RESUMO
Poor tumor infiltration, development of exhaustion, and antigen insufficiency are common mechanisms that limit chimeric antigen receptor (CAR)-T cell efficacy. Delivery of pattern recognition receptor agonists is one strategy to improve immune function; however, targeting these agonists to immune cells is challenging, and off-target signaling in cancer cells can be detrimental. Here, we engineer CAR-T cells to deliver RN7SL1, an endogenous RNA that activates RIG-I/MDA5 signaling. RN7SL1 promotes expansion and effector-memory differentiation of CAR-T cells. Moreover, RN7SL1 is deployed in extracellular vesicles and selectively transferred to immune cells. Unlike other RNA agonists, transferred RN7SL1 restricts myeloid-derived suppressor cell (MDSC) development, decreases TGFB in myeloid cells, and fosters dendritic cell (DC) subsets with costimulatory features. Consequently, endogenous effector-memory and tumor-specific T cells also expand, allowing rejection of solid tumors with CAR antigen loss. Supported by improved endogenous immunity, CAR-T cells can now co-deploy peptide antigens with RN7SL1 to enhance efficacy, even when heterogenous CAR antigen tumors lack adequate neoantigens.
Assuntos
Fatores Imunológicos/farmacologia , RNA/farmacologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Antígenos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Proteína DEAD-box 58/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Imunidade/efeitos dos fármacos , Imunocompetência , Memória Imunológica , Imunoterapia , Interferons/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Peptídeos/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Linfócitos T/efeitos dos fármacosRESUMO
Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach, intratumoral TSA-reactive CD4+, CD8+ T cells, and TSA-suppressive CD4+ T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype, TSA-reactive CD8+ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle, FucoID should have the potential of accelerating the pace of personalized cancer treatment.
Assuntos
Antígenos de Neoplasias/metabolismo , Comunicação Celular , Fucose/metabolismo , Linfócitos T/imunologia , Linfócitos T/patologia , Adulto , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais/metabolismo , Biotinilação , Efeito Espectador/imunologia , Linfócitos T CD8-Positivos/imunologia , Membrana Celular/metabolismo , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Fucosiltransferases/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Helicobacter pylori/enzimologia , Humanos , Imunidade , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Peptídeos/química , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Baço/metabolismoRESUMO
Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.
Assuntos
Inibidores de Checkpoint Imunológico/uso terapêutico , Imunidade , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Nanotecnologia , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Animais , Comportamento Animal , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Proliferação de Células/efeitos dos fármacos , Colesterol/metabolismo , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Imunidade/efeitos dos fármacos , Imunoterapia , Lipoproteínas HDL/metabolismo , Camundongos Endogâmicos C57BL , Primatas , Distribuição Tecidual/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Mammals evolved in the face of fluctuating food availability. How the immune system adapts to transient nutritional stress remains poorly understood. Here, we show that memory T cells collapsed in secondary lymphoid organs in the context of dietary restriction (DR) but dramatically accumulated within the bone marrow (BM), where they adopted a state associated with energy conservation. This response was coordinated by glucocorticoids and associated with a profound remodeling of the BM compartment, which included an increase in T cell homing factors, erythropoiesis, and adipogenesis. Adipocytes, as well as CXCR4-CXCL12 and S1P-S1P1R interactions, contributed to enhanced T cell accumulation in BM during DR. Memory T cell homing to BM during DR was associated with enhanced protection against infections and tumors. Together, this work uncovers a fundamental host strategy to sustain and optimize immunological memory during nutritional challenges that involved a temporal and spatial reorganization of the memory pool within "safe haven" compartments.
