RESUMO
Crohn's disease (CD) is a kind of refractory intestinal inflammatory diseases. Pyroptosis was recently identified as a gasdermin-mediated proinflammatory cell death. However, it is unclear whether gasdermin-mediated pyroptosis participates in the pathogenesis of CD. Here, we show that the pyroptosis-inducing fragment GSDME N-terminal is obviously detected in the inflamed colonic mucosa but not in the uninflamed mucosa of patients with CD, suggesting that GSDME-mediated pyroptosis may be correlated with intestinal mucosal inflammation in CD. To investigate the role of GSDME in colitis development, Gsdme-/- mice and wild-type (WT) littermate controls were treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce colitis. We found that Gsdme-/- mice exhibit less-severe intestinal inflammation than WT controls do. Furthermore, our results indicate that GSDME-mediated epithelial-cell pyroptosis induces intestinal inflammation through the release of proinflammatory intracellular contents. In summary, we show that GSDME participates in the pathogenesis of CD through GSDME-mediated pyroptosis to release proinflammatory cytokines.
Assuntos
Doença de Crohn/patologia , Inflamação/patologia , Intestinos/patologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose , Animais , Colite/induzido quimicamente , Colite/imunologia , Colite/metabolismo , Colite/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteína HMGB1/metabolismo , Hematopoese , Humanos , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas Citotóxicas Formadoras de Poros/deficiência , Índice de Gravidade de Doença , Ácido TrinitrobenzenossulfônicoRESUMO
Cellular inflammasome activation causes caspase-1 cleavage of the pore-forming protein gasdermin D (GSDMD) with subsequent pyroptotic cell death and cytokine release. Here, we clarify the ambiguous role of the related family member gasdermin E (GSDME) in this process. Inflammasome stimulation in GSDMD-deficient cells led to apoptotic caspase cleavage of GSDME. Endogenous GSDME activation permitted sublytic, continuous interleukin-1ß (IL-1ß) release and membrane leakage, even in GSDMD-sufficient cells, whereas ectopic expression led to pyroptosis with GSDME oligomerization and complete liberation of IL-1ß akin to GSDMD pyroptosis. We find that NLRP3 and NLRP1 inflammasomes ultimately rely concurrently on both gasdermins for IL-1ß processing and release separately from their ability to induce cell lysis. Our study thus identifies GSDME as a conduit for IL-1ß release independent of its ability to cause cell death.
Assuntos
Inflamassomos/genética , Interleucina-1beta/genética , Macrófagos/imunologia , Proteínas de Ligação a Fosfato/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Piroptose/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Caspase 1/genética , Caspase 1/imunologia , Caspase 3/genética , Caspase 3/imunologia , Linhagem Celular Transformada , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Inflamassomos/imunologia , Interleucina-1beta/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/imunologia , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/imunologia , Piroptose/efeitos dos fármacos , Piroptose/imunologia , Salmonella typhimurium/química , Salmonella typhimurium/patogenicidade , Transdução de Sinais , Células THP-1RESUMO
Immunotherapeutic strategies are increasingly important in neuro-oncology, and the elucidation of escape mechanisms that lead to treatment resistance is crucial. We investigated the impact of immune pressure on the clonal dynamics and immune escape signature by comparing glioma growth in immunocompetent versus immunodeficient mice. Glioma-bearing WT and Pd-1-/- mice survived significantly longer than immunodeficient Pfp-/- Rag2-/- mice. While tumors in Pfp-/- Rag2-/- mice were highly polyclonal, immunoedited tumors in WT and Pd-1-/- mice displayed reduced clonality with emergence of immune escape clones. Tumor cells in WT mice were distinguished by an IFN-γ-mediated response signature with upregulation of genes involved in immunosuppression. Tumor-infiltrating stromal cells, which include macrophages/microglia, contributed even more strongly to the immunosuppressive signature than the actual tumor cells. The identified murine immune escape signature was reflected in human patients and correlated with poor survival. In conclusion, immune pressure profoundly shapes the clonal composition and gene regulation in malignant gliomas.
