RESUMO
It is well established that extracellular proteins that negatively regulate T cell function, such as Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) and Programmed Cell Death protein 1 (PD-1), can be effectively targeted to enhance cancer immunotherapies and Chimeric Antigen Receptor T cells (CAR-T cells). Intracellular proteins that inhibit T cell receptor (TCR) signal transduction, though less well studied, are also potentially useful therapeutic targets to enhance T cell activity against tumor. Four major classes of enzymes that attenuate TCR signaling include E3 ubiquitin kinases such as the Casitas B-lineage lymphoma proteins (Cbl-b and c-Cbl), and Itchy (Itch), inhibitory tyrosine phosphatases, such as Src homology region 2 domain-containing phosphatases (SHP-1 and SHP-2), inhibitory protein kinases, such as C-terminal Src kinase (Csk), and inhibitory lipid kinases such as Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase (SHIP) and Diacylglycerol kinases (DGKs). This review describes the mechanism of action of eighteen intracellular inhibitory regulatory proteins in T cells within these four classes, and assesses their potential value as clinical targets to enhance the anti-tumor activity of endogenous T cells and CAR-T cells.
Assuntos
Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Linfócitos T/imunologia , Animais , HumanosRESUMO
Regulatory T cells (Tregs) are a subset of CD4(+) T cells that maintain immune tolerance in part by their ability to inhibit the proliferation of conventional CD4(+) T cells (Tconvs). The role of the TCR and the downstream signaling pathways required for this suppressive function of Tregs are not fully understood. To yield insight into how TCR-mediated signals influence Treg suppressive function, we assessed the ability of Tregs with altered TCR-mediated signaling capacity to inhibit Tconv proliferation. Mature Tregs deficient in Src homology 2 domain containing leukocyte protein of 76 kDa (SLP-76), an adaptor protein that nucleates the proximal signaling complex downstream of the TCR, were unable to inhibit Tconv proliferation, suggesting that TCR signaling is required for Treg suppressive function. Moreover, Tregs with defective phospholipase C γ (PLCγ) activation due to a Y145F mutation of SLP-76 were also defective in their suppressive function. Conversely, enhancement of diacylglycerol-mediated signaling downstream of PLCγ by genetic ablation of a negative regulator of diacylglycerol kinase ζ increased the suppressive ability of Tregs. Because SLP-76 is also important for integrin activation and signaling, we tested the role of integrin activation in Treg-mediated suppression. Tregs lacking the adaptor proteins adhesion and degranulation promoting adapter protein or CT10 regulator of kinase/CT10 regulator of kinase-like, which are required for TCR-mediated integrin activation, inhibited Tconv proliferation to a similar extent as wild-type Tregs. Together, these data suggest that TCR-mediated PLCγ activation, but not integrin activation, is required for Tregs to inhibit Tconv proliferation.
Assuntos
Imunomodulação , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Diglicerídeos/metabolismo , Integrinas/metabolismo , Camundongos , Camundongos Transgênicos , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
Modulation of T cell receptor signal transduction in CD8(+) T cells represents a novel strategy toward enhancing the immune response to tumor. Recently, levels of guanine exchange factors, RasGRP and SOS, within T cells have been shown to represent a key determinant in the regulation of the analog to the digital activation threshold of Ras. One important for regulating activation levels of RasGRP is diacylglycerol (DAG), and its levels are influenced by diacylglycerol kinase-ζ (DGKζ), which metabolizes DAG into phosphatidic acid, terminating DAG-mediated Ras signaling. We sought to determine whether DGKζ-deficient CD8(+) T cells demonstrated enhanced in vitro responses in a manner predicted by the current model of Ras activation and to evaluate whether targeting this threshold confers enhanced CD8(+) T cell responsiveness to tumor. We observed that DGKζ-deficient CD8(+) T cells conform to most predictions of the current model of how RasGRP levels influence Ras activation. But our results differ in that the EC(50) value of stimulation is not altered for any T cell receptor stimulus, a finding that suggests a further degree of complexity to how DGKζ deficiency affects signals important for Ras and ERK activation. Additionally, we found that DGKζ-deficient CD8(+) T cells demonstrate enhanced responsiveness in a subcutaneous lymphoma model, implicating the analog to a digital conversion threshold as a novel target for potential therapeutic manipulation.