RESUMO
Despite the impressive rates of clinical response to programmed death 1 (PD-1) blockade in multiple cancers, the majority of patients still fail to respond to this therapy. The CT26 tumor in mice showed similar heterogeneity, with most tumors unaffected by anti-PD-1. As in humans, response of CT26 to anti-PD-1 correlated with increased T- and B-cell infiltration and IFN expression. We show that intratumoral injection of a highly interferogenic TLR9 agonist, SD-101, in anti-PD-1 nonresponders led to a complete, durable rejection of essentially all injected tumors and a majority of uninjected, distant-site tumors. Therapeutic efficacy of the combination was also observed with the TSA mammary adenocarcinoma and MCA38 colon carcinoma tumor models that show little response to PD-1 blockade alone. Intratumoral SD-101 substantially increased leukocyte infiltration and IFN-regulated gene expression, and its activity was dependent on CD8+ T cells and type I IFN signaling. Anti-PD-1 plus intratumoral SD-101 promoted infiltration of activated, proliferating CD8+ T cells and led to a synergistic increase in total and tumor antigen-specific CD8+ T cells expressing both IFN-γ and TNF-α. Additionally, PD-1 blockade could alter the CpG-mediated differentiation of tumor-specific CD8+ T cells into CD127lowKLRG1high short-lived effector cells, preferentially expanding the CD127highKLRG1low long-lived memory precursors. Tumor control and intratumoral T-cell proliferation in response to the combined treatment is independent of T-cell trafficking from secondary lymphoid organs. These findings suggest that a CpG oligonucleotide given intratumorally may increase the response of cancer patients to PD-1 blockade, increasing the quantity and the quality of tumor-specific CD8+ T cells.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Oligodesoxirribonucleotídeos/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Injeções Intralesionais , Interferon Tipo I/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/farmacologia , Receptor Toll-Like 9/agonistas , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
To achieve a durable adaptive immune response, lymphocytes must undergo clonal expansion and induce a survival program that enables the persistence of Ag-experienced cells and the development of memory. During the priming phase of this response, CD4(+)T lymphocytes either remain tolerized or undergo clonal expansion. In this article, we show that Usp9X functions as a positive regulatory switch during T lymphocyte priming through removal of inhibitory monoubiquitination from ZAP70. In the absence of Usp9X, an increased amount of ZAP70 localized to early endosomes consistent with the role of monoubiquitin in endocytic sorting. Usp9X becomes competent to deubiquitinate ZAP70 through TCR-dependent phosphorylation and enhancement of its catalytic activity and association with the LAT signalosome. In B lymphocytes, Usp9X is required for the induction of PKCß kinase activity after BCR-dependent activation. Accordingly, inUsp9Xknockout B cells, there was a significant reduction in phospho-CARMA1 levels that resulted in reduced CARMA1/Bcl-10/MALT-1 complex formation and NF-κB-dependent cell survival. The pleiotropic effect of Usp9X during Ag-receptor signaling highlights its importance for the development of an effective and durable adaptive immune response.
Assuntos
Linfócitos B/imunologia , Endopeptidases/genética , Ativação Linfocitária/imunologia , Proteína Quinase C beta/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Imunidade Adaptativa/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Caspases/imunologia , Linhagem Celular , Sobrevivência Celular , Endopeptidases/imunologia , Ativação Enzimática/imunologia , Humanos , Células Jurkat , Proteínas de Membrana/imunologia , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/genética , Proteínas de Neoplasias/imunologia , Fosforilação , Receptores de Antígenos de Linfócitos T/imunologia , Ubiquitina Tiolesterase , UbiquitinaçãoRESUMO
The T cell hyperproliferation and autoimmune phenotypes that manifest in mice lacking E3 ubiquitin ligases such as Cbl, ITCH, or GRAIL highlight the importance of ubiquitination for the maintenance of peripheral T cell tolerance. Less is known, however, about the deubiquitinating enzymes that regulate T cell proliferation and effector function. Here, we define a cell intrinsic role for the deubiquitinase Usp9X during proximal TCR signaling. Usp9X-deficient T cells were hypoproliferative, yet mice with T cell-specific Usp9x deletion had elevated numbers of antigen-experienced T cells and expanded PD-1 and OX40-expressing populations consistent with immune hyperactivity. Aged Usp9x KO mice developed lupus-like autoimmunity and lymphoproliferative disease, indicating that ubiquitin ligases and deubiquitinases maintain the delicate balance between effective immunity and self-tolerance.