Assuntos
Neoplasias Encefálicas/imunologia , Glioma/imunologia , Evasão Tumoral/imunologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Evolução Clonal/genética , Evolução Clonal/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Glioma/genética , Glioma/patologia , Humanos , Imunocompetência , Hospedeiro Imunocomprometido , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Evasão Tumoral/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity.
Assuntos
Autoimunidade/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Inflamação/imunologia , Síndrome Metabólica/imunologia , Proteínas Citotóxicas Formadoras de Poros/análise , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Transferência Adotiva , Animais , Antígenos de Diferenciação/análise , Antígeno CD11c/análise , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Células Clonais/imunologia , Grânulos Citoplasmáticos/química , Células Dendríticas/classificação , Células Dendríticas/ultraestrutura , Dieta Hiperlipídica/efeitos adversos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Inflamação/patologia , Depleção Linfocítica , Masculino , Síndrome Metabólica/etiologia , Síndrome Metabólica/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/imunologia , Obesidade/patologia , Fenótipo , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Quimera por Radiação , Tolerância a Antígenos Próprios/imunologiaRESUMO
Exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone-marrow. A hematological response dependent on endogenous glucocorticoids underlies bone-marrow eosinophilia induced by trauma or allergic sensitization/challenge. We detected a defect in granulopoiesis in nonsensitized, perforin-deficient mice. In steady-state conditions, perforin- (Pfp-) deficient mice showed significantly decreased bone-marrow and blood eosinophil and neutrophil counts, and colony formation in response to GM-CSF, relative to wild-type controls of comparable age and/or weight. By contrast, peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency. Dexamethasone enhanced colony formation by GM-CSF-stimulated progenitors from wild-type controls, but not Pfp mice. Dexamethasone injection increased bone-marrow eosinophil and neutrophil counts in wild-type controls, but not Pfp mice. Because perforin is expressed in effector lymphocytes, we examined whether this defect would be corrected by transferring wild-type lymphocytes into perforin-deficient recipients. Short-term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild-type donors. Transfer of the same amount of splenic lymphocytes from perforin-deficient donors was ineffective. This demonstrates that the perforin-dependent, granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations.
Assuntos
Dexametasona/farmacologia , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Linfócitos/imunologia , Proteínas Citotóxicas Formadoras de Poros/deficiência , Animais , Dexametasona/administração & dosagem , Eosinófilos/citologia , Eosinófilos/imunologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Granulócitos/citologia , Linfócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/imunologia , Proteínas Citotóxicas Formadoras de Poros/genéticaRESUMO
Multiple myeloma (MM) is an age-dependent hematological malignancy. Evaluation of immune interactions that drive MM relies on in vitro experiments that do not reflect the complex cellular stroma involved in MM pathogenesis. Here we used Vk*MYC transgenic mice, which spontaneously develop MM, and demonstrated that the immune system plays a critical role in the control of MM progression and the response to treatment. We monitored Vk*MYC mice that had been crossed with Cd226 mutant mice over a period of 3 years and found that CD226 limits spontaneous MM development. The CD226-dependent anti-myeloma immune response against transplanted Vk*MYC MM cells was mediated both by NK and CD8+ T cells through perforin and IFN-γ pathways. Moreover, CD226 expression was required for optimal antimyeloma efficacy of cyclophosphamide (CTX) and bortezomib (Btz), which are both standardly used to manage MM in patients. Activation of costimulatory receptor CD137 with mAb (4-1BB) exerted strong antimyeloma activity, while inhibition of coinhibitory receptors PD-1 and CTLA-4 had no effect. Taken together, the results of this study provide in vivo evidence that CD226 is important for MM immunosurveillance and indicate that specific immune components should be targeted for optimal MM treatment efficacy. As progressive immunosuppression associates with MM development, strategies aimed to increase immune functions may have important therapeutic implications in MM.