RESUMEN
CD8+ T cells responding to chronic infections or tumors acquire an 'exhausted' state associated with elevated expression of inhibitory receptors, including PD-1, and impaired cytokine production. Exhausted T cells are continuously replenished by T cells with precursor characteristics that self-renew and depend on the transcription factor TCF1; however, their developmental requirements are poorly understood. In the present study, we demonstrate that high antigen load promoted the differentiation of precursor T cells, which acquired hallmarks of exhaustion within days of infection, whereas early effector cells retained polyfunctional features. Early precursor T cells showed epigenetic imprinting characteristic of T cell receptor-dependent transcription factor binding and were restricted to the generation of cells displaying exhaustion characteristics. Transcription factors BACH2 and BATF were key regulators with opposing functions in the generation of early precursor T cells. Overall, we demonstrate that exhaustion manifests first in TCF1+ precursor T cells and is propagated subsequently to the pool of antigen-specific T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Células Precursoras de Linfocitos T/inmunología , Animales , Diferenciación Celular , Autorrenovación de las Células , Células Cultivadas , Enfermedad Crónica , Anergia Clonal , Epigénesis Genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Tolerancia Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos TRESUMEN
Antigen-specific CD8+ T cells in chronic viral infections and tumors functionally deteriorate, a process known as exhaustion. Exhausted T cells are sustained by precursors of exhausted (Tpex) cells that self-renew while continuously generating exhausted effector (Tex) cells. However, it remains unknown how Tpex cells maintain their functionality. Here, we demonstrate that Tpex cells sustained mitochondrial fitness, including high spare respiratory capacity, while Tex cells deteriorated metabolically over time. Tpex cells showed early suppression of mTOR kinase signaling but retained the ability to activate this pathway in response to antigen receptor signals. Early transient mTOR inhibition improved long-term T cell responses and checkpoint inhibition. Transforming growth factor-ß repressed mTOR signaling in exhausted T cells and was a critical determinant of Tpex cell metabolism and function. Overall, we demonstrate that the preservation of cellular metabolism allows Tpex cells to retain long-term functionality to sustain T cell responses during chronic infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Metabolismo Energético/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Retículo Endoplásmico/metabolismo , Metabolismo Energético , Memoria Inmunológica , Mitocondrias/metabolismo , Transducción de Señal , Respiración de la Célula , Retículo Endoplásmico/ultraestructura , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucólisis , Membranas Intracelulares/metabolismo , Activación de Linfocitos , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Mitocondrias/ultraestructura , Modelos Biológicos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/deficienciaRESUMEN
Antigen-experienced memory T cells acquire effector function with innate-like kinetics; however, the metabolic requirements of these cells are unknown. Here we show that rapid interferon-γ (IFN-γ) production of effector memory (EM) CD8(+) T cells, activated through stimulation mediated by the T cell antigen receptor (TCR) and the costimulatory receptor CD28 or through cognate interactions, was linked to increased glycolytic flux. EM CD8(+) T cells exhibited more glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity at early time points, before proliferation commenced, than did naive cells activated under similar conditions. CD28 signaling via the serine-threonine kinase Akt and the metabolic-checkpoint kinase mTORC2 was needed to sustain TCR-mediated immediate-early glycolysis. Unlike glycolysis in proliferating cells, immediate-early glycolysis in memory CD8(+) T cells was rapamycin insensitive. Thus, CD8(+) memory T cells have an Akt-dependent 'imprinted' glycolytic potential that is required for efficient immediate-early IFN-γ recall responses.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Memoria Inmunológica , Ensamble y Desensamble de Cromatina , Epítopos de Linfocito T/inmunología , Glucólisis , Herpesvirus Humano 4/inmunología , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Activación de Linfocitos , Diana Mecanicista del Complejo 2 de la Rapamicina , Metaboloma , Metabolómica , Complejos Multiproteicos/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Tumor cells steal methionine from CD8 T cells. High expression of the methionine transporters SLC7A5 and SLC43A2 allows tumor cells to outcompete CD8 T cells for methionine uptake. Lower methionine concentrations in CD8 T cells lead to reduced levels of dimethylated H3K79, an active epigenetic mark, which in turn results in reduced STAT5 expression and activity.
Asunto(s)
Histonas , Neoplasias , Linfocitos T CD8-positivos/metabolismo , Humanos , Metionina/metabolismo , Metilación , Factor de Transcripción STAT5/metabolismoRESUMEN
The role of mitochondrial biogenesis during naïve to effector differentiation of CD8+ T cells remains ill explored. In this study, we describe a critical role for early mitochondrial biogenesis in supporting cytokine production of nascent activated human naïve CD8+ T cells. Specifically, we found that prior to the first round of cell division activated naïve CD8+ T cells rapidly increase mitochondrial mass, mitochondrial respiration, and mitochondrial reactive oxygen species (mROS) generation, which were all inter-linked and important for CD8+ T cell effector maturation. Inhibition of early mitochondrial biogenesis diminished mROS dependent IL-2 production - as well as subsequent IL-2 dependent TNF, IFN-γ, perforin, and granzyme B production. Together, these findings point to the importance of mitochondrial biogenesis during early effector maturation of CD8+ T cells.