Assuntos
Autoimunidade/genética , Endopeptidases/metabolismo , Transtornos Linfoproliferativos/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Tolerância a Antígenos Próprios/imunologia , Transdução de Sinais/imunologia , Animais , Cálcio/metabolismo , Proliferação de Células , Primers do DNA/genética , Endopeptidases/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Linfócitos/metabolismo , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , Tolerância a Antígenos Próprios/genética , Ubiquitina TiolesteraseRESUMO
For malignant growth, solid cancers must stimulate the formation of new blood vessels by producing vascular endothelial growth factor (VEGF-A), which is required for the survival of tumor-associated vessels. Novel anticancer agents that block VEGF-A signaling trigger endothelial cell (EC) apoptosis and vascular regression preferentially within tumors, but how the ECs die is not understood. In this study, we demonstrate that VEGF-A deprivation, provoked either by drug-induced tumor shrinkage or direct VEGF-A blockade, up-regulates the proapoptotic BH3 (Bcl-2 homology 3)-only Bcl-2 family member Bim in ECs. Importantly, the tumor growth inhibitory activity of a VEGF-A antagonist required Bim-induced apoptosis of ECs. These findings thus reveal the mechanism by which VEGF-A blockade induces EC apoptosis and impairs tumor growth. They also indicate that drugs mimicking BH3-only proteins may be exploited to kill tumor cells not only directly but also indirectly by ablating the tumor vasculature.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/fisiopatologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/fisiopatologia , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Ganciclovir/farmacologia , Expressão Gênica/efeitos dos fármacos , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/patologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Timidina Quinase/genética , Timidina Quinase/metabolismo , Fator A de Crescimento do Endotélio Vascular/deficiência , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
High levels of reactive oxygen species (ROS) are observed in chronic human diseases such as neurodegeneration, Crohn's disease, and cancer. In addition to the presence of oxidative stress, these diseases are also characterized by deregulated inflammatory responses, including but not limited to proinflammatory cytokine production. New work exploring the mechanisms linking ROS and inflammation find that ROS derived from mitochondria act as signal-transducing molecules that provoke the up-regulation of inflammatory cytokine subsets via distinct molecular pathways.
Assuntos
Citocinas/fisiologia , Inflamação/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Citocinas/biossíntese , Humanos , Inflamação/fisiopatologia , Mitocôndrias/fisiologia , Transdução de SinaisRESUMO
INTRODUCTION: The innate immune system orchestrates inflammatory responses to microorganisms or danger-associated molecular patterns generated, for example, by the deposition of uric acid in the joints of gout patients. The innate immune system comprises multiple germ-line encoded receptors, of which the nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs) are crucial for the maturation of pro-inflammatory cytokines. NLRs oligomerize to form large multi-protein complexes termed inflammasomes that generate active caspase-1 fragments leading to the cleavage and secretion of mature cytokines such as IL-1beta and IL-18. THE REGULATION OF MULTIPLE INFLAMMASOMES: At least four independent inflammasomes have been identified, NLRP1, NLRP3, IPAF, and AIM2. These inflammasomes assemble in response to different stimuli to confer specificity and are also subject to negative regulatory mechanisms to ensure that once a productive inflammatory response has been mounted, inflammatory cytokine production is restrained. TREATMENT OF AUTO-INFLAMMATORY DISORDERS: A number of human conditions are characterized by unrestrained inflammasome activation. As much is now known about how inflammasomes are regulated, it is hoped that this can be channeled into the development of novel therapeutics, for example, those that may block the upstream activation and assembly of inflammasomes.
