RESUMO
Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Saco Vitelino , Células-Tronco Hematopoéticas , Organoides , Atividades CotidianasRESUMO
BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. METHODS: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. FINDINGS: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo. CONCLUSIONS: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.
Assuntos
Linfócitos do Interstício Tumoral , Linfócitos T , Transferência Adotiva , Animais , Citocinas/metabolismo , Humanos , Imunoterapia Adotiva/métodos , CamundongosRESUMO
Adoptive T cell therapies (ACT) have been curative for a limited number of cancer patients. The sensitization of cancer cells to T cell killing may expand the benefit of these therapies for more patients. To this end, we use a three-step approach to identify cancer genes that disfavor T cell immunity. First, we profile gene transcripts upregulated by cancer under selection pressure from T cell killing. Second, we identify potential tumor gene targets and pathways that disfavor T cell killing using signaling pathway activation libraries and genome-wide loss-of-function CRISPR-Cas9 screens. Finally, we implement pharmacological perturbation screens to validate these targets and identify BIRC2, ITGAV, DNPEP, BCL2, and ERRα as potential ACT-drug combination candidates. Here, we establish that BIRC2 limits antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity and provide evidence that BIRC2 inhibition in combination with ACT is an effective strategy to increase efficacy.
Assuntos
Neoplasias , Linfócitos T , Apresentação de Antígeno , Sistemas CRISPR-Cas/genética , Humanos , Neoplasias/genética , Oncogenes , Análise de SistemasRESUMO
The administration of T cells as cellular therapy against advanced cancers has brought clinical benefit to many patients and has progressed the field of cancer research. However, current cell therapy treatments are not curative in most patients, particularly in those with solid tumors, and it remains to be seen how broadly and efficaciously they may be applied going forward. Recent research has begun to elucidate key factors that regulate the efficacy of cell therapy in cancer patients, including T cell stemness and the ability to effectively target tumor antigens and overcome tumor heterogeneity. In this review, we discuss key properties of clinically effective anti-cancer T cell therapies along with strategies to improve T cell characteristics to augment clinical efficacy in solid tumors.
Assuntos
Neoplasias , Linfócitos T , Antígenos de Neoplasias , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Imunoterapia Adotiva , Neoplasias/patologiaRESUMO
T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here, we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor-driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions.
Assuntos
Neoplasias da Mama/terapia , Sistemas CRISPR-Cas , Imunoterapia Adotiva/métodos , Melanoma Experimental/terapia , Fenótipo , Linfócitos T/transplante , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Diferenciação Celular , Feminino , Engenharia Genética , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Small molecule based targeted therapies for the treatment of metastatic melanoma hold promise but responses are often not durable, and tumors frequently relapse. Response to adoptive cell transfer (ACT)-based immunotherapy in melanoma patients are durable but patients develop resistance primarily due to loss of antigen expression. The combination of small molecules that sustain T cell effector function with ACT could lead to long lasting responses. Here, we have developed a novel co-culture cell-based high throughput assay system to identify compounds that could potentially synergize or enhance ACT-based immunotherapy of melanoma. A BRAFV600E mutant melanoma cell line, SB-3123p which is resistant to Pmel-1-directed ACT due to low gp100 expression levels was used to develop a homogenous time resolve fluorescence (HTRF), screening assay. This high throughput screening assay quantitates IFNγ released upon recognition of the SB-3123p melanoma cells by Pmel-1 CD8+ T-cells. A focused collection of approximately 500 small molecules targeting a broad range of cellular mechanisms was screened, and four active compounds that increased melanoma antigen expression leading to enhanced IFNγ production were identified and their in vitro activity was validated. These four compounds may provide a basis for enhanced immune recognition and design of novel therapeutic approaches for patients with BRAF mutant melanoma resistant to ACT due to antigen downregulation.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Imunoterapia/métodos , Melanoma/tratamento farmacológico , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Fatores Imunológicos/metabolismo , Imunoterapia Adotiva/métodos , Melanoma/fisiopatologia , Recidiva Local de Neoplasia/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismoRESUMO
Although immunotherapy has revolutionized cancer treatment, only a subset of patients demonstrate durable clinical benefit. Definitive predictive biomarkers and targets to overcome resistance remain unidentified, underscoring the urgency to develop reliable immunocompetent models for mechanistic assessment. Here we characterize a panel of syngeneic mouse models, representing a variety of molecular and phenotypic subtypes of human melanomas and exhibiting their diverse range of responses to immune checkpoint blockade (ICB). Comparative analysis of genomic, transcriptomic and tumor-infiltrating immune cell profiles demonstrated alignment with clinical observations and validated the correlation of T cell dysfunction and exclusion programs with resistance. Notably, genome-wide expression analysis uncovered a melanocytic plasticity signature predictive of patient outcome in response to ICB, suggesting that the multipotency and differentiation status of melanoma can determine ICB benefit. Our comparative preclinical platform recapitulates melanoma clinical behavior and can be employed to identify mechanisms and treatment strategies to improve patient care.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais , Imunoterapia , Melanoma/patologia , Melanoma/terapia , Animais , Antineoplásicos Imunológicos/uso terapêutico , Antígeno CTLA-4/imunologia , Células Cultivadas , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Heterogeneidade Genética , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Ipilimumab/uso terapêutico , Melanoma/diagnóstico , Melanoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Prognóstico , Receptor de Morte Celular Programada 1/imunologia , RNA-Seq , Resultado do Tratamento , Sequenciamento Completo do GenomaRESUMO
Host conditioning has emerged as an important component of effective adoptive cell transfer-based immunotherapy for cancer. High levels of IL-1ß are induced by host conditioning, but its impact on the antitumor function of T cells remains unclear. We found that the administration of IL-1ß increased the population size and functionality of adoptively transferred T cells within the tumor. Most importantly, IL-1ß enhanced the ability of tumor-specific T cells to trigger the regression of large, established B16 melanoma tumors in mice. Mechanistically, we showed that the increase in T cell numbers was associated with superior tissue homing and survival abilities and was largely mediated by IL-1ß-stimulated host cells. In addition, IL-1ß enhanced T cell functionality indirectly via its actions on radio-resistant host cells in an IL-2- and IL-15-dependent manner. Our findings not only underscore the potential of provoking inflammation to enhance antitumor immunity but also uncover novel host regulations of T cell responses.
Assuntos
Imunoterapia Adotiva/métodos , Interleucina-1beta/imunologia , Ativação Linfocitária/imunologia , Melanoma Experimental/terapia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Citocinas/sangue , Interleucina-1beta/metabolismo , Interleucina-6/sangue , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Linfócitos T/metabolismo , Linfócitos T/transplante , Fator de Necrose Tumoral alfa/sangueRESUMO
Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity.
Assuntos
Imunidade Adaptativa , Doenças Autoimunes do Sistema Nervoso/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Malformações do Sistema Nervoso/genética , Ribonuclease H/genética , Animais , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/mortalidade , Doenças Autoimunes do Sistema Nervoso/patologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Metástase Linfática , Camundongos , Camundongos Nus , Mutação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Malformações do Sistema Nervoso/imunologia , Malformações do Sistema Nervoso/mortalidade , Malformações do Sistema Nervoso/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/imunologia , Análise de Sequência de RNA , Transdução de Sinais , Análise de Sobrevida , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
A paradox of tumor immunology is that tumor-infiltrating lymphocytes are dysfunctional in situ, yet are capable of stem cell-like behavior including self-renewal, expansion, and multipotency, resulting in the eradication of large metastatic tumors. We find that the overabundance of potassium in the tumor microenvironment underlies this dichotomy, triggering suppression of T cell effector function while preserving stemness. High levels of extracellular potassium constrain T cell effector programs by limiting nutrient uptake, thereby inducing autophagy and reduction of histone acetylation at effector and exhaustion loci, which in turn produces CD8+ T cells with improved in vivo persistence, multipotency, and tumor clearance. This mechanistic knowledge advances our understanding of T cell dysfunction and may lead to novel approaches that enable the development of enhanced T cell strategies for cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Tolerância Imunológica , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Potássio/metabolismo , Células-Tronco/imunologia , Acetilcoenzima A/metabolismo , Acetilação , Animais , Autofagia/imunologia , Restrição Calórica , Diferenciação Celular/genética , Epigênese Genética , Histonas/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Microambiente TumoralRESUMO
Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.