Asunto(s)
Linfocitos T CD8-positivos/citología , Diferenciación Celular/inmunología , Mitocondrias/fisiología , Biogénesis de Organelos , Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Humanos , Interleucina-2/inmunología , Activación de Linfocitos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
ATP-binding cassette (ABC) transporters, including ABC-transporter B1 (ABCB1), extrude drugs, metabolites, and other compounds (such as mitotracker green (MTG)) from cells. Susceptibility of CD4(+) regulatory T (Treg) cells to the ABCB1-substrate cyclophosphamide (CPA) has been reported. Here, we characterized ABCB1 expression and function in human CD4(+) T-cell subsets. Naïve, central memory, and effector-memory CD4(+) T cells, but not Treg cells, effluxed MTG in an ABCB1-dependent manner. In line with this, ABCB1 mRNA and protein was expressed by nonregulatory CD4(+) T-cell subsets, but not Treg cells. In vitro, the ABCB1-substrate CPA was cytotoxic for Treg cells at a 100-fold lower dose than for nonregulatory counterparts, and, inversely, verapamil, an inhibitor of ABC transporters, increased CPA-toxicity in nonregulatory CD4(+) T cells but not Treg cells. Thus, Treg cells lack expression of ABCB1, rendering them selectively susceptible to CPA. Our findings provide mechanistic support for therapeutic strategies using CPA to boost anti-tumor immunity by selectively depleting Treg cells.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Apoptosis/efectos de los fármacos , Ciclofosfamida/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Antineoplásicos Alquilantes/farmacocinética , Apoptosis/inmunología , Ciclofosfamida/farmacocinética , Citotoxinas/farmacocinética , Citotoxinas/farmacología , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Depleción Linfocítica/métodos , Masculino , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/terapia , Linfocitos T Reguladores/citologíaRESUMEN
Tumor cells undergo uncontrolled proliferation driven by enhanced anabolic metabolism including glycolysis and glutaminolysis. Targeting these pathways to inhibit cancer growth is a strategy for cancer treatment. Critically, however, tumor-responsive T cells share metabolic features with cancer cells, making them susceptible to these treatments as well. Here, we assess the impact on anti-tumor T cell immunity and T cell exhaustion by genetic ablation of lactate dehydrogenase A (LDHA) and glutaminase1 (GLS1), key enzymes in aerobic glycolysis and glutaminolysis. Loss of LDHA severely impairs expansion of T cells in response to tumors and chronic infection. In contrast, T cells lacking GLS1 can compensate for impaired glutaminolysis by engaging alternative pathways, including upregulation of asparagine synthetase, and thus efficiently respond to tumor challenge and chronic infection as well as immune checkpoint blockade. Targeting GLS1-dependent glutaminolysis, but not aerobic glycolysis, may therefore be a successful strategy in cancer treatment, particularly in combination with immunotherapy.
RESUMEN
The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.
Asunto(s)
Quinasas de la Proteína-Quinasa Activada por el AMP , Adenocarcinoma del Pulmón , Linfocitos T CD8-positivos , Glutaminasa , Neoplasias Pulmonares , Quinasas de la Proteína-Quinasa Activada por el AMP/deficiencia , Quinasas de la Proteína-Quinasa Activada por el AMP/inmunología , Quinasas de la Proteína-Quinasa Activada por el AMP/metabolismo , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Glutaminasa/antagonistas & inhibidores , Glutaminasa/inmunología , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Activación de Linfocitos , Ratones , Mutación , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas p21(ras)/inmunología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Microambiente TumoralRESUMEN
Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is widely used for mRNA quantification. To accurately measure changing gene transcript levels under different experimental conditions, the use of appropriate reference gene transcripts is instrumental. In T cell immunology, suitable reference genes have been reported for bulk CD4+ and CD8+ T cells. However, many CD4+ and CD8+ T cell subsets have been described in the past. Although they respond differently to given activation stimuli, proper validation of suitable reference genes in these subsets is lacking. In this study, we evaluated twelve commonly used reference gene products in human naïve (NV) and effector memory (EM) CD8+ T cells under non-activated and activated (2 h, 10 h and 20 h) conditions. We used five different statistical approaches for data analysis. Our results show that a number of widely used reference transcripts become differentially expressed under activating conditions. Using them as references markedly alters results as exemplified with IFNG mRNA expression. The only candidate reference gene products that remained stable during the activation process were 18S rRNA and SDHA mRNA, encouraging their usage as reference gene products for RT-qPCR experiments, when quantifying mRNA levels in human NV and EM CD8+ T cells.