Assuntos
Medula Óssea/metabolismo , Memória Imunológica , Animais , Medula Óssea/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Restrição Calórica/veterinária , Linhagem Celular Tumoral , Quimiocina CXCL12/metabolismo , Dieta Redutora/veterinária , Metabolismo Energético , Regulação da Expressão Gênica , Glucocorticoides , Melanoma Experimental/mortalidade , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Taxa de Sobrevida , Linfócitos T/imunologia , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Differentiation of proinflammatory CD4+ conventional T cells (Tconv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4+ Tconv, but then fail to support antitumor CD4+ Tconv differentiation. Regulatory T cell (Treg) depletion enhanced their capacity to elicit strong CD4+ Tconv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to Treg predicts protective ICOS+ PD-1lo CD4+ Tconv phenotypes and survival. Further, in melanoma patients with low Treg abundance, intratumoral cDC2 density alone correlates with abundant CD4+ Tconv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4+ Tconv abundance and controls tumor growth.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Toxina Diftérica/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Ativação Linfocitária , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Quimiocinas/metabolismo , Linfócitos T Reguladores/imunologia , Microambiente TumoralRESUMO
Cancer and chronic infections induce T cell exhaustion, a hypofunctional fate carrying distinct epigenetic, transcriptomic and metabolic characteristics. However, drivers of exhaustion remain poorly understood. As intratumoral exhausted T cells experience severe hypoxia, we hypothesized that metabolic stress alters their responses to other signals, specifically, persistent antigenic stimulation. In vitro, although CD8+ T cells experiencing continuous stimulation or hypoxia alone differentiated into functional effectors, the combination rapidly drove T cell dysfunction consistent with exhaustion. Continuous stimulation promoted Blimp-1-mediated repression of PGC-1α-dependent mitochondrial reprogramming, rendering cells poorly responsive to hypoxia. Loss of mitochondrial function generated intolerable levels of reactive oxygen species (ROS), sufficient to promote exhausted-like states, in part through phosphatase inhibition and the consequent activity of nuclear factor of activated T cells. Reducing T cell-intrinsic ROS and lowering tumor hypoxia limited T cell exhaustion, synergizing with immunotherapy. Thus, immunologic and metabolic signaling are intrinsically linked: through mitigation of metabolic stress, T cell differentiation can be altered to promote more functional cellular fates.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Metabolismo Energético , Ativação Linfocitária , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/metabolismo , Mitocôndrias/metabolismo , Microambiente Tumoral , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Células HEK293 , Humanos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/imunologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Hipóxia TumoralRESUMO
Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.
Assuntos
Linfócitos T CD8-Positivos/enzimologia , Neoplasias do Colo/enzimologia , Metabolismo Energético , Ativação Linfocitária , Linfócitos do Interstício Tumoral/enzimologia , Melanoma Experimental/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Citotoxicidade Imunológica , Estabilidade Enzimática , Feminino , Glucosefosfato Desidrogenase/metabolismo , Glicólise , Hexoquinase/genética , Hexoquinase/metabolismo , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/transplante , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADP/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Microambiente Tumoral , Quinase Induzida por NF-kappaBRESUMO
CD8+ T cell exhaustion dampens antitumor immunity. Although several transcription factors have been identified that regulate T cell exhaustion, the molecular mechanisms by which CD8+ T cells are triggered to enter an exhausted state remain unclear. Here, we show that interleukin-2 (IL-2) acts as an environmental cue to induce CD8+ T cell exhaustion within tumor microenvironments. We find that a continuously high level of IL-2 leads to the persistent activation of STAT5 in CD8+ T cells, which in turn induces strong expression of tryptophan hydroxylase 1, thus catalyzing the conversion to tryptophan to 5-hydroxytryptophan (5-HTP). 5-HTP subsequently activates AhR nuclear translocation, causing a coordinated upregulation of inhibitory receptors and downregulation of cytokine and effector-molecule production, thereby rendering T cells dysfunctional in the tumor microenvironment. This molecular pathway is not only present in mouse tumor models but is also observed in people with cancer, identifying IL-2 as a novel inducer of T cell exhaustion.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Interleucina-2/metabolismo , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Neoplasias/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Microambiente Tumoral , 5-Hidroxitriptofano/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Interleucina-2/antagonistas & inibidores , Interleucina-2/genética , Células Jurkat , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Células MCF-7 , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais , Triptofano Hidroxilase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metabolic programming controls immune cell lineages and functions, but little is known about γδ T cell metabolism. Here, we found that γδ T cell subsets making either interferon-γ (IFN-γ) or interleukin (IL)-17 have intrinsically distinct metabolic requirements. Whereas IFN-γ+ γδ T cells were almost exclusively dependent on glycolysis, IL-17+ γδ T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17+ γδ T cells selectively showed high lipid uptake and intracellular lipid storage and were expanded in obesity and in tumors of obese mice. Conversely, glucose supplementation enhanced the antitumor functions of IFN-γ+ γδ T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector γδ T cells and their manipulation in cancer immunotherapy.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias do Colo/metabolismo , Metabolismo Energético , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/metabolismo , Timo/metabolismo , Microambiente Tumoral , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Linhagem da Célula , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Feminino , Glucose/metabolismo , Glicólise , Humanos , Imunoterapia Adotiva , Interferon gama/metabolismo , Interleucina-17/metabolismo , Metabolismo dos Lipídeos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/transplante , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Obesidade/imunologia , Obesidade/metabolismo , Técnicas de Cultura de Órgãos , Fenótipo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/transplante , Timo/imunologia , Carga TumoralRESUMO