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Vigilância Imunológica/imunologia , Mieloma Múltiplo/imunologia , Proteínas de Neoplasias/fisiologia , Animais , Anticorpos Monoclonais/uso terapêutico , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antineoplásicos/uso terapêutico , Ácidos Borônicos/uso terapêutico , Bortezomib , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Cruzamentos Genéticos , Ciclofosfamida/uso terapêutico , Progressão da Doença , Genes myc , Predisposição Genética para Doença , Imunoterapia , Interferon gama/deficiência , Interferon gama/genética , Interferon gama/fisiologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Transplante de Neoplasias , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/fisiologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Pirazinas/uso terapêutico , Receptores Virais/deficiência , Receptores Virais/genética , Receptores Virais/fisiologia , Carga Tumoral , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Immunosuppressive pretransplantation conditioning is essential for donor cell engraftment in allogeneic bone marrow transplantation (BMT). The role of residual postconditioning recipient immunity in determining engraftment is poorly understood. We examined the role of recipient perforin in the kinetics of donor cell engraftment. MHC-mismatched BMT mouse models demonstrated that both the rate and proportion of donor lymphoid cell engraftment and expansion of effector memory donor T cells in both spleen and BM were significantly increased within 5 to 7 days post-BMT in perforin-deficient (pfn(-/-)) recipients, compared with wild-type. In wild-type recipients, depletion of natural killer (NK) cells before BMT enhanced donor lymphoid cell engraftment to that seen in pfn(-/-) recipients. This demonstrated that a perforin-dependent, NK-mediated, host-versus-graft (HVG) effect limits the rate of donor engraftment and T cell activation. Radiation-resistant natural killer T (NKT) cells survived in the BM of lethally irradiated mice and may drive NK cell activation, resulting in the HVG effect. Furthermore, reduced pretransplant irradiation doses in pfn(-/-) recipients permitted long-term donor lymphoid cell engraftment. These findings suggest that suppression of perforin activity or selective depletion of recipient NK cells before BMT could be used to improve donor stem cell engraftment, in turn allowing for the reduction of pretransplant conditioning.
Assuntos
Transplante de Medula Óssea , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/prevenção & controle , Células Matadoras Naturais/efeitos da radiação , Proteínas Citotóxicas Formadoras de Poros/imunologia , Linfócitos T/imunologia , Animais , Feminino , Expressão Gênica , Reação Hospedeiro-Enxerto/efeitos da radiação , Memória Imunológica , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Tolerância a Radiação , Linfócitos T/citologia , Transplante Homólogo , Irradiação Corporal TotalRESUMO
Defects in perforin and related genes lead to a loss of normal immune regulation and underlie hemophagocytic lymphohistiocytosis (HLH), which requires hematopoietic cell transplantation for long-term cure. However, transplantation may be complicated by the development of mixed chimerism and uncertainty regarding the risk of HLH recurrence. To help clarify this risk and investigate how perforin influences immune activation, we studied perforin-mediated immune regulation in the context of mixed chimerism using a murine model of HLH. We found that there is a distinct threshold of â¼10% to 20% perforin expression with either mixed hematopoietic or CD8(+) T cell chimerism, above which immune regulation was reestablished. These findings demonstrate that perforin-mediated immunoregulation functions in trans and are consistent with a feedback model in which cytotoxic T cells control immune activation by killing dendritic cells. These findings also suggest rational targets for maintenance of minimal posttransplant chimerism and for therapeutic strategies involving gene correction.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células-Tronco Hematopoéticas/imunologia , Linfo-Histiocitose Hemofagocítica/imunologia , Linfo-Histiocitose Hemofagocítica/terapia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Células-Tronco Hematopoéticas/citologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Linfo-Histiocitose Hemofagocítica/genética , Camundongos , Camundongos Transgênicos , Perforina , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Quimeras de TransplanteRESUMO
We performed gene-expression profiling of PBMCs obtained from patients with familial hemophagocytic lymphohistiocytosis (FHL) to screen for biologic correlates with the genetic and/or clinical forms of this disease. Unsupervised hierarchical clustering of 167 differentially expressed probe sets, representing 143 genes, identified 3 groups of patients corresponding to the genetic forms and clinical presentations of the disease. Two clusters of up- and down-regulated genes separated patients with perforin-deficient FHL from those with unidentified genetic cause(s) of the disease. The clusterscomprised genes involved in defense/immune responses, apoptosis, zinc homeostasis, and systemic inflammation. Unsupervised hierarchical clustering partitioned patients with unknown genetic cause(s) of FHL into 2 well-distinguished subgroups. Patterns of up- and down-regulated genes separated patients with "late-onset" and "relapsing" forms of FHL from patients with an "early onset and rapidly evolving" form of the disease. A cluster was identified in patients with "late onset and relapsing" form of FHL related to B- and T-cell differentiation/survival, T-cell activation, and vesicular transport. The resulting data suggest that unique gene-expression signatures can distinguish between genetic and clinical subtypes of FHL. These differentially expressed genes may represent biomarkers that can be used as predictors of disease progression.