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diacilglicerol Quinase/imunologia , Diglicerídeos/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Linfoma/imunologia , Modelos Imunológicos , Neoplasias Experimentais/imunologia , Animais , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/metabolismo , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Diglicerídeos/genética , Diglicerídeos/metabolismo , Ativação Enzimática/genética , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Linfoma/enzimologia , Linfoma/genética , Linfoma/terapia , Camundongos , Camundongos Knockout , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/terapia , Proteínas ras/imunologia , Proteínas ras/metabolismoRESUMO
The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T-cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of WT SLP-76 protein in thymocytes. Here, we show that following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR-generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T-cell-mediated autoimmune disease experimental autoimmune encephalomyelitis. Altogether, our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T-cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos T CD4-Positivos/imunologia , Fosfoproteínas/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Tamoxifeno/farmacologiaRESUMO
Two isoforms of diacylglycerol kinases (DGKs), DGKα and DGKζ, are primarily responsible for terminating DAG-mediated activation of Ras and PKCθ pathways in T cells. A direct comparison of tumor growth between mice lacking each isoform has not been undertaken. We evaluated the growth of three syngeneic tumor cell lines in mice lacking either DGKα or DGKζ in the presence or absence of treatment with anti-PD1 and determined that (i) mice deficient in DGKζ conferred enhanced control of tumor relative to mice deficient in DGKα and (ii) deficiency of DGKζ acted additively with anti-PD1 in tumor control. Consistent with this finding, functional and RNA-sequencing analyses revealed greater changes in stimulated DGKζ-deficient T cells compared with DGKα-deficient T cells, which were enhanced relative to wildtype T cells. DGKζ also imparted greater regulation than DGKα in human T cells. Together, these data support targeting the ζ isoform of DGKs to therapeutically enhance T cell anti-tumor activity.
Assuntos
Diacilglicerol Quinase , Linfócitos T , Animais , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Diacilglicerol Quinase/genética , CamundongosRESUMO
The signaling adapter MyD88 is critical for immune cell activation in response to viral or bacterial pathogens via several TLRs, IL-1ßR and IL-18R. However, the essential role of MyD88 during activations mediated by germline-encoded NK cell receptors (NKRs), such as Ly49H or NKG2D, has yet to be investigated. To define the NK cell-intrinsic function of MyD88, we generated a novel NK cell conditional knockout mouse for MyD88 (Myd88fl/flNcr1Cre/+). Phenotypic characterization of these mice demonstrated that MyD88 is dispensable for NK cell development and maturation. However, the MyD88-deficient NK cells exhibited significantly reduced cytotoxic potentials in vivo. In addition, the lack of MyD88 significantly reduced the NKG2D-mediated inflammatory cytokine production in vitro. Consistent with this, mice lacking MyD88 were unable to respond and clear MCMV infection. Transcriptomic analyses of splenic NK cells following MCMV infection revealed that inflammatory gene signatures were upregulated in Ly49H+. In contrast, Ly49H- NK cells have significant enrichment in G2M checkpoint genes, revealing distinct transcriptomic profiles of these subsets. Our results identify a central role for MyD88 in Ly49H-dependent gene signatures, including alterations in genes regulating proliferation in Ly49H+ NK cells. In summary, our study reveals a previously unknown function of MyD88 in Ly49H-dependent signaling and in vivo functions of NK cells.