Identifying signals in the tumor microenvironment (TME) that shape CD8+ T cell phenotype can inform novel therapeutic approaches for cancer. Here, we identified a gradient of increasing glucocorticoid receptor (GR) expression and signaling from naïve to dysfunctional CD8+ tumor-infiltrating lymphocytes (TILs). Conditional deletion of the GR in CD8+ TILs improved effector differentiation, reduced expression of the transcription factor TCF-1, and inhibited the dysfunctional phenotype, culminating in tumor growth inhibition. GR signaling transactivated the expression of multiple checkpoint receptors and promoted the induction of dysfunction-associated genes upon T cell activation. In the TME, monocyte-macrophage lineage cells produced glucocorticoids and genetic ablation of steroidogenesis in these cells as well as localized pharmacologic inhibition of glucocorticoid biosynthesis improved tumor growth control. Active glucocorticoid signaling associated with failure to respond to checkpoint blockade in both preclinical models and melanoma patients. Thus, endogenous steroid hormone signaling in CD8+ TILs promotes dysfunction, with important implications for cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Glucocorticoides/metabolismo , Macrófagos/metabolismo , Melanoma Experimental/patologia , Microambiente Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linhagem Celular Tumoral , Hematopoese/imunologia , Fator 1-alfa Nuclear de Hepatócito/biossíntese , Inibidores de Checkpoint Imunológico , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/imunologiaRESUMO
The primary mechanisms supporting immunoregulatory polarization of myeloid cells upon infiltration into tumors remain largely unexplored. Elucidation of these signals could enable better strategies to restore protective anti-tumor immunity. Here, we investigated the role of the intrinsic activation of the PKR-like endoplasmic reticulum (ER) kinase (PERK) in the immunoinhibitory actions of tumor-associated myeloid-derived suppressor cells (tumor-MDSCs). PERK signaling increased in tumor-MDSCs, and its deletion transformed MDSCs into myeloid cells that activated CD8+ T cell-mediated immunity against cancer. Tumor-MDSCs lacking PERK exhibited disrupted NRF2-driven antioxidant capacity and impaired mitochondrial respiratory homeostasis. Moreover, reduced NRF2 signaling in PERK-deficient MDSCs elicited cytosolic mitochondrial DNA elevation and, consequently, STING-dependent expression of anti-tumor type I interferon. Reactivation of NRF2 signaling, conditional deletion of STING, or blockade of type I interferon receptor I restored the immunoinhibitory potential of PERK-ablated MDSCs. Our findings demonstrate the pivotal role of PERK in tumor-MDSC functionality and unveil strategies to reprogram immunosuppressive myelopoiesis in tumors to boost cancer immunotherapy.
Assuntos
Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Epitelial do Ovário/imunologia , Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/imunologia , Proteínas de Membrana/imunologia , Neoplasias Cutâneas/imunologia , eIF-2 Quinase/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/patologia , Feminino , Humanos , Terapia de Imunossupressão , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/patologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/imunologia , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Resposta a Proteínas não Dobradas/imunologia , eIF-2 Quinase/deficiência , eIF-2 Quinase/genéticaRESUMO
The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour growth1,2. Here, using high-throughput flow cytometry as well as bulk and single-cell RNA-sequencing and B-cell-receptor-sequencing analysis of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumour-bearing mice. The expanding B cell subset expresses the cell surface molecule T cell immunoglobulin and mucin domain 1 (TIM-1, encoded by Havcr1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. Although conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumour burden, selective deletion of Havcr1 in B cells both substantially inhibited tumour growth and enhanced effector T cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumour-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing B cells enables engagement of the second arm of adaptive immunity to promote anti-tumour immunity and inhibit tumour growth.
Assuntos
Linfócitos B , Melanoma , Animais , Camundongos , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Ativação Linfocitária , Melanoma/imunologia , Melanoma/patologia , Melanoma/prevenção & controle , Linfócitos T/citologia , Linfócitos T/imunologia , Citometria de Fluxo , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Linfonodos/citologia , Linfonodos/imunologia , Apresentação de Antígeno , Receptores de Antígenos de Linfócitos B/genética , Análise da Expressão Gênica de Célula Única , Carga Tumoral , Interferon Tipo IRESUMO
Missense mutations of the tumor suppressor Neurofibromin 2 (NF2/Merlin/schwannomin) result in sporadic to frequent occurrences of tumorigenesis in multiple organs. However, the underlying pathogenicity of NF2-related tumorigenesis remains mostly unknown. Here we found that NF2 facilitated innate immunity by regulating YAP/TAZ-mediated TBK1 inhibition. Unexpectedly, patient-derived individual mutations in the FERM domain of NF2 (NF2m) converted NF2 into a potent suppressor of cGAS-STING signaling. Mechanistically, NF2m gained extreme associations with IRF3 and TBK1 and, upon innate nucleic acid sensing, was directly induced by the activated IRF3 to form cellular condensates, which contained the PP2A complex, to eliminate TBK1 activation. Accordingly, NF2m robustly suppressed STING-initiated antitumor immunity in cancer cell-autonomous and -nonautonomous murine models, and NF2m-IRF3 condensates were evident in human vestibular schwannomas. Our study reports phase separation-mediated quiescence of cGAS-STING signaling by a mutant tumor suppressor and reveals gain-of-function pathogenesis for NF2-related tumors by regulating antitumor immunity.