Assuntos
Linfo-Histiocitose Hemofagocítica/genética , Adolescente , Idade de Início , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Lactente , Linfo-Histiocitose Hemofagocítica/classificação , Linfo-Histiocitose Hemofagocítica/imunologia , Família Multigênica , Mutação , Perforina , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Liver parenchymal cell allografts initiate both CD4-dependent and CD4-independent, CD8(+) T cell-mediated acute rejection pathways. The magnitude of allospecific CD8(+) T cell in vivo cytotoxic effector function is maximal when primed in the presence of CD4(+) T cells. The current studies were conducted to determine if and how CD4(+) T cells might influence cytotoxic effector mechanisms. METHODS: Mice were transplanted with allogeneic hepatocytes. In vivo cytotoxicity assays and various gene-deficient recipient mice and target cells were used to determine the development of Fas-, TNF-α-, and perforin-dependent cytotoxic effector mechanisms after transplantation. RESULTS: CD8(+) T cells maturing in CD4-sufficient hepatocyte recipients develop multiple (Fas-, TNF-α-, and perforin-mediated) cytotoxic mechanisms. However, CD8(+) T cells, maturing in the absence of CD4(+) T cells, mediate cytotoxicity and transplant rejection that is exclusively TNF-α/TNFR-dependent. To determine the kinetics of CD4-mediated help, CD4(+) T cells were adoptively transferred into CD4-deficient mice at various times posttransplant. The maximal influence of CD4(+) T cells on the magnitude of CD8-mediated in vivo allocytotoxicityf occurs within 48 hours. CONCLUSION: The implication of these studies is that interference of CD4(+) T cell function by disease or immunotherapy will have downstream consequences on both the magnitude of allocytotoxicity as well as the cytotoxic effector mechanisms used by allospecific CD8(+) cytolytic T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Rejeição de Enxerto/imunologia , Hepatócitos/transplante , Transplante de Fígado/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Transferência Adotiva , Animais , Antígenos CD4/genética , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/metabolismo , Sobrevivência de Enxerto , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Transplante de Fígado/efeitos adversos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais , Fatores de Tempo , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Contact hypersensitivity (CHS) is a T cell response to hapten skin challenge of sensitized individuals proposed to be mediated by hapten-primed CD8 cytolytic T cells. Effector CD8 T cell recruitment into hapten challenge sites to elicit CHS requires prior CXCL1- and CXCL2-mediated neutrophil infiltration into the site. We investigated whether neutrophil activities directing hapten-primed CD8 T cell skin infiltration in response to 2,4-dinitro-1-fluorobenzene (DNFB) required Fas ligand (FasL) and perforin expression. Although DNFB sensitization of gld/perforin-/- mice induced hapten-specific CD8 T cells producing IFN-γ and IL-17, these T cells did not infiltrate the DNFB challenge site to elicit CHS but did infiltrate the challenge site and elicit CHS when transferred to hapten-challenged naive wild-type recipients. Hapten-primed wild-type CD8 T cells, however, did not elicit CHS when transferred to naive gld/perforin-/- recipients. Wild-type bone marrow neutrophils expressed FasL and perforin, and when transferred to sensitized gld/perforin-/- mice, they restored hapten-primed CD8 T cell infiltration into the challenge site and CHS. The FasL/perforin-mediated activity of wild-type neutrophils induced the expression of T cell chemoattractants, CCL1, CCL2, and CCL5, within the hapten-challenged skin. These results indicate FasL/perforin-independent functions of hapten-primed CD8 T cells in CHS and identify new functions for neutrophils in regulating effector CD8 T cell recruitment and immune responses in the skin.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Movimento Celular/imunologia , Dermatite de Contato/imunologia , Dinitrofluorbenzeno/administração & dosagem , Proteína Ligante Fas/genética , Neutrófilos/imunologia , Proteínas Citotóxicas Formadoras de Poros/genética , Pele/imunologia , Animais , Antígenos/administração & dosagem , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Movimento Celular/genética , Dermatite de Contato/patologia , Dinitrofluorbenzeno/imunologia , Modelos Animais de Doenças , Orelha Externa , Proteína Ligante Fas/biossíntese , Feminino , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/biossíntese , Proteínas Citotóxicas Formadoras de Poros/deficiênciaRESUMO