Assuntos
Transferência Adotiva , Engenharia Genética , Neoplasias Experimentais/terapia , Receptores de Antígenos Quiméricos , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia , Animais , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/uso terapêutico , Transdução de Sinais/genética , Microambiente Tumoral/genética , Receptor fas/genética , Receptor fas/imunologiaRESUMO
Tumor-derived soluble factors promote the production of Gr-1+CD11b+ immature myeloid cells, and TGFß signaling is critical in their immune suppressive function. Here, we report that miR-130a and miR-145 directly target TGFß receptor II (TßRII) and are down-regulated in these myeloid cells, leading to increased TßRII. Ectopic expression of miR-130a and miR-145 in the myeloid cells decreased tumor metastasis. This is mediated through a downregulation of type 2 cytokines in myeloid cells and an increase in IFNγ-producing cytotoxic CD8 T lymphocytes. miR-130a- and miR-145-targeted molecular networks including TGFß and IGF1R pathways were correlated with higher tumor stages in cancer patients. Lastly, miR-130a and miR-145 mimics, as well as IGF1R inhibitor NT157 improved anti-tumor immunity and inhibited metastasis in preclinical mouse models. These results demonstrated that miR-130a and miR-145 can reprogram tumor-associated myeloid cells by altering the cytokine milieu and metastatic microenvironment, thus enhancing host antitumor immunity.
Assuntos
Regulação Neoplásica da Expressão Gênica , Imunidade Inata/efeitos dos fármacos , Neoplasias Pulmonares/genética , Neoplasias Mamárias Experimentais/genética , MicroRNAs/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Animais , Antineoplásicos/farmacologia , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Injeções Intravenosas , Interferon gama/genética , Interferon gama/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Transgênicos , MicroRNAs/imunologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/patologia , Oligorribonucleotídeos/administração & dosagem , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Pirogalol/análogos & derivados , Pirogalol/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo II/imunologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Transdução de Sinais , Sulfonamidas/farmacologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologiaRESUMO
Cancer recurrence after initial diagnosis and treatment is a major cause of breast cancer (BC) mortality, which results from the metastatic outbreak of dormant tumour cells. Alterations in the tumour microenvironment can trigger signalling pathways in dormant cells leading to their proliferation. However, processes involved in the initial and the long-term survival of disseminated dormant BC cells remain largely unknown. Here we show that autophagy is a critical mechanism for the survival of disseminated dormant BC cells. Pharmacologic or genetic inhibition of autophagy in dormant BC cells results in significantly decreased cell survival and metastatic burden in mouse and human 3D in vitro and in vivo preclinical models of dormancy. In vivo experiments identify autophagy gene autophagy-related 7 (ATG7) to be essential for autophagy activation. Mechanistically, inhibition of the autophagic flux in dormant BC cells leads to the accumulation of damaged mitochondria and reactive oxygen species (ROS), resulting in cell apoptosis.
Assuntos
Proteína 7 Relacionada à Autofagia/genética , Autofagia/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/genética , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia/antagonistas & inibidores , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/antagonistas & inibidores , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Feminino , Humanos , Hidroxicloroquina/farmacologia , Metástase Linfática , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Recidiva , Transdução de SinaisRESUMO
Adoptive immunotherapies using T cells genetically redirected with a chimeric antigen receptor (CAR) or T cell receptor (TCR) are entering mainstream clinical practice. Despite encouraging results, some patients do not respond to current therapies. In part, this phenomenon has been associated with infusion of reduced numbers of early memory T cells. Herein, we report that AKT signaling inhibition is compatible with CAR and TCR retroviral transduction of human T cells while promoting a CD62L-expressing central memory phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT signaling preserved MAPK activation and promoted the intranuclear localization of FOXO1, a transcriptional regulator of T cell memory. Consequently, AKT signaling inhibition synchronized the transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKT signaling inhibition, while addition of AKT signaling inhibition to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, treatment of established B cell acute lymphoblastic leukemia was superior using anti-CD19 CAR-modified T cells transduced and expanded in the presence of an AKT inhibitor compared with conventionally grown T cells. Thus, inhibition of signaling along the PI3K/AKT axis represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype and superior antitumor efficacy.