Assuntos
Imunidade Inata , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Neoplasias/metabolismo , Neurofibromina 2/metabolismo , Nucleotidiltransferases/metabolismo , Evasão Tumoral , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neurofibromina 2/genética , Nucleotidiltransferases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems1-5. Whether the neo-innervation of tumours by pain-initiating sensory neurons affects cancer immunosurveillance remains unclear. Here we show that melanoma cells interact with nociceptor neurons, leading to increases in their neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. Calcitonin gene-related peptide (CGRP)-one such nociceptor-produced neuropeptide-directly increases the exhaustion of cytotoxic CD8+ T cells, which limits their capacity to eliminate melanoma. Genetic ablation of the TRPV1 lineage, local pharmacological silencing of nociceptors and antagonism of the CGRP receptor RAMP1 all reduced the exhaustion of tumour-infiltrating leukocytes and decreased the growth of tumours, nearly tripling the survival rate of mice that were inoculated with B16F10 melanoma cells. Conversely, CD8+ T cell exhaustion was rescued in sensory-neuron-depleted mice that were treated with local recombinant CGRP. As compared with wild-type CD8+ T cells, Ramp1-/- CD8+ T cells were protected against exhaustion when co-transplanted into tumour-bearing Rag1-deficient mice. Single-cell RNA sequencing of biopsies from patients with melanoma revealed that intratumoral RAMP1-expressing CD8+ T cells were more exhausted than their RAMP1-negative counterparts, whereas overexpression of RAMP1 correlated with a poorer clinical prognosis. Overall, our results suggest that reducing the release of CGRP from tumour-innervating nociceptors could be a strategy to improve anti-tumour immunity by eliminating the immunomodulatory effects of CGRP on cytotoxic CD8+ T cells.
Assuntos
Linfócitos T CD8-Positivos , Melanoma , Nociceptores , Animais , Camundongos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Melanoma/imunologia , Melanoma/patologia , Nociceptores/fisiologia , Células Receptoras Sensoriais/metabolismo , Neuritos/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Taxa de Sobrevida , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Genes RAG-1/genética , Humanos , Biópsia , PrognósticoRESUMO
CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.
Assuntos
Cisteína Endopeptidases/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Colite/imunologia , Colite/patologia , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/patologia , Vírus da Coriomeningite Linfocítica/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/citologia , Células Th1/citologiaRESUMO
Circular RNAs (circRNAs) are prevalent in eukaryotic cells and viral genomes. Mammalian cells possess innate immunity to detect foreign circRNAs, but the molecular basis of self versus foreign identity in circRNA immunity is unknown. Here, we show that N6-methyladenosine (m6A) RNA modification on human circRNAs inhibits innate immunity. Foreign circRNAs are potent adjuvants to induce antigen-specific T cell activation, antibody production, and anti-tumor immunity in vivo, and m6A modification abrogates immune gene activation and adjuvant activity. m6A reader YTHDF2 sequesters m6A-circRNA and is essential for suppression of innate immunity. Unmodified circRNA, but not m6A-modified circRNA, directly activates RNA pattern recognition receptor RIG-I in the presence of lysine-63-linked polyubiquitin chain to cause filamentation of the adaptor protein MAVS and activation of the downstream transcription factor IRF3. CircRNA immunity has considerable parallel to prokaryotic DNA restriction modification system that transforms nucleic acid chemical modification into organismal innate immunity.
Assuntos
Adenosina/análogos & derivados , Imunidade Inata , Melanoma Experimental/terapia , RNA Circular/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina/administração & dosagem , Adenosina/imunologia , Adenosina/metabolismo , Adjuvantes Imunológicos/administração & dosagem , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Imunização , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferons/imunologia , Interferons/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Poliubiquitina/imunologia , Poliubiquitina/metabolismo , Multimerização Proteica , RNA Circular/administração & dosagem , RNA Circular/metabolismo , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/metabolismo , Receptores Imunológicos , UbiquitinaçãoRESUMO
Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.
