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The inhibitory receptor PD-1 suppresses T cell activation by recruiting the phosphatase SHP-2. However, mice with a T-cell-specific deletion of SHP-2 do not have improved antitumor immunity. Here we showed that mice with conditional targeting of SHP-2 in myeloid cells, but not in T cells, had diminished tumor growth. RNA sequencing (RNA-seq) followed by gene set enrichment analysis indicated the presence of polymorphonuclear myeloid-derived suppressor cells and tumor-associated macrophages (TAMs) with enriched gene expression profiles of enhanced differentiation, activation and expression of immunostimulatory molecules. In mice with conditional targeting of PD-1 in myeloid cells, which also displayed diminished tumor growth, TAMs had gene expression profiles enriched for myeloid differentiation, activation and leukocyte-mediated immunity displaying >50% overlap with enriched profiles of SHP-2-deficient TAMs. In bone marrow, GM-CSF induced the phosphorylation of PD-1 and recruitment of PD-1-SHP-2 to the GM-CSF receptor. Deletion of SHP-2 or PD-1 enhanced GM-CSF-mediated phosphorylation of the transcription factors HOXA10 and IRF8, which regulate myeloid differentiation and monocytic-moDC lineage commitment, respectively. Thus, SHP-2 and PD-1-SHP-2 signaling restrained myelocyte differentiation resulting in a myeloid landscape that suppressed antitumor immunity.
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Fator Estimulador de Colônias de Granulócitos e Macrófagos , Neoplasias , Animais , Camundongos , Diferenciação Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Células Mieloides , Receptor de Morte Celular Programada 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Transdução de SinaisRESUMO
In the version of this article initially published, the author surname citing the linked article (Miyama) was incorrect in the seventh and eighth paragraphs. The correct name is Miyajima.
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During the past decade there has been a revolution in cancer therapeutics by the emergence of antibody-based and cell-based immunotherapies that modulate immune responses against tumors. These new therapies have extended and improved the therapeutic efficacy of chemo-radiotherapy and have offered treatment options to patients who are no longer responding to these classic anti-cancer treatments. Unfortunately, tumor eradication and long-lasting responses are observed in a small fraction of patients, whereas the majority of patients respond only transiently. These outcomes indicate that the maximum potential of immunotherapy has not been reached due to incomplete knowledge of the cellular and molecular mechanisms that guide the development of successful anti-tumor immunity and its failure. In this review, we discuss recent discoveries about the immune cellular composition of the tumor microenvironment (TME) and the role of key signaling mechanisms that compromise the function of immune cells leading to cancer immune escape.
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Neoplasias , Microambiente Tumoral , Humanos , Imunoterapia , Neoplasias/patologia , Transdução de Sinais , Fatores ImunológicosRESUMO
Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.
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Antígeno B7-H1/genética , Imunossupressores/farmacologia , Multimerização Proteica , Splicing de RNA , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/química , Antígeno B7-H1/farmacologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Células Supressoras Mieloides/fisiologia , Placenta/metabolismo , Gravidez , Microambiente TumoralRESUMO
Graft versus host disease (GvHD), mediated by donor T cells, remains the primary cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation and novel therapeutic approaches are required. Cdk2 is a critical node of signal integration and programming of T cell responses towards immunity versus anergy but is dispensable for hematopoiesis and thymocyte development. We examined the effects of pharmacologic Cdk2 inhibition on alloreactive human T cells. Inhibition of Cdk2 blocked expansion of alloreactive T cells upon culture with HLA-mismatched dendritic cells and prevented generation of IFN-γ-producing alloantigen-specific effectors. In contrast, Cdk2 inhibition preserved effectors specific for Wilms' tumor 1 (WT1) leukemia antigen and for CMV as determined by WT1-specific and CMV-specific pentamers. Cdk2 inhibition preserved Treg cells, which have the ability to prevent GvHD while maintaining GvL. Thus, Cdk inhibitors may improve allogeneic HSCT by reducing alloreactivity and GvHD without loss of pathogen-specific and leukemia-specific immunity.