Assuntos
Doenças Hereditárias Autoinflamatórias/tratamento farmacológico , Imunidade Inata , Inflamação , Receptores de Reconhecimento de Padrão/imunologia , Doenças Hereditárias Autoinflamatórias/imunologia , Humanos , Inflamação/imunologia , Interleucina-1beta/imunologia , Transdução de Sinais/imunologiaRESUMO
Interleukin 15 (IL-15) promotes the survival of natural killer (NK) cells by preventing apoptosis through mechanisms unknown at present. Here we identify Bim, Noxa and Mcl-1 as key regulators of IL-15-dependent survival of NK cells. IL-15 suppressed apoptosis by limiting Bim expression through the kinases Erk1 and Erk2 and mechanisms dependent on the transcription factor Foxo3a, while promoting expression of Mcl-1, which was necessary and sufficient for the survival of NK cells. Withdrawal of IL-15 led to upregulation of Bim and, accordingly, both Bim-deficient and Foxo3a-/- NK cells were resistant to cytokine deprivation. Finally, IL-15-mediated inactivation of Foxo3a and cell survival were dependent on phosphotidylinositol-3-OH kinase. Thus, IL-15 regulates the survival of NK cells at multiple steps, with Bim and Noxa being key antagonists of Mcl-1, the critical survivor factor in this process.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/imunologia , Interleucina-15/metabolismo , Células Matadoras Naturais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/imunologia , Proteína 11 Semelhante a Bcl-2 , Sobrevivência Celular/imunologia , Citometria de Fluxo , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Immunoblotting , Interleucina-15/imunologia , Células Matadoras Naturais/imunologia , Proteínas de Membrana/imunologia , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/imunologiaRESUMO
To identify the mechanisms of ultraviolet radiation (UVR)-induced cell death, for which the tumor suppressor p53 is essential, we have analyzed mouse embryonic fibroblasts (MEFs) and keratinocytes in mouse skin that have specific apoptotic pathways blocked genetically. Blocking the death receptor pathway provided no protection to MEFs, whereas UVR-induced apoptosis was potently inhibited by Bcl-2 overexpression, implicating the mitochondrial pathway. Indeed, Bcl-2 overexpression boosted cell survival more than p53 loss, revealing a p53-independent pathway controlled by the Bcl-2 family. Analysis of primary MEFs lacking individual members of its BH3-only subfamily identified major initiating roles for the p53 targets Noxa and Puma. In the transformed derivatives, where Puma, unexpectedly, was not induced by UVR, Noxa had the dominant role and Bim a minor role. Furthermore, loss of Noxa suppressed the formation of apoptotic keratinocytes in the skin of UV-irradiated mice. Collectively, these results demonstrate that UVR activates the Bcl-2-regulated apoptotic pathway predominantly through activation of Noxa and, depending on cellular context, Puma.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pele/metabolismo , Animais , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/genética , Células Cultivadas , Fibroblastos/efeitos da radiação , Queratinócitos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de Morte Celular/antagonistas & inibidores , Receptores de Morte Celular/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Pele/citologia , Pele/efeitos da radiação , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/fisiopatologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Raios UltravioletaRESUMO
Commitment of cells to apoptosis is governed largely by the interaction between members of the Bcl-2 protein family. Its three subfamilies have distinct roles: The BH3-only proteins trigger apoptosis by binding via their BH3 domain to prosurvival relatives, while the proapoptotic Bax and Bak have an essential downstream role involving permeabilization of organellar membranes and induction of caspase activation. We have investigated the regulation of Bak and find that, in healthy cells, Bak associates with Mcl-1 and Bcl-x(L) but surprisingly not Bcl-2, Bcl-w, or A1. These interactions require the Bak BH3 domain, which is also necessary for Bak dimerization and killing activity. When cytotoxic signals activate BH3-only proteins that can engage both Mcl-1 and Bcl-x(L) (such as Noxa plus Bad), Bak is displaced and induces cell death. Accordingly, the BH3-only protein Noxa could bind to Mcl-1, displace Bak, and promote Mcl-1 degradation, but Bak-mediated cell death also required neutralization of Bcl-x(L) by other BH3-only proteins. The results indicate that Bak is held in check solely by Mcl-1 and Bcl-x(L) and induces apoptosis only if freed from both. The finding that different prosurvival proteins have selective roles has notable implications for the design of anti-cancer drugs that target the Bcl-2 family.
Assuntos
Apoptose/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Sequência de Aminoácidos , Apoptose/efeitos da radiação , Células HeLa , Humanos , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/química , Raios Ultravioleta , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína bcl-XRESUMO
The Lyn tyrosine kinase plays essential inhibitory signaling roles within hematopoietic cells by recruiting inhibitory phosphatases such as SH2-domain containing phosphatase-1 (SHP-1), SHP-2, and SH2-domain containing 5'-inositol phosphatase (SHIP-1) to the plasma membrane in response to specific stimuli. Lyn-deficient mice display a collection of hematopoietic defects, including autoimmune disease as a result of autoantibody production, and perturbations in myelopoiesis that ultimately lead to splenomegaly and myeloid neoplasia. In this study, we demonstrate that loss of Lyn results in a stem/progenitor cell-intrinsic defect leading to an age-dependent increase in myeloid, erythroid, and primitive hematopoietic progenitor numbers that is independent of autoimmune disease. Despite possessing increased numbers of erythroid progenitors, and a more robust expansion of these cells following phenylhydrazine challenge, Lyn-deficient mice are more severely affected by the chemotherapeutic drug 5-fluorouracil, revealing a greater proportion of cycling progenitors. We also show that mice lacking SHIP-1 have defects in the erythroid and myeloid compartments similar to those in mice lacking Lyn or SHP-1, suggesting an intimate relationship between Lyn, SHP-1, and SHIP-1 in regulating hematopoiesis.