Assuntos
Infecções por Herpesviridae/imunologia , Células Matadoras Naturais/imunologia , Muromegalovirus/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Animais , Proliferação de Células/fisiologia , Citocinas/imunologia , Feminino , Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores de Células Matadoras Naturais/imunologia , Transdução de Sinais/imunologia , Transcriptoma/imunologiaRESUMO
BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is an emerging option for cancer treatment, but its efficacy is limited, especially in solid tumors. This is partly because the CAR T cells become dysfunctional and exhausted in the tumor microenvironment. However, the key pathways responsible for impaired function of exhausted cells remain unclear, which is essential to overcome CAR T-cell exhaustion. METHODS: Analysis of RNA-sequencing data from CD8+ tumor-infiltrating lymphocytes (TILs) led to identification of Cbl-b as a potential target. The sequencing data were validated using a syngeneic MC38 colon cancer model. To analyze the in vivo role of Cbl-b in T-cell exhaustion, tumor growth, % PD1+Tim3+ cells, and expression of effector cytokines were analyzed in cbl-b+/+ and cbl-b-/- mice. To evaluate the therapeutic potential of Cbl-b depletion, we generated a new CAR construct, hCEAscFv-CD28-CD3ζ.GFP, that recognizes human carcinoembryonic antigen (CEA). cbl-b+/+ and cbl-b-/- CEA-CAR T cells were generated by retroviral transduction. Rag-/- mice bearing MC38-CEA cells were injected with cbl-b+/+ and cbl-b-/- ; CEA-CAR T cells, tumor growth, % PD1+Tim3+ cells and expression of effector cytokines were analyzed. RESULTS: Our results show that the E3 ubiquitin ligase Cbl-b is upregulated in exhausted (PD1+Tim3+) CD8+ TILs. CRISPR-Cas9-mediated inhibition of Cbl-b restores the effector function of exhausted CD8+ TILs. Importantly, the reduced growth of syngeneic MC38 tumors in cbl-b-/- mice was associated with a marked reduction of PD1+Tim3+ CD8+ TILs. Depletion of Cbl-b inhibited CAR T-cell exhaustion, resulting in reduced MC38-CEA tumor growth, reduced PD1+Tim3+ cells and increased expression of interferon gamma, tumor necrosis factor alpha, and increased tumor cell killing. CONCLUSION: Our studies demonstrate that deficiency of Cbl-b overcomes endogenous CD8+ T-cell exhaustion, and deletion of Cbl-b in CAR T cells renders them resistant to exhaustion. Our results could facilitate the development of efficient CAR T-cell therapy for solid tumors by targeting Cbl-b.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias do Colo/genética , Neoplasias do Colo/terapia , Proteínas Proto-Oncogênicas c-cbl/genética , Receptores de Antígenos Quiméricos/metabolismo , Regulação para Cima , Animais , Linfócitos T CD8-Positivos/imunologia , Antígeno Carcinoembrionário/metabolismo , Neoplasias do Colo/imunologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia Adotiva , Interferon gama/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Transplante de Neoplasias , Análise de Sequência de RNA , Microambiente Tumoral , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) is a lethal chemoresistant cancer that exhibits early metastatic spread. The highly immunosuppressive PDA tumor microenvironment renders patients resistant to emerging immune-targeted therapies. Building from our prior work, we evaluated stimulator of interferon genes (STING) agonist activation of PDA cell interferon-α/ß-receptor (IFNAR) signaling in systemic antitumor immune responses. METHODS: PDA cells were implanted subcutaneously to wild-type, IFNAR-, or CXCR3-knockout mice. Tumor growth was monitored, and immune responses were comprehensively profiled. RESULTS: Human and mouse STING agonist ADU-S100 reduced local and distal tumor burden and activated systemic antitumor immune responses in PDA-bearing mice. Effector T-cell infiltration and inflammatory cytokine and chemokine production, including IFN-dependent CXCR3-agonist chemokines, were elevated, whereas suppressive immune populations were decreased in treated tumors. Intratumoral STING agonist treatment also generated inflammation in distal noninjected tumors and peripheral immune tissues. STING agonist treatment of type I IFN-responsive PDA tumors engrafted to IFNAR-/- recipient mice was sufficient to contract tumors and stimulate local and systemic T-cell activation. Tumor regression and CD8+ T-cell infiltration were abolished in PDA engrafted to CXCR3-/- mice treated with STING agonist. CONCLUSIONS: These data indicate that STING agonists promote T-cell infiltration and counteract immune suppression in locally treated and distant tumors. Tumor-intrinsic type I IFN signaling initiated systemic STING-mediated antitumor inflammation and required CXCR3 expression. STING-mediated induction of systemic immune responses provides an approach to harness the immune system to treat primary and disseminated pancreatic cancers.