Assuntos
Imunoterapia Adotiva/métodos , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores de Antígenos Quiméricos/imunologia , Subpopulações de Linfócitos T/imunologia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Feminino , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Memória Imunológica , Selectina L/metabolismo , Ativação Linfocitária/imunologia , Camundongos Endogâmicos NOD , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/imunologia , Transdução Genética/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.
Assuntos
Genes Essenciais/genética , Imunoterapia , Neoplasias/genética , Neoplasias/terapia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Transferência Adotiva , Animais , Apresentação de Antígeno/genética , Apelina/metabolismo , Receptores de Apelina/genética , Receptores de Apelina/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Feminino , Genoma/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interferon gama/imunologia , Janus Quinase 1/metabolismo , Bases de Conhecimento , Melanoma/genética , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/terapia , Camundongos , Mutação , Neoplasias/imunologia , Neoplasias/metabolismo , Reprodutibilidade dos Testes , Linfócitos T Citotóxicos/metabolismoRESUMO
Tumours progress despite being infiltrated by tumour-specific effector T cells. Tumours contain areas of cellular necrosis, which are associated with poor survival in a variety of cancers. Here, we show that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumours, causing profound suppression of T cell effector function. Elevation of the extracellular potassium concentration ([K+]e) impairs T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programmes. Potassium-mediated suppression of Akt-mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A. Although the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it requires an increase in intracellular potassium ([K+]i). Accordingly, augmenting potassium efflux in tumour-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo and enhances tumour clearance and survival in melanoma-bearing mice. These results uncover an ionic checkpoint that blocks T cell function in tumours and identify potential new strategies for cancer immunotherapy.
Assuntos
Cátions Monovalentes/metabolismo , Melanoma/imunologia , Potássio/metabolismo , Linfócitos T/imunologia , Evasão Tumoral/imunologia , Microambiente Tumoral/imunologia , Animais , Humanos , Tolerância Imunológica/imunologia , Imunoterapia/métodos , Canal de Potássio Kv1.3/metabolismo , Masculino , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Potenciais da Membrana , Camundongos , Necrose , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Análise de Sobrevida , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Nitric oxide (NO) generated by inducible NO synthase (iNOS) is critical for defense against intracellular pathogens but may mediate inflammatory tissue damage. To elucidate the role of iNOS in neuroinflammation, infections with encephalitogenic Trypanosoma brucei parasites were compared in inos(-/-) and wild-type mice. Inos(-/-) mice showed enhanced brain invasion by parasites and T cells, and elevated protein permeability of cerebral vessels, but similar parasitemia levels. Trypanosome infection stimulated T cell- and TNF-mediated iNOS expression in perivascular macrophages. NO nitrosylated and inactivated pro-inflammatory molecules such as NF-κΒp65, and reduced TNF expression and signalling. iNOS-derived NO hampered both TNF- and T cell-mediated parasite brain invasion. In inos(-/-) mice, TNF stimulated MMP, including MMP9 activity that increased cerebral vessel permeability. Thus, iNOS-generated NO by perivascular macrophages, strategically located at sites of leukocyte brain penetration, can serve as a negative feed-back regulator that prevents unlimited influx of inflammatory cells by restoring the integrity of the blood-brain barrier.
Assuntos
Barreira Hematoencefálica/metabolismo , Encefalite/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Animais , Citocinas/metabolismo , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMO
Novel methods for treatment of African trypanosomiasis, caused by infection with Trypanosoma brucei are needed. Cordycepin (3'-deoxyadenosine, 1a) is a powerful trypanocidal compound in vitro but is ineffective in vivo because of rapid metabolic degradation by adenosine deaminase (ADA). We elucidated the structural moieties of cordycepin required for trypanocidal activity and designed analogues that retained trypanotoxicity while gaining resistance to ADA-mediated metabolism. 2-Fluorocordycepin (2-fluoro-3'-deoxyadenosine, 1b) was identified as a selective, potent, and ADA-resistant trypanocidal compound that cured T. brucei infection in mice. Compound 1b is transported through the high affinity TbAT1/P2 adenosine transporter and is a substrate of T. b. brucei adenosine kinase. 1b has good preclinical properties suitable for an oral drug, albeit a relatively short plasma half-life. We present a rapid and efficient synthesis of 2-halogenated cordycepins, also useful synthons for the development of additional novel C2-substituted 3'-deoxyadenosine analogues to be evaluated in development of experimental therapeutics.