RESUMO
Natural killer (NK) cells often become dysfunctional during tumor progression, but the molecular mechanisms underlying this phenotype remain unclear. To explore this phenomenon, we set up mouse lymphoma models activating or not activating NK cells. Both tumor types elicited type I interferon production, leading to the expression of a T cell exhaustion-like signature in NK cells, which included immune checkpoint proteins (ICPs). However, NK cell dysfunction occurred exclusively in the tumor model that triggered NK cell activation. Moreover, ICP-positive NK cells demonstrated heightened reactivity compared to negative ones. Furthermore, the onset of NK cell dysfunction was swift and temporally dissociated from ICPs induction, which occurred as a later event during tumor growth. Last, NK cell responsiveness was restored when stimulation was discontinued, and interleukin-15 had a positive impact on this reversion. Therefore, our data demonstrate that the reactivity of NK cells is dynamically controlled and that NK cell dysfunction is a reversible process uncoupled from the expression of ICPs.
Assuntos
Células Matadoras Naturais , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Animais , Camundongos , Proteínas de Checkpoint Imunológico/metabolismo , Proteínas de Checkpoint Imunológico/genética , Ativação Linfocitária/imunologia , Linhagem Celular Tumoral , Interleucina-15/metabolismo , Linfoma/imunologia , Linfoma/patologia , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , HumanosRESUMO
OBJECTIVE: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC). DESIGN: To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumours in mice. RESULTS: Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immunoevasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer. CONCLUSION: One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.
Assuntos
ATPases Associadas a Diversas Atividades Celulares , Carcinoma Ductal Pancreático , DNA Helicases , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas c-myc , Animais , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Camundongos , Humanos , DNA Helicases/genética , DNA Helicases/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Linhagem Celular Tumoral , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genéticaRESUMO
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that have the ability to orchestrate adaptive and innate immune responses by antigen phagocytosis and T cell activation across different inflammatory settings such as tumor development. As specific DC identity and how these cells interact with their neighbors is still not fully understood, it remains a challenge to unravel DC heterogeneity, particularly in human cancers. In this chapter, we describe a protocol to isolate and characterize tumor-infiltrating DCs.
Assuntos
Células Dendríticas , Neoplasias , Humanos , Camundongos , Animais , Ativação Linfocitária , Neoplasias/patologia , FagocitoseRESUMO
NK cell receptors allow NK cells to recognize targets such as tumor cells. Many of them are expressed on a subset of NK cells, independently of each other, which creates a vast diversity of receptor combinations. Whether these combinations influence NK cell antitumor responses is not well understood. We addressed this question in the C57BL/6 mouse model and analyzed the individual effector response of 444 mouse NK cell subsets, defined by combinations of 12 receptors, against tumor cell lines originating from different tissues and mouse strains. We found a wide range of reactivity among NK subsets, but the same hierarchy of responses was observed for the different tumor types, showing that the repertoire of NK cell receptors does not encode for different tumor specificities but for different intrinsic reactivities. The coexpression of CD27, NKG2A, and DNAM-1 identified subsets with relative cytotoxic specialization, whereas reciprocally, CD11b and KLRG1 defined the best IFN-γ producers. The expression of educating receptors Ly49C, Ly49I, and NKG2A was also strongly correlated with IFN-γ production, but this effect was suppressed by unengaged receptors Ly49A, Ly49F, and Ly49G2. Finally, IL-15 coordinated NK cell effector functions, but education and unbound inhibitory receptors retained some influence on their response. Collectively, these data refine our understanding of the mechanisms governing NK cell reactivity, which could help design new NK cell therapy protocols.
Assuntos
Interferon gama , Células Matadoras Naturais , Animais , Linhagem Celular Tumoral , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Células Matadoras Naturais/metabolismoRESUMO
NK cells are cytotoxic lymphocytes displaying strong antimetastatic activity. Mouse models and in vitro studies suggest a prominent role of the mechanistic target of rapamycin (mTOR) kinase in the control of NK cell homeostasis and antitumor functions. However, mTOR inhibitors are used as chemotherapies in several cancer settings. The impact of such treatments on patients' NK cells is unknown. We thus performed immunophenotyping of circulating NK cells from metastatic breast cancer patients treated with the mTOR inhibitor everolimus over a three-month period. Everolimus treatment resulted in inhibition of mTORC1 activity in peripheral NK cells, whereas mTORC2 activity was preserved. NK cell homeostasis was profoundly altered with a contraction of the NK cell pool and an overall decrease in their maturation. Phenotype and function of the remaining NK cell population was less affected. This is, to our knowledge, the first in vivo characterization of the role of mTOR in human NK cells.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Everolimo/administração & dosagem , Células Matadoras Naturais/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/patologia , Feminino , Seguimentos , França/epidemiologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Pessoa de Meia-Idade , Metástase Neoplásica , Estudos Prospectivos , Serina-Treonina Quinases TOR/metabolismo , Resultado do TratamentoRESUMO
The clinical outcome of colorectal cancer (CRC) is associated with the immune response; thus, these tumors could be responsive to different immune therapy approaches. Natural killer (NK) cells are key antitumor primary effectors that can eliminate CRC cells without prior immunization. We previously determined that NK cells from the local tumor environment of CRC tumors display a profoundly altered phenotype compared with circulating NK cells from healthy donors (HD). In this study, we evaluated peripheral blood NK cells from untreated patients and their possible role in metastasis progression. We observed profound deregulation in receptor expression even in early stages of disease compared with HD. CRC-NK cells displayed underexpression of CD16, NKG2D, DNAM-1, CD161, NKp46, and NKp30 activating receptors, while inhibitory receptors CD85j and NKG2A were overexpressed. This inhibited phenotype affected cytotoxic functionality against CRC cells and interferon-γ production. We also determined that NKp30 and NKp46 are the key receptors involved in detriment of CRC-NK cells' antitumor activity. Moreover, NKp46 expression correlated with relapse-free survival of CRC patients with a maximum follow-up of 71 months. CRC-NK cells also exhibited altered antibody-dependent cellular cytotoxicity function responding poorly to cetuximab. IL-2 and IL-15 in combination with cetuximab stimulated NK cell, improving cytotoxicity. These results show potential strategies to enhance CRC-NK cell activity.