Assuntos
Proteínas de Membrana/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Receptores CXCR3/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/deficiência , Receptores CXCR3/deficiência , Transdução de SinaisRESUMO
Immunotherapy with monoclonal antibodies targeting immune checkpoint molecules, including programmed death-1 (PD-1), PD ligand-1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen (CTLA)-4, has become prominent in the treatment of many types of cancer. However, a significant number of patients treated with immune checkpoint inhibitors (ICIs) develop immune-related adverse events (irAEs). irAEs can affect any organ system, and although most are clinically manageable, irAEs can result in mortality or long-term morbidity. Factors that can predict irAEs remain elusive. Understanding the etiology of ICI-induced irAEs and ways to limit these adverse events are needed. In this review, we provide basic science and clinical insights on the mechanisms responsible for ICI efficacy and ICI-induced irAEs. We further provide insights into approaches that may uncouple irAEs from the ability of ICIs to kill tumor cells.
RESUMO
The transcriptional activation and repression during NK cell ontology are poorly understood. Here, using single-cell RNA-sequencing, we reveal a novel role for T-bet in suppressing the immature gene signature during murine NK cell development. Based on transcriptome, we identified five distinct NK cell clusters and define their relative developmental maturity in the bone marrow. Transcriptome-based machine-learning classifiers revealed that half of the mTORC2-deficient NK cells belongs to the least mature NK cluster. Mechanistically, loss of mTORC2 results in an increased expression of signature genes representing immature NK cells. Since mTORC2 regulates the expression of T-bet through AktS473-FoxO1 axis, we further characterized the T-bet-deficient NK cells and found an augmented immature transcriptomic signature. Moreover, deletion of Foxo1 restores the expression of T-bet and corrects the abnormal expression of immature NK genes. Collectively, our study reveals a novel role for mTORC2-AktS473-FoxO1-T-bet axis in suppressing the transcriptional signature of immature NK cells.
Assuntos
Células da Medula Óssea/metabolismo , Perfilação da Expressão Gênica , Células Matadoras Naturais/metabolismo , Aprendizado de Máquina , RNA-Seq , Análise de Célula Única , Proteínas com Domínio T/genética , Transcriptoma , Animais , Células da Medula Óssea/imunologia , Análise por Conglomerados , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica , Genótipo , Células Matadoras Naturais/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina/deficiência , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/deficiência , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/genética , Proteínas com Domínio T/metabolismoRESUMO
During an immune response, natural killer (NK) cells activate specific metabolic pathways to meet the increased energetic and biosynthetic demands associated with effector functions. Here, we found in vivo activation of NK cells during Listeria monocytogenes infection-augmented transcription of genes encoding mitochondria-associated proteins in a manner dependent on the transcriptional coactivator PGC-1α. Using an Ncr1Cre-based conditional knockout mouse, we found that PGC-1α was crucial for optimal NK cell effector functions and bioenergetics, as the deletion of PGC-1α was associated with decreased cytotoxic potential and cytokine production along with altered ADP/ATP ratios. Lack of PGC-1α also significantly impaired the ability of NK cells to control B16F10 tumor growth in vivo, and subsequent gene expression analysis showed that PGC-1α mediates transcription required to maintain mitochondrial activity within the tumor microenvironment. Together, these data suggest that PGC-1α-dependent transcription of specific target genes is required for optimal NK cell function during the response to infection or tumor growth.