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Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Doença Enxerto-Hospedeiro/imunologia , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Linfócitos T Reguladores/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas , Humanos , Interferon gama/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Complexo Repressor Polycomb 2/biossíntese , Complexo Repressor Polycomb 2/imunologia , Roscovitina , Linfócitos T Reguladores/efeitos dos fármacos , Transplante Homólogo , Proteínas WT1/imunologiaRESUMO
Cell-based immunotherapy is a promising approach to cancer treatment. However, the metabolically hostile tumor microenvironment (TME) poses a major barrier to this therapeutic approach. Metabolic reprogramming may enhance T cell effector function and support longevity and persistence within the TME. Metabolic processes lead reactive oxygen species (ROS) production, which are mandatory mediators of signaling and immune cell functions, but detrimental when present in excess. Catalase (CAT) is an intracellular antioxidant enzyme that scavenges hydrogen peroxide (H2 O2 ), a central ROS member with a plethora of biological effects. H2 O2 is produced intracellularly and extracellularly, diffusing freely between the two compartments. In this study, it is found that scavenging extracellular H2 O2 by CAT supplementation has a major impact on the cell redox state, decreased intracellular ROS, but enhanced activation and altered memory differentiation. Under in vitro chronic activation conditions, CAT treatment favors CD8 T cells with less exhausted phenotype, increased activation and memory markers, and high bioenergetic capacity. Under in vitro acute activation conditions, CAT treatment selectively prevents differentiation transition from the stem cell memory/naive (TSCM /TN )- to the central memory (TCM )-like phenotype, while enhancing activation and polyfunctionality. The study highlights the critical role of H2 O2 as a "hidden player" in T cell fitness and memory differentiation.
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Antioxidantes , Linfócitos T CD8-Positivos , Catalase/metabolismo , Espécies Reativas de Oxigênio , Linfócitos T CD8-Positivos/metabolismo , Antioxidantes/metabolismo , Diferenciação CelularRESUMO
Programmed Death-1 (PD-1; CD279) is an inhibitory receptor induced in several activated immune cells and, after engagement with its ligands PD-L1 and PD-L2, serves as a key mediator of peripheral tolerance. However, PD-1 signaling also has detrimental effects on T cell function by posing breaks on antitumor and antiviral immunity. PD-1 blocking immunotherapy either alone or in combination with other therapeutic modalities has shown great promise in cancer treatment. However, it is unclear why only a small fraction of patients responds to this type of therapy. For this reason, efforts to better understand the mechanisms of PD-1 function have recently been intensified, with the goal to reveal new strategies to overcome current limitations. The signaling pathways that are inhibited by PD-1 impact key regulators of metabolism. Here, we provide an overview of the current knowledge about the effects of PD-1 on metabolic reprogramming of immune cells and their consequences on systemic metabolism.
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Leukocytes, the leading players of immune system, are involved in innate and adaptive immune responses. Leukocyte adhesion to endothelial cells during transmigration or to antigen presenting cells during T cell activation, requires integrin activation through a process termed inside-out integrin signaling. In hematopoietic cells, Rap1 and its downstream effector RIAM (Rap1-interacting adaptor molecule) form a cornerstone for inside-out integrin activation. The Rap1/RIAM pathway is involved in signal integration for activation, actin remodeling and cytoskeletal reorganization in T cells, as well as in myeloid cell differentiation and function. RIAM is instrumental for phagocytosis, a process requiring particle recognition, cytoskeletal remodeling and membrane protrusion for engulfment and digestion. In the present review, we discuss the structural and molecular properties of RIAM and the recent discoveries regarding the functional role of the Rap1/RIAM module in hematopoietic cells.
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Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Membrana/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adesão Celular , Células Endoteliais/metabolismo , Humanos , Integrinas/metabolismo , Proteínas de Membrana/metabolismoRESUMO
T cell activation is a fine-tuned process that involves T cell receptor and costimulation signals. To prevent undue activation of T cells, inhibitory molecules including PD-1 (programmed death 1) are induced and function as brakes for T cell signaling. In a steady state, the interaction of PD-1 with its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) maintains peripheral immune tolerance. However, the expression of PD-L1 on tumor cells and interaction with PD-1 on T cells dampen anti-tumor immunity. Therapeutic inhibitors of the PD-1 pathway have revolutionized tumor immunotherapy. Unfortunately, the majority of patients do not develop sustained anti-tumor responses. However, the knowledge about unique PD-1 interactions and their role in mediating PD-1 inhibitory signals is currently limited. Advances in the mechanistic understanding of the molecular and signaling integration of the PD-1 pathway could unleash the great potential in tumor immunotherapy by allowing the development of combinatorial approaches that target not only PD-1 and its ligands but also its unique downstream signal mediators. In this review, the current advances in understanding the mechanisms of extracellular and intracellular PD-1 interactions and their significance in potential future therapeutic approaches are discussed.