BACKGROUND: Pancreatic cancer is still associated with devastating prognosis. Real progress in treatment options has still not been achieved. Therefore new models are urgently needed to investigate this deadly disease. As a part of this process we have established and characterized a new human pancreatic cancer cell line. METHODS: The newly established pancreatic cancer cell line PaCa 5061 was characterized for its morphology, growth rate, chromosomal analysis and mutational analysis of the K-ras, EGFR and p53 genes. Gene-amplification and RNA expression profiles were obtained using an Affymetrix microarray, and overexpression was validated by IHC analysis. Tumorigenicity and spontaneous metastasis formation of PaCa 5061 cells were analyzed in pfp-/-/rag2-/- mice. Sensitivity towards chemotherapy was analysed by MTT assay. RESULTS: PaCa 5061 cells grew as an adhering monolayer with a doubling time ranging from 30 to 48 hours. M-FISH analyses showed a hypertriploid complex karyotype with multiple numerical and unbalanced structural aberrations. Numerous genes were overexpressed, some of which have previously been implicated in pancreatic adenocarcinoma (GATA6, IGFBP3, IGFBP6), while others were detected for the first time (MEMO1, RIOK3). Specifically highly overexpressed genes (fold change > 10) were identified as EGFR, MUC4, CEACAM1, CEACAM5 and CEACAM6. Subcutaneous transplantation of PaCa 5061 into pfp-/-/rag2-/- mice resulted in formation of primary tumors and spontaneous lung metastasis. CONCLUSION: The established PaCa 5061 cell line and its injection into pfp-/-/rag2-/- mice can be used as a new model for studying various aspects of the biology of human pancreatic cancer and potential treatment approaches for the disease.
Assuntos
Adenocarcinoma/secundário , Neoplasias Pulmonares/secundário , Neoplasias Pancreáticas/patologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Forma Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Cariotipagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Fatores de Tempo , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Natural killer (NK) cells suppress graft-versus-host disease (GVHD) without causing GVHD themselves. Our previous studies demonstrated that allogeneic T cells and NK cells traffic similarly after allogeneic bone marrow transplantation (BMT). We therefore investigated the impact of donor NK cells on donor alloreactive T cells in GVHD induction. Animals receiving donor NK and T cells showed improved survival and decreased GVHD score compared with controls receiving donor T cells alone. Donor T cells exhibited less proliferation, lower CD25 expression, and decreased interferon-gamma (IFN-gamma) production in the presence of NK cells. In vivo, we observed perforin- and Fas ligand (FasL)-mediated reduction of donor T cell proliferation and increased T cell apoptosis in the presence of NK cells. Further, activated NK cells mediated direct lysis of reisolated GVHD-inducing T cells in vitro. The graft-versus-tumor (GVT) effect was retained in the presence of donor NK cells. We demonstrate a novel mechanism of NK cell-mediated GVHD reduction whereby donor NK cells inhibit and lyse autologous donor T cells activated during the initiation of GVHD.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Tumor/imunologia , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Animais , Apoptose , Transplante de Medula Óssea , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/biossíntese , Proteína Ligante Fas/deficiência , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Feminino , Mediadores da Inflamação/metabolismo , Interferon gama/deficiência , Interferon gama/genética , Interferon gama/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Células Matadoras Naturais/transplante , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Linfócitos T/citologia , Linfócitos T/transplante , Linfócitos T Reguladores/imunologia , Transplante HomólogoRESUMO