RESUMO
Adenosine mediates immunosuppression within the tumor microenvironment through triggering adenosine 2A receptors (A2AR) on immune cells. To determine whether this pathway could be targeted as an immunotherapy, we performed a phase I clinical trial with a small-molecule A2AR antagonist. We find that this molecule can safely block adenosine signaling in vivo. In a cohort of 68 patients with renal cell cancer (RCC), we also observe clinical responses alone and in combination with an anti-PD-L1 antibody, including subjects who had progressed on PD-1/PD-L1 inhibitors. Durable clinical benefit is associated with increased recruitment of CD8+ T cells into the tumor. Treatment can also broaden the circulating T-cell repertoire. Clinical responses are associated with an adenosine-regulated gene-expression signature in pretreatment tumor biopsies. A2AR signaling, therefore, represents a targetable immune checkpoint distinct from PD-1/PD-L1 that restricts antitumor immunity. SIGNIFICANCE: This first-in-human study of an A2AR antagonist for cancer treatment establishes the safety and feasibility of targeting this pathway by demonstrating antitumor activity with single-agent and anti-PD-L1 combination therapy in patients with refractory RCC. Responding patients possess an adenosine-regulated gene-expression signature in pretreatment tumor biopsies.See related commentary by Sitkovsky, p. 16.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Renais/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Receptor A2A de Adenosina/química , Terapia de Salvação , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Carcinoma de Células Renais/patologia , Feminino , Seguimentos , Furanos/administração & dosagem , Humanos , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Prognóstico , Piridinas/administração & dosagem , Pirimidinas/administração & dosagem , Receptor A2A de Adenosina/metabolismo , Taxa de SobrevidaRESUMO
Germ cell tumors (GCTs) are the most common solid malignancy in young adult males. Approximately 28% of metastatic non-seminomatous GCTs have features that confer a poor prognosis, as defined by the International Germ Cell Consensus Classification. Five-year survival rates in poor prognosis GCTs remain about 50%. Despite numerous clinical trials testing a range of approaches, four cycles of bleomycin, etoposide, and cisplatin remain the standard of care for patients with poor-risk disease. Further improvements in the outcomes of patients with poor-risk GCTs might be achieved by incorporating targeted therapies into existing regimens, and by improving our understanding of the mechanism of cisplatin insensitivity.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Neoplasias Testiculares/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Bleomicina/administração & dosagem , Cisplatino/administração & dosagem , Etoposídeo/administração & dosagem , Humanos , Ifosfamida/administração & dosagem , Masculino , Neoplasias Embrionárias de Células Germinativas/diagnóstico , Prognóstico , Neoplasias Testiculares/diagnóstico , Vimblastina/administração & dosagemRESUMO
Diacylglycerol kinases (DGKs) are a family of enzymes that convert diacylglycerol (DAG) into phosphatidic acid (PA). The ζ isoform of DGK (DGKζ) has been reported to inhibit T-cell responsiveness by downregulating intracellular levels of DAG. However, its role in platelet function remains undefined. In this study, we show that DGKζ was expressed at significant levels in both platelets and megakaryocytes and that DGKζ-knockout (DGKζ-KO) mouse platelets were hyperreactive to glycoprotein VI (GPVI) agonists, as assessed by aggregation, spreading, granule secretion, and activation of relevant signal transduction molecules. In contrast, they were less responsive to thrombin. Platelets from DGKζ-KO mice accumulated faster on collagen-coated microfluidic surfaces under conditions of arterial shear and stopped blood flow faster after ferric chloride-induced carotid artery injury. Other measures of hemostasis, as measured by tail bleeding time and rotational thromboelastometry analysis, were normal. Interestingly, DGKζ deficiency led to increased GPVI expression on the platelet and megakaryocyte surfaces without affecting the expression of other platelet surface receptors. These results implicate DGKζ as a novel negative regulator of GPVI-mediated platelet activation that plays an important role in regulating thrombus formation in vivo.