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Ativação Linfocitária , Receptor de Morte Celular Programada 1 , Humanos , Imunoterapia , Ligantes , Linfócitos TRESUMO
Programmed Death-1 (PD-1; CD279) is an inhibitory receptor induced in activated T cells. PD-1 engagement by its ligands, PD-L1 and PD-L2, maintains peripheral tolerance but also compromises anti-tumor immunity. Blocking antibodies against PD-1 or its ligands have revolutionized cancer immunotherapy. However, only a fraction of patients develop durable antitumor responses. Clinical outcomes have reached a plateau without substantial advances by combinatorial approaches. Thus, great interest has recently emerged to investigate, in depth, the mechanisms by which the PD-1 pathway transmits inhibitory signals with the goal to identify molecular targets for improvement of the therapeutic success. These efforts have revealed unpredictable dimensions of the pathway and uncovered novel mechanisms involved in PD-1 and PD-L1 regulation and function. Here, we provide an overview of the recent advances on the mechanistic aspects of the PD-1 pathway and discuss the implications of these new discoveries and the gaps that remain to be filled.
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Immune checkpoint therapies aiming to enhance T cell responses have revolutionized cancer immunotherapy. However, although a small fraction of patients develops durable anti-tumor responses, the majority of patients display only transient responses, underlying the need for finding auxiliary approaches. Tumor microenvironment poses a major metabolic barrier to efficient anti-tumor T cell activity. As it is now well accepted that metabolism regulates T cell fate and function, harnessing metabolism may be a new strategy to potentiate T cell-based immunotherapies.
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PD-1, a T cell checkpoint receptor and target of cancer immunotherapy, is also expressed on myeloid cells. The role of myeloid-specific versus T cell-specific PD-1 ablation on antitumor immunity has remained unclear because most studies have used either PD-1-blocking antibodies or complete PD-1 KO mice. We generated a conditional allele, which allowed myeloid-specific (PD-1f/fLysMcre) or T cell-specific (PD-1f/fCD4cre) targeting of Pdcd1 gene. Compared with T cell-specific PD-1 ablation, myeloid cell-specific PD-1 ablation more effectively decreased tumor growth. We found that granulocyte/macrophage progenitors (GMPs), which accumulate during cancer-driven emergency myelopoiesis and give rise to myeloid-derived suppressor cells (MDSCs), express PD-1. In tumor-bearing PD-1f/fLysMcre but not PD-1f/fCD4cre mice, accumulation of GMP and MDSC was prevented, whereas systemic output of effector myeloid cells was increased. Myeloid cell-specific PD-1 ablation induced an increase of T effector memory cells with improved functionality and mediated antitumor protection despite preserved PD-1 expression in T cells. In PD-1-deficient myeloid progenitors, growth factors driving emergency myelopoiesis induced increased metabolic intermediates of glycolysis, pentose phosphate pathway, and TCA cycle but, most prominently, elevated cholesterol. Because cholesterol is required for differentiation of inflammatory macrophages and DC and promotes antigen-presenting function, our findings indicate that metabolic reprogramming of emergency myelopoiesis and differentiation of effector myeloid cells might be a key mechanism of antitumor immunity mediated by PD-1 blockade.
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Neoplasias do Colo/imunologia , Melanoma/imunologia , Células Mieloides/imunologia , Receptor de Morte Celular Programada 1/imunologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/genéticaRESUMO
Programmed cell death-1 (PD-1) inhibits T cell responses. This function relies on interaction with SHP-2. PD-1 has one immunoreceptor tyrosine-based inhibitory motif (ITIM) at Y223 and one immunoreceptor tyrosine-based switch motif (ITSM) at Y248. Only ITSM-Y248 is indispensable for PD-1-mediated inhibitory function but how SHP-2 enzymatic activation is mechanistically regulated by one PD-1 phosphotyrosine remains a puzzle. We found that after PD-1 phosphorylation, SHP-2 can bridge phosphorylated ITSM-Y248 residues on two PD-1 molecules via its amino terminal (N)-SH2 and carboxyterminal (C)-SH2 domains forming a PD-1: PD-1 dimer in live cells. The biophysical ability of SHP-2 to interact with two ITSM-pY248 residues was documented by isothermal titration calorimetry. SHP-2 interaction with two ITSM-pY248 phosphopeptides induced robust enzymatic activation. Our results unravel a mechanism of PD-1: SHP-2 interaction that depends only on ITSM-Y248 and explain how a single docking site within the PD-1 cytoplasmic tail can activate SHP-2 and PD-1-mediated inhibitory function.