Cytotoxic lymphocytes (CLs) are the killer cells that destroy intracellular pathogen-infected and transformed cells, predominantly through the cytotoxic granule-mediated death pathway. Soluble cytotoxic granule components, including pore-forming perforin and pro-apoptotic serine proteases, granzymes, synergize to induce unscheduled apoptosis of the target cell. A complete loss of CL function results in an aggressive immunoregulatory disorder, familial hemophagocytic lymphohistiocytosis, whereas a partial loss of function seems to be a factor strongly predisposing to hematological malignancies. This review discusses the pathological manifestations of CL deficiencies due to impaired perforin function and describes novel aspects of perforin biology.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Vigilância Imunológica/fisiologia , Neoplasias/enzimologia , Neoplasias/imunologia , Proteínas Citotóxicas Formadoras de Poros/deficiência , Linfócitos T Citotóxicos/enzimologia , Animais , Proteínas Reguladoras de Apoptose/genética , Predisposição Genética para Doença/genética , Granzimas/metabolismo , Humanos , Sistema Imunitário/enzimologia , Sistema Imunitário/fisiopatologia , Linfo-Histiocitose Hemofagocítica/enzimologia , Linfo-Histiocitose Hemofagocítica/genética , Linfo-Histiocitose Hemofagocítica/imunologia , Camundongos , Neoplasias/genética , Perforina , Proteínas Citotóxicas Formadoras de Poros/genética , Linfócitos T Citotóxicos/imunologiaRESUMO
When developing malaria vaccines, the most crucial step is to elucidate the mechanisms involved in protective immunity against the parasites. We found that CD8(+) T cells contribute to protective immunity against infection with blood-stage parasites of Plasmodium yoelii. Infection of C57BL/6 mice with P. yoelii 17XL was lethal, while all mice infected with a low-virulence strain of the parasite 17XNL acquired complete resistance against re-infection with P. yoelii 17XL. However, the host mice transferred with CD8(+) T cells from mice primed only with P. yoelii 17XNL failed to acquire protective immunity. On the other hand, the irradiated host mice were completely resistant to P. yoelii 17XL infection, showing no grade of parasitemia when adoptively transferred with CD8(+) T cells from immune mice that survived infection with both P. yoelii XNL and, subsequently, P. yoelii 17XL. These protective CD8(+) T cells from immune WT mice had the potential to generate IFN-gamma, perforin (PFN) and granzyme B. When mice deficient in IFN-gamma were used as donor mice for CD8(+) T cells, protective immunity in the host mice was fully abrogated, and the immunity was profoundly attenuated in PFN-deficient mice. Thus, CD8(+) T cells producing IFN-gamma and PFN appear to be involved in protective immunity against infection with blood-stage malaria.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Malária/imunologia , Parasitemia/imunologia , Plasmodium yoelii/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Convalescença , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Granzimas/biossíntese , Interferon gama/biossíntese , Interferon gama/deficiência , Interferon gama/genética , Macrófagos/imunologia , Malária/prevenção & controle , Vacinas Antimaláricas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parasitemia/prevenção & controle , Plasmodium yoelii/patogenicidade , Proteínas Citotóxicas Formadoras de Poros/biossíntese , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Quimera por Radiação , VirulênciaRESUMO