Assuntos
Antineoplásicos/farmacologia , Antígeno B7-H1/genética , Antígeno CTLA-4/genética , Regulação Neoplásica da Expressão Gênica , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/imunologia , Animais , Antígeno B7-H1/imunologia , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Feminino , Glicosilação , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/imunologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos NOD , Fosforilação , Serina/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaRESUMO
Interferon-γ (IFNγ) is a critical antitumor cytokine that has varied effects on different cell types. The global effect of IFNγ in the tumor depends on which cells it acts upon and the spatial extent of its spread. Reported measurements of IFNγ spread vary dramatically in different contexts, ranging from nearest-neighbor signaling to perfusion throughout the entire tumor. Here, we apply theoretical considerations to experiments both in vitro and in vivo to study the spread of IFNγ in melanomas. We observe spatially confined niches of IFNγ signaling in 3-D mouse melanoma cultures and human tumors that generate cellular heterogeneity in gene expression and alter the susceptibility of affected cells to T cell killing. Widespread IFNγ signaling only occurs when niches overlap due to high local densities of IFNγ-producing T cells. We measured length scales of ~30 to 40 µm for IFNγ spread in B16 mouse melanoma cultures and human primary cutaneous melanoma. Our results are consistent with IFNγ spread being governed by a simple diffusion-consumption model and offer insight into how the spatial organization of T cells contributes to intratumor heterogeneity in inflammatory signaling, gene expression, and immune-mediated clearance. Solid tumors are often viewed as collections of diverse cellular "neighborhoods": Our work provides a general explanation for such nongenetic cellular variability due to confinement in the spread of immune mediators.
Assuntos
Interferon gama , Melanoma Experimental , Neoplasias Cutâneas , Animais , Humanos , Camundongos , Interferon gama/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Transdução de Sinais , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Técnicas de Cultura de CélulasRESUMO
Cytotoxic CD8+ T cells are central to the antitumor immune response by releasing cytotoxic granules that kill tumor cells. They are activated by antigen-presenting cells, which become activated by DAMPs (damage associated molecular patterns) through MyD88. However, the suppressive tumor microenvironment promotes T-cell tolerance to tumor antigens, in part by enhancing the activity of immune checkpoint molecules that prevent CD8+ T-cell activation and cytotoxicity. MyD88 limits CD4+ T-cell activation during cardiac adaptation to stress. A similar mechanism is hypothesized to exist in CD8+ T cells that could be modulated to improve antitumor immunity. Herein, adoptive transfer of MyD88-/- CD8+ T cells in melanoma-bearing T-cell-deficient mice resulted in slower tumor growth, greater intratumoral T-cell accumulation, and higher melanoma cell death compared with transfer of wild-type CD8+ T cells. These findings were also observed in T-cell-specific MyD88-/- mice compared with wild-type littermates implanted with melanoma. Mechanistically, deletion of MyD88 enhanced CD8+ T-cell activation and survival, and T-cell receptor induced degranulation of cytotoxic molecules, overall improving the killing of melanoma cells. This enhanced cytotoxicity was retained in mice bearing tumors expressing the specific antigen for which cytotoxic T-cells were restricted. This study's results demonstrate a conserved mechanism for MyD88 in modulating CD8+ T-cell activation and represent a novel target in improving cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos , Fator 88 de Diferenciação Mieloide , Animais , Camundongos , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Melanoma/imunologia , Melanoma/patologia , Melanoma/genética , Melanoma/terapia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Melanoma Experimental/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Microambiente Tumoral/imunologiaRESUMO
Cytolytic activity by CD8(+) cytotoxic T lymphocytes (CTLs) is a powerful strategy for the elimination of intracellular pathogens and tumor cells. The destructive capacity of CTLs is progressively dampened during chronic infection, yet the environmental cues and molecular pathways that influence immunological 'exhaustion' remain unclear. Here we found that CTL immunity was regulated by the central transcriptional response to hypoxia, which is controlled in part by hypoxia-inducible factors (HIFs) and the von Hippel-Lindau tumor suppressor VHL. Loss of VHL, the main negative regulator of HIFs, led to lethal CTL-mediated immunopathology during chronic infection, and VHL-deficient CTLs displayed enhanced control of persistent viral infection and neoplastic growth. We found that HIFs and oxygen influenced the expression of pivotal transcription, effector and costimulatory-inhibitory molecules of CTLs, which was relevant to strategies that promote the clearance of viruses and tumors.