Assuntos
Diacilglicerol Quinase/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/farmacologia , Animais , Plaquetas/metabolismo , Diacilglicerol Quinase/deficiência , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Hemostasia , Humanos , Megacariócitos/metabolismo , Camundongos , Camundongos Knockout , Glicoproteínas da Membrana de Plaquetas/efeitos dos fármacos , Trombose/etiologiaRESUMO
Pancreatic cancer is characterized by an immune suppressive stromal reaction that creates a barrier to therapy. A murine transgenic pancreatic cancer cell line that recapitulates human disease was used to test whether a STimulator of Interferon Genes (STING) agonist could reignite immunologically inert pancreatic tumors. STING agonist treatment potently changed the tumor architecture, altered the immune profile, and increased the survival of tumor-bearing mice. Notably, STING agonist increased numbers and activity of cytotoxic T cells within tumors and decreased levels of suppressive regulatory T cells. Further, STING agonist treatment upregulated costimulatory molecule expression on cross-presenting dendritic cells and reprogrammed immune-suppressive macrophages into immune-activating subtypes. STING agonist promoted the coordinated and differential cytokine production by dendritic cells, macrophages, and pancreatic cancer cells. Cumulatively, these data demonstrate that pancreatic cancer progression is potently inhibited by STING agonist, which reignited immunologically cold pancreatic tumors to promote trafficking and activation of tumor-killing T cells.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Membrana/agonistas , Neoplasias Pancreáticas/tratamento farmacológico , Evasão Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral/transplante , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/genética , Humanos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia , Microambiente Tumoral/imunologia , Xantonas/farmacologia , Xantonas/uso terapêuticoRESUMO
Natural killer (NK) cells generate proinflammatory cytokines that are required to contain infections and tumor growth. However, the posttranscriptional mechanisms that regulate NK cell functions are not fully understood. Here, we define the role of the microRNA cluster known as Mirc11 (which includes miRNA-23a, miRNA-24a, and miRNA-27a) in NK cell-mediated proinflammatory responses. Absence of Mirc11 did not alter the development or the antitumor cytotoxicity of NK cells. However, loss of Mirc11 reduced generation of proinflammatory factors in vitro and interferon-γ-dependent clearance of Listeria monocytogenes or B16F10 melanoma in vivo by NK cells. These functional changes resulted from Mirc11 silencing ubiquitin modifiers A20, Cbl-b, and Itch, allowing TRAF6-dependent activation of NF-κB and AP-1. Lack of Mirc11 caused increased translation of A20, Cbl-b, and Itch proteins, resulting in deubiquitylation of scaffolding K63 and addition of degradative K48 moieties on TRAF6. Collectively, our results describe a function of Mirc11 that regulates generation of proinflammatory cytokines from effector lymphocytes.
Assuntos
Inflamação/imunologia , Células Matadoras Naturais/imunologia , Melanoma Experimental/imunologia , MicroRNAs/genética , Linfócitos T Citotóxicos/imunologia , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/imunologia , MicroRNAs/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Asthma is a chronic allergic inflammatory airway disease caused by aberrant immune responses to inhaled allergens, which leads to airway hyperresponsiveness (AHR) to contractile stimuli and airway obstruction. Blocking T helper 2 (TH2) differentiation represents a viable therapeutic strategy for allergic asthma, and strong TCR-mediated ERK activation blocks TH2 differentiation. Here, we report that targeting diacylglycerol (DAG) kinase zeta (DGKζ), a negative regulator of DAG-mediated cell signaling, protected against allergic asthma by simultaneously reducing airway inflammation and AHR though independent mechanisms. Targeted deletion of DGKζ in T cells decreased type 2 inflammation without reducing AHR. In contrast, loss of DGKζ in airway smooth muscle cells decreased AHR but not airway inflammation. T cell-specific enhancement of ERK signaling was only sufficient to limit type 2 airway inflammation, not AHR. Pharmacological inhibition of DGK diminished both airway inflammation and AHR in mice and also reduced bronchoconstriction of human airway samples in vitro. These data suggest that DGK is a previously unrecognized therapeutic target for asthma and reveal that the inflammatory and AHR components of asthma are not as interdependent as generally believed.