RESUMO
Clinical studies suggest that triple negative breast cancer (TNBC) patients with epidermal growth factor receptor (EGFR)-expressing tumors could benefit from therapy with Cetuximab, which targets EGFR. NK cells are the primary effectors of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) and thus play a role in Ab-based therapies. We have previously described diminished levels of Cetuximab-mediated ADCC in vitro in patients with advanced breast cancer. Here, we investigated the potential causes of this NK-cell functional deficiency. We characterized NK-cell activating/inhibitory receptors in the peripheral blood of breast cancer patients and found CD85j inhibitory receptor overexpression. The capacity of NK cells to perform Cetuximab-triggered ADCC against TNBC cells correlated inversely with CD85j expression, even in the presence of the stimulatory cytokines IL-2 or IL-15. Hence, patients expressing high levels of CD85j had an impaired ability to lyse TNBC cells in the presence of Cetuximab. We also found that CD85j overexpression was associated with HLA-I and soluble HLA-G expression by tumors. A CD85j functional blockade with a CD85j antagonist Ab restored ADCC levels in breast cancer patients and reverted this negative effect. Our data suggest that strategies that overcome the hurdles of immune activation could improve Cetuximab clinical efficacy.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD/metabolismo , Células Matadoras Naturais/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Adulto , Antineoplásicos/farmacologia , Estudos de Casos e Controles , Cetuximab , Receptores ErbB/antagonistas & inibidores , Feminino , Antígenos HLA/metabolismo , Antígenos HLA-G/metabolismo , Humanos , Células K562 , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Pessoa de Meia-Idade , Adulto JovemRESUMO
Despite NK cells being originally identified because of their ability to kill tumor cells in vitro, only limited information is available on NK cells infiltration of malignant tumors, especially in humans. NK cells infiltrating human colorectal carcinomas (CRCs) were analyzed to identify their potential protective role in an antitumor immune response. The expression and function of relevant molecules were analyzed from different sources, comparing tumor-associated NK cells (TANKs) with autologous peripheral blood NK cells (PB-NKs) from CRC patients-the latter in comparison with PB-NKs from normal donors. TANKs displayed a profound alteration of their phenotype with a drastic reduction of NK cell receptor expression. Co-culture experiments showed that CRC cells produce modulation in NK phenotype and functionality. Moreover, PB-NKs from CRC patients also exhibited an altered phenotype and profound defects in the ability to activate degranulation and IFN-γ production. For the first time, TANK and PB-NK cells from CRC patients have been characterized. It is shown that they are not capable of producing relevant cytokines and degranulate. Taken together, our results suggest that NK cells from CRC patients present alterations of phenotype and function therefore supporting the progression of cancer.
Assuntos
Células Sanguíneas/imunologia , Neoplasias Colorretais/imunologia , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptores de Células Matadoras Naturais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Degranulação Celular , Transformação Celular Neoplásica , Células Cultivadas , Feminino , Humanos , Imunofenotipagem , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de Células Matadoras Naturais/genéticaRESUMO
Triple-negative breast cancer (TNBC) patients do not benefit from target-specific treatments and is associated with a high relapse rate. Epidermal growth factor receptor is frequently expressed in TNBC and is a candidate for new therapies. In this work, we studied Cetuximab-mediated immune activity by NK cells. Thirteen activating/inhibitory receptors were examined on peripheral blood and tumor infiltrating NK cells. NK-cell functionality was evaluated using as effectors tumor-modulated NK cells and NK cells from patients. We evaluated the treatment with Cetuximab plus IL-2 or IL-15 in vivo in TNBC xenografts. Tumor NK-cells receptor profile showed upregulation of inhibitory receptors and downregulation of activating ones. Tumor-modulated NK cells were less cytotoxic. They could perform antibody-dependent cellular cytotoxicity (ADCC) triggered by Cetuximab, although impaired, it could still be restored by stimulation with IL-2 or IL-15. Patients with advanced disease displayed diminished levels of ADCC compared to healthy volunteers. ADCC was restored and potentiated with both cytokines, which were also effective in enhancing the therapeutic activity of Cetuximab in vivo. The combination of Cetuximab with IL-15 and IL-2 may be considered an attractive therapeutic approach to enhance the clinical efficacy of Cetuximab in TNBC.