Understanding the dual participation of the immune response in controlling the invader and at the same time causing tissue damage might contribute to the design of effective new vaccines and therapies for Chagas disease. Perforin, a cytolytic protein product of killer cells, is involved in resistance to acute Trypanosoma cruzi infection. However, the contribution of perforin in parasite control and chronic chagasic cardiomyopathy is unclear. Perforin-positive cells were detected in the heart tissue during the acute and chronic phases of infection of C57BL/6 mice inoculated with low dose (10(2) parasites) of the Colombian T. cruzi strain. This protocol led to acute phase survival in both wild-type and perforin null (pfp(-/-)) mice lineages. During the chronic infection, parasitism and inducible nitric oxide synthase (iNOS) as well as interleukin (IL)-4+ and, mainly, interferon (IFN)-gamma+ cells were more elevated in the heart tissue of pfp(-/-) mice. Higher levels of circulating NO and anti-parasite immunoglobulin (Ig)G2c and IgG3, paralleled by a prominent frequency of IFN-gamma+ and IL-10+ splenocytes, were present in pfp(-/-)-infected mice. Therefore, although the perforin-dependent pathway plays a role, it is not crucial for anti-T. cruzi immunity and acute phase survival of mice infected with a low inoculum. Further, perforin deficiency resulted in lower activity of creatine kinase-muscle brain isoform (CK-MB) isoenzyme in serum and a more restricted connexin 43 loss, both of which are markers of the cardiomyocyte lesion. Moreover, perforin deficiency hampered the development of severe electrocardiographic abnormalities. Hence, our results corroborate that perforin-bearing cytotoxic cells might contribute to cardiomyocyte lesion and heart dysfunction during chronic T. cruzi infection, shedding light on immunopathogenesis of chronic chagasic cardiomyopathy.
Assuntos
Cardiomiopatia Chagásica/imunologia , Citotoxicidade Imunológica , Miocardite/imunologia , Miocárdio/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Linfócitos T Citotóxicos/imunologia , Trypanosoma cruzi/imunologia , Doença Aguda , Animais , Anticorpos Antiprotozoários/sangue , Células Cultivadas , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/fisiopatologia , Cardiomiopatia Chagásica/prevenção & controle , Doença Crônica , Conexina 43/metabolismo , Creatina Quinase Forma MB/sangue , Modelos Animais de Doenças , Eletrocardiografia , Feminino , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/parasitologia , Miocardite/fisiopatologia , Miocardite/prevenção & controle , Miocárdio/metabolismo , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , Linfócitos T Citotóxicos/parasitologia , Fatores de Tempo , Trypanosoma cruzi/patogenicidadeRESUMO
BACKGROUND: Patients with inflammatory bowel disease (IBD) have a markedly increased risk to develop colon cancer, but there are only limited data about the host antitumor response in such colitis-associated cancer. In the present study we aimed at assessing the role of perforin-dependent effector mechanisms in the immune response in a murine model of colitis-associated colon cancer. METHODS: Wildtype and perforin-deficient mice were analyzed in a mouse model of colitis-associated colon cancer using azoxymethane (AOM) and dextran sodium sulfate (DSS). RESULTS: Tumors of wildtype mice showed infiltration of CD4+, CD8+ T cells, natural killer (NK) cells, high numbers of apoptotic cells, and expression of the transcription factor eomesodermin and cytotoxic effector proteins, suggesting a potential role of the antitumor immune response in AOM/DSS tumorigenesis. Furthermore, perforin deficiency resulted in reduced apoptosis of epithelial cells as compared to wildtype mice, whereas tumor infiltration by NK cells, CD8+, and CD4+ T cells was unchanged. However, perforin-deficient mice surprisingly developed significantly fewer tumors than wildtype mice. Subsequent studies identified an important role of perforin in regulating colitis activity, as perforin deficiency caused a significant reduction of DSS colitis activity and proinflammatory cytokine production as compared to wildtype controls. CONCLUSIONS: Perforin is involved in both the antitumor immune response and the regulation of activity of mucosal inflammation in colitis-associated cancer. Our data emphasize the possible consequences for therapeutic strategies targeting colitis-associated colon cancer.