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Receptor de Morte Celular Programada 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Linfócitos T/enzimologia , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática , Células HEK293 , Humanos , Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Células Jurkat , Camundongos Knockout , Fosforilação , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/genética , Ligação Proteica , Multimerização Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Domínios de Homologia de srcRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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PD-1 is a target of cancer immunotherapy but responses are limited to a fraction of patients. Identifying patients with T cells subjected to PD-1-mediated inhibition will allow selection of suitable candidates for PD-1-blocking therapy and will improve the therapeutic success. We sought to develop an approach to detect PD-1-mediated inhibitory signaling. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) encompassing Y223 and an immunoreceptor tyrosine-based switch motif (ITSM) encompassing Y248, which is indispensable for interaction of SHP-2 and delivery of PD-1 inhibitory function. We generated an antibody specific for phosphorylated PD-1-Y248 and examined PD-1pY248+ (pPD-1) expression in human T cells. pPD-1 was upregulated by TCR/CD3 + CD28 stimulation and simultaneous PD-1 ligation. pPD-1+CD8+ T cells were identified in human peripheral blood and had impaired effector function. pPD-1+ T cells were also detected in tumor-draining lymph nodes of tumor bearing mice and in biopsies of patients with glioblastoma multiform. Detection of pPD-1+ T cells might serve as a biomarker for identification of T cells subjected to PD-1-mediated immunosuppression.
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Linfócitos T CD8-Positivos/metabolismo , Motivo de Inibição do Imunorreceptor Baseado em Tirosina/fisiologia , Receptor de Morte Celular Programada 1/metabolismo , Animais , Antígenos CD/metabolismo , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores/sangue , Antígenos CD28/metabolismo , Feminino , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Motivo de Inibição do Imunorreceptor Baseado em Tirosina/genética , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Cultura Primária de Células , Receptor de Morte Celular Programada 1/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Tirosina/metabolismoRESUMO
In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer's patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes. Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on TFH and GC B cell subsets.
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Linfócitos B/imunologia , Citometria de Fluxo/métodos , Imunidade Humoral/fisiologia , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Diferenciação Celular/imunologia , Centro Germinativo/imunologia , Imunidade nas Mucosas/fisiologia , CamundongosRESUMO
There has been significant progress in utilizing our immune system against cancer, mainly by checkpoint blockade and T cell-mediated therapies. The field of cancer immunotherapy is growing rapidly but durable clinical benefits occur only in a small subset of responding patients. It is currently recognized that cancer creates a suppressive metabolic microenvironment, which contributes to ineffective immune function. Metabolism is a common cellular feature, and although there has been significant progress in understanding the detrimental role of metabolic changes of the tumor microenvironment (TEM) in immune cells, there is still much to be learned regarding unique targetable pathways. Elucidation of cancer and immune cell metabolic profiles is critical for identifying mechanisms that regulate metabolic reprogramming within the TEM. Metabolic targets that mediate immunosuppression and are fundamental in sustaining tumor growth can be exploited therapeutically for the development of approaches to increase the efficacy of immunotherapies. Here, we will highlight the importance of metabolism on the function of tumor-associated immune cells and will address the role of key metabolic determinants that might be targets of therapeutic intervention for improvement of tumor immunotherapies.
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Host immunity provides wide spectrum protection that serves to eradicate pathogens and cancer cells, while maintaining self-tolerance and immunological homeostasis. Ligation of the T cell receptor (TCR) by antigen activates signaling pathways that coordinately induce aerobic glycolysis, mitochondrial activity, anabolic metabolism, and T effector cell differentiation. Activation of PI3K, Akt, and mTOR triggers the switch to anabolic metabolism by inducing transcription factors such as Myc and HIF1, and the glucose transporter Glut1, which is pivotal for the increase of glucose uptake after T cell activation. Activation of MAPK signaling is required for glucose and glutamine utilization, whereas activation of AMPK is critical for energy balance and metabolic fitness of T effector and memory cells. Coinhibitory receptors target TCR-proximal signaling and generation of second messengers. Imbalanced activation of such signaling pathways leads to diminished rates of aerobic glycolysis and impaired mitochondrial function resulting in defective anabolic metabolism and altered T cell differentiation. The coinhibitory receptors mediate distinct and synergistic effects on the activation of signaling pathways thereby modifying metabolic programs of activated T cells and resulting in altered immune functions. Understanding and therapeutic targeting of metabolic programs impacted by coinhibitory receptors might have significant clinical implications for the treatment of chronic infections, cancer, and autoimmune diseases.