitivity to lysis by NK cells and their ability to chemoattract and regulate the activity of NK cells. In addition, fresh tumor samples and peripheral blood from six patients with ATC were analyzed for NK cell infiltration and phenotype. RESULTS: We observed that ATC cell lines are sensitive to lysis by ex vivo expanded NK cells and that the lysis was abrogated upon blockade of NKG2D. Sensitivity of thyroid cancer cell lines to NK cell-mediated lysis correlated with surface expression of UL16-binding protein 2 on tumor cells. Moreover, ATC cell lines produced high levels of CXCL10 and stimulated migration of expanded NK cells and ATC tumors were enriched for NK cells expressing the cognate chemokine receptor CXCR3. However, compared with NK cells in peripheral blood, ATC tumor-derived NK cells displayed a suppressed phenotype with a downregulated expression of NKG2D. In vitro, suppression of NK cell-mediated lysis and NKG2D expression by ATC cells was restored upon neutralization of prostaglandin-E2. CONCLUSIONS: ATC cell lines are sensitive to NK cell-mediated lysis via ULBP2/5/6 and chemoattract CXCR3-positive NK cells. Patients with ATC may benefit from NK cell-based immunotherapy.
Assuntos
Citotoxicidade Imunológica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Carcinoma Anaplásico da Tireoide/imunologia , Carcinoma Anaplásico da Tireoide/metabolismo , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Quimiocina CXCL10/biossíntese , Quimiotaxia/imunologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Dinoprostona/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Humanos , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intercelular/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Fenótipo , Receptores CXCR3/genética , Receptores CXCR3/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Carcinoma Anaplásico da Tireoide/genéticaRESUMO
Although the mechanisms underlying the cytotoxic effect of NK cells on tumor cells and intracellular bacteria have been studied extensively, it remains unclear how these cells kill extracellular bacterial pathogens. In this study, we examine how human NK cells kill Mycobacterium kansasii and M.tb. The underlying mechanism is contact dependent and requires two cytolytic proteins: perforin and granulysin. Mycobacteria induce enhanced expression of the cytolytic proteins via activation of the NKG2D/NCR cell-surface receptors and intracellular signaling pathways involving ERK, JNK, and p38 MAPKs. These results suggest that NK cells use similar cellular mechanisms to kill both bacterial pathogens and target host cells. This report reveals a novel role for NK cells, perforin, and granulysin in killing mycobacteria and highlights a potential alternative defense mechanism that the immune system can use against mycobacterial infection.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Bacteriólise , Células Matadoras Naturais/imunologia , Mycobacterium kansasii , Mycobacterium tuberculosis , Perforina/metabolismo , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/farmacologia , Bacteriólise/efeitos dos fármacos , Bacteriólise/fisiologia , Linhagem Celular Tumoral , Parede Celular/efeitos dos fármacos , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/ultraestrutura , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Nanotubos , Receptor 2 Desencadeador da Citotoxicidade Natural/antagonistas & inibidores , Receptor 2 Desencadeador da Citotoxicidade Natural/biossíntese , Receptor 2 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/antagonistas & inibidores , Receptor 3 Desencadeador da Citotoxicidade Natural/biossíntese , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Perforina/biossíntese , Perforina/genética , Perforina/farmacologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The natural killer group 2 membrane D (NKG2D) receptor is an NK-activating receptor that plays an important role in host defense against tumors and viral infections. Although the marmoset is an important and reliable animal model, especially for the study of human-specific viral infections, functional characterization of NKG2D on marmoset NK cells has not previously been conducted. In the present study, we investigated a subpopulation of marmoset NK cells that express NKG2D and exhibit cytolytic potential. On the basis of their CD16 and CD56 expression patterns, marmoset NK cells can be classified into three subpopulations: CD16(+) CD56(-), CD16(-) CD56(+) and CD16(-) CD56(-) cells. NKG2D expression on marmoset CD16(+) CD56(-) and CD16(-) CD56(+) splenocytes was confirmed using an NKG2D ligand composed of an MHC class I chain-related molecule A (MICA)-Fc fusion protein. When marmoset splenocytes were cultured with IL-2 for 4 days, NKG2D expression was retained on CD16(+) CD56(-) and CD16(-) CD56(+). In addition, CD16(+) CD56(+) cells within the marmoset NK population appeared which expressed NKG2D after IL-2 stimulation. IL-2-activated marmoset NK cells showed strong cytolytic activity against K562 target cells and target cells stably expressing MICA. Further, the cytolytic activity of marmoset splenocytes was significantly reduced after addition of MICA-Fc fusion protein. Thus, NKG2D functions as an activating receptor on marmoset NK cells that possesses cytotoxic potential, and phenotypic profiles of marmoset NK cell subpopulations are similar to those seen in humans.