Assuntos
Colite/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Proteínas Citotóxicas Formadoras de Poros/deficiência , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença Crônica , Colite/induzido quimicamente , Colite/patologia , Neoplasias do Colo/etiologia , Citotoxicidade Imunológica , Sulfato de Dextrana/toxicidade , Mucosa Intestinal/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Citotóxicas Formadoras de Poros/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos Específicos , Linfócitos T/imunologiaRESUMO
A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4(+) and CD8(+) T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4(+) and CD8(+) T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-gamma) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8(+) T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-gamma or IFN-gamma/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-gamma in the presence of highly cytotoxic T cells. Vaccinated IFN-gamma-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-gamma in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/prevenção & controle , Interferon gama/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Animais , Feminino , Imunização Secundária/métodos , Interferon gama/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Neuraminidase/genética , Neuraminidase/imunologia , Parasitemia/prevenção & controle , Proteínas Citotóxicas Formadoras de Poros/deficiência , Análise de Sobrevida , Vacinação/métodos , Vacinas de DNA/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Cytotoxic T lymphocytes and their granule components, such as perforin and granzyme, play an important role in the defense of hepatic infections caused by different pathogens. Moreover, it has been shown in vitro that hepatocytes can initiate cell death via a perforin-dependent mechanism. Although it is well known that hepatocellular apoptosis in D-galactosamine/lipopolysaccharide (D-Gal/LPS)-associated liver failure is mediated by TNF-alpha-dependent Fas/FasL cytotoxicity, there is no information on the role of perforin-mediated mechanisms in vivo. Therefore, we studied whether the cytolytic perforin/granzyme pathway contributes to the D-Gal/LPS-associated hepatotoxicity. Perforin knockout (Pko) mice showed significantly higher hepatic TNF-alpha and IL-6 mRNA expression as well as plasma TNF-alpha and IL-6 concentrations within the first hour upon D-Gal/LPS challenge compared with perforin wild-type (Pwt) mice. At 6 h upon D-Gal/LPS challenge, Pko mice further presented with higher transaminase release and onconecrotic tissue damage, whereas hepatocellular apoptosis and caspase-3 cleavage remained unaffected by the perforin deficiency. Pretreatment with a recombinant human TNF-alpha receptor fusion protein attenuated necrotic and apoptotic tissue damage and reduced plasma transaminase activities as well as cytokine release, thereby preventing acute liver failure in Pko mice as effectively as in Pwt mice. These data do not only confirm the significance of TNF-alpha as distal mediator of hepatic injury in this model but simultaneously reveal a contribution of a perforin-dependent immunoregulation, limiting the D-Gal/LPS-induced overwhelming cytokine release and onconecrotic tissue injury.
Assuntos
Apoptose , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Granzimas/metabolismo , Falência Hepática Aguda/imunologia , Fígado/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Transdução de Sinais , Linfócitos T Citotóxicos/imunologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Etanercepte , Galactosamina , Imunoglobulina G/farmacologia , Interleucina-6/sangue , Lipopolissacarídeos , Fígado/efeitos dos fármacos , Fígado/patologia , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/genética , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/enzimologia , Fatores de Tempo , Transaminases/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/sangueRESUMO
Hereditary haemophagocytic lymphohistiocytosis (HLH) is a fatal inflammatory disease and treatments currently may lead to serious side effects. There is a pressing need for effective, less toxic treatments for this disease. Previous reports have suggested that interferon gamma (IFNgamma) has a role in the pathogenesis of HLH. Here, we report that blocking IFNgamma had a therapeutic effect in two different murine models of human hereditary HLH (perforin-deficient and Rab27a-deficient mice, both infected with lymphocytic choriomeningitis virus). Therapeutic administration of an anti-IFNgamma antibody induced recovery from haemophagocytosis in both genetic models, as evidenced by increased survival in perforin-deficient mice and correction of blood cytopenia, moderation of body temperature changes, decreased cytokinaemia, restoration of splenic architecture and reduced haemophagocytosis in the liver of both murine models. Involvement of the central nervous system in Rab27a-deficient mice was prevented by anti-IFNgamma therapy. Hepatic T-cell infiltrates and virus persisted, with no detectable harm during the time course of these studies. These data strongly suggest that neutralization of IFNgamma could be used in humans to safely alleviate the clinical manifestations of haemophagocytosis.