Assuntos
Asma/imunologia , Diacilglicerol Quinase/imunologia , Inflamação/imunologia , Hipersensibilidade Respiratória/imunologia , Animais , Asma/enzimologia , Asma/genética , Broncoconstrição/efeitos dos fármacos , Broncoconstrição/genética , Broncoconstrição/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Inflamação/enzimologia , Inflamação/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/imunologia , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Hipersensibilidade Respiratória/enzimologia , Hipersensibilidade Respiratória/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Th2/efeitos dos fármacos , Células Th2/enzimologia , Células Th2/imunologiaRESUMO
Natural killer (NK) cells are critical to both innate and adaptive immunity. However, the development and heterogeneity of human NK cells are yet to be fully defined. Using single-cell RNA-sequencing technology, here we identify distinct NK populations in human bone marrow and blood, including one population expressing higher levels of immediate early genes indicative of a homeostatic activation. Functionally matured NK cells with high expression of CX3CR1, HAVCR2 (TIM-3), and ZEB2 represents terminally differentiated status with the unique transcriptional profile. Transcriptomic and pseudotime analyses identify a transitional population between CD56bright and CD56dim NK cells. Finally, a donor with GATA2T354M mutation exhibits reduced percentage of CD56bright NK cells with altered transcriptome and elevated cell death. These data expand our understanding of the heterogeneity and development of human NK cells.
Assuntos
Medula Óssea/metabolismo , Células Matadoras Naturais/metabolismo , Análise de Célula Única/métodos , Transcriptoma/genética , Células da Medula Óssea/metabolismo , Antígeno CD56/genética , Antígeno CD56/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Heterogeneidade Genética , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismoRESUMO
Targeting negative regulators downstream of the T cell receptor (TCR) represents a novel strategy to improve cancer immunotherapy. Two proteins that serve as critical inhibitory regulators downstream of the TCR are diacylglycerol kinase ζ (DGKζ), a regulator of Ras and PKC-θ signaling, and Casitas b-lineage proto-oncogene b (Cbl-b), an E3 ubiquitin ligase that predominantly regulates PI(3)K signaling. We sought to compare the signaling and functional effects that result from deletion of DGKζ, Cbl-b, or both (double knockout, DKO) in T cells, and to evaluate tumor responses generated in a clinically relevant orthotopic pancreatic tumor model. We found that whereas deletion of Cbl-b primarily served to enhance NF-κB signaling, deletion of DGKζ enhanced TCR-mediated signal transduction downstream of Ras/Erk and NF-κB. Deletion of DGKζ or Cbl-b comparably enhanced CD8+ T cell functional responses, such as proliferation, production of IFNγ, and generation of granzyme B when compared with WT T cells. DKO T cells demonstrated enhanced function above that observed with single knockout T cells after weak, but not strong, stimulation. Deletion of DGKζ, but not Cbl-b, however, resulted in significant increases in numbers of activated (CD44hi) CD8+ T cells in both non-treated and tumor-bearing mice. DGKζ-deficient mice also had enhanced control of pancreatic tumor cell growth compared to Cbl-b-deficient mice. This represents the first direct comparison between mice of these genotypes and suggests that T cell immunotherapies may be better improved by targeting TCR signaling molecules that are regulated by DGKζ as opposed to molecules regulated by Cbl-b.
RESUMO
Inhibitory cell surface proteins on T cells are often dynamically regulated, which contributes to their physiologic function. PECAM-1 (CD31) is an inhibitory receptor that facilitates TGF-ß-mediated suppression of T cell activity. It is well established in CD4+ T cells that PECAM-1 is expressed in naïve recent thymic emigrants, but is down-regulated after acute T cell activation and absent from memory cells. The extent to which PECAM-1 expression is similarly regulated in CD8+ T cells is much less well characterized. We evaluated T cells recovered from mice after infection with a model intracellular pathogen and determined that, in CD8+ T cells, PECAM-1 expression was strongly down-regulated during acute infection but re-expressed to intermediate levels in memory cells. Down-regulation of PECAM-1 expression in CD8+ T cells was transcriptionally regulated and affected by the strength and nature of TCR signaling. PECAM-1 was also detected on the surface of human activated/memory CD8+ , but not CD4+ T cells. These data demonstrate that PECAM-1 expression is dynamically regulated, albeit differently, in both CD4+ and CD8+ T cells. Furthermore, unlike memory CD4+ T cells, memory CD8+ T cells retain PECAM-1 expression and have the potential to be modulated by this inhibitory receptor.