RESUMO
Telomerase enables replicative immortality in most cancers including acute myeloid leukemia (AML). Imetelstat is a first-in-class telomerase inhibitor with clinical efficacy in myelofibrosis and myelodysplastic syndromes. Here, we develop an AML patient-derived xenograft resource and perform integrated genomics, transcriptomics and lipidomics analyses combined with functional genetics to identify key mediators of imetelstat efficacy. In a randomized phase II-like preclinical trial in patient-derived xenografts, imetelstat effectively diminishes AML burden and preferentially targets subgroups containing mutant NRAS and oxidative stress-associated gene expression signatures. Unbiased, genome-wide CRISPR/Cas9 editing identifies ferroptosis regulators as key mediators of imetelstat efficacy. Imetelstat promotes the formation of polyunsaturated fatty acid-containing phospholipids, causing excessive levels of lipid peroxidation and oxidative stress. Pharmacological inhibition of ferroptosis diminishes imetelstat efficacy. We leverage these mechanistic insights to develop an optimized therapeutic strategy using oxidative stress-inducing chemotherapy to sensitize patient samples to imetelstat causing substantial disease control in AML.
Assuntos
Ferroptose , Leucemia Mieloide Aguda , Oligonucleotídeos , Telomerase , Humanos , Telomerase/genética , Telomerase/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Ácidos GraxosRESUMO
Acute myeloid leukemia (AML) is a genetically heterogeneous, aggressive hematological malignancy induced by distinct oncogenic driver mutations. The effect of specific AML oncogenes on immune activation or suppression is unclear. Here, we examine immune responses in genetically distinct models of AML and demonstrate that specific AML oncogenes dictate immunogenicity, the quality of immune response and immune escape through immunoediting. Specifically, expression of NrasG12D alone is sufficient to drive a potent anti-leukemia response through increased MHC Class II expression that can be overcome with increased expression of Myc. These data have important implications for the design and implementation of personalized immunotherapies for patients with AML.
Assuntos
Neoplasias Hematológicas , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/patologia , Oncogenes , Neoplasias Hematológicas/genéticaRESUMO
Murine models offer a valuable tool to recapitulate genetically defined subtypes of AML, and to assess the potential of compound mutations and clonal evolution during disease progression. This is of particular importance for difficult to treat leukemias such as FLT3 internal tandem duplication (ITD) positive AML. While conditional gene targeting by Cre recombinase is a powerful technology that has revolutionized biomedical research, consequences of Cre expression such as lack of fidelity, toxicity or off-target effects need to be taken into consideration. We report on a transgenic murine model of FLT3-ITD induced disease, where Cre recombinase expression alone, and in the absence of a conditional allele, gives rise to an aggressive leukemia phenotype. Here, expression of various Cre recombinases leads to polyclonal expansion of FLT3ITD/ITD progenitor cells, induction of a differentiation block and activation of Myc-dependent gene expression programs. Our report is intended to alert the scientific community of potential risks associated with using this specific mouse model and of unexpected effects of Cre expression when investigating cooperative oncogenic mutations in murine models of cancer.
Assuntos
Leucemia Mieloide Aguda , Animais , Camundongos , Modelos Animais de Doenças , Tirosina Quinase 3 Semelhante a fms/genética , Duplicação Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos Transgênicos , MutaçãoRESUMO
Chemotherapy-resistant acute myeloid leukemia (AML), frequently driven by clonal evolution, has a dismal prognosis. A genome-wide CRISPR knockout screen investigating resistance to doxorubicin and cytarabine (Dox/AraC) in human AML cell lines identified gene knockouts involving AraC metabolism and genes that regulate cell cycle arrest (cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53) as contributing to resistance. In human AML cohorts, reduced expression of CDKN2A conferred inferior overall survival and CDKN2A downregulation occurred at relapse in paired diagnosis-relapse samples, validating its clinical relevance. Therapeutically targeting the G1S cell cycle restriction point (with CDK4/6 inhibitor, palbociclib and KAT6A inhibitor, WM-1119, to upregulate CDKN2A) synergized with chemotherapy. Additionally, direct promotion of apoptosis with venetoclax, showed substantial synergy with chemotherapy, overcoming resistance mediated by impaired cell cycle arrest. Altogether, we identify defective cell cycle arrest as a clinically relevant contributor to chemoresistance and identify rationally designed therapeutic combinations that enhance response in AML, potentially circumventing chemoresistance.
Assuntos
Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ciclo Celular , Citarabina/farmacologia , Citarabina/uso terapêutico , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular TumoralRESUMO
The caudal-related homeobox transcription factor CDX2 is expressed in leukemic cells but not during normal blood formation. Retroviral overexpression of Cdx2 induces AML in mice, however the developmental stage at which CDX2 exerts its effect is unknown. We developed a conditionally inducible Cdx2 mouse model to determine the effects of in vivo, inducible Cdx2 expression in hematopoietic stem and progenitor cells (HSPCs). Cdx2-transgenic mice develop myelodysplastic syndrome with progression to acute leukemia associated with acquisition of additional driver mutations. Cdx2-expressing HSPCs demonstrate enrichment of hematopoietic-specific enhancers associated with pro-differentiation transcription factors. Furthermore, treatment of Cdx2 AML with azacitidine decreases leukemic burden. Extended scheduling of low-dose azacitidine shows greater efficacy in comparison to intermittent higher-dose azacitidine, linked to more specific epigenetic modulation. Conditional Cdx2 expression in HSPCs is an inducible model of de novo leukemic transformation and can be used to optimize treatment in high-risk AML.
Assuntos
Fator de Transcrição CDX2/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicas/metabolismo , Animais , Fator de Transcrição CDX2/genética , Transformação Celular Neoplásica , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/fisiopatologiaRESUMO
JAK2V617F is the most common mutation in patients with BCR-ABL negative myeloproliferative neoplasms (MPNs). The eradication of JAK2V617F hematopoietic stem cells (HSCs) is critical for achieving molecular remissions and cure. We investigate the distinct effects of two therapies, ruxolitinib (JAK1/2 inhibitor) and interferon-alpha (IFN-α), on the disease-initiating HSC population. Whereas ruxolitinib inhibits Stat5 activation in erythroid progenitor populations, it fails to inhibit this same pathway in HSCs. In contrast, IFN-α has direct effects on HSCs. Furthermore, STAT1 phosphorylation and pathway activation is greater after IFN-α stimulation in Jak2V617F murine HSCs with increased induction of reactive oxygen species, DNA damage and reduction in quiescence after chronic IFN-α treatment. Interestingly, ruxolitinib does not block IFN-α induced reactive oxygen species and DNA damage in Jak2V617F murine HSCs in vivo. This work provides a mechanistic rationale informing how pegylated IFN-α reduces JAK2V617F allelic burden in the clinical setting and may inform future clinical efforts to combine ruxolitinib with pegylated IFN-α in patients with MPN.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Interferon-alfa/farmacologia , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Pirazóis/farmacologia , Fator de Transcrição STAT1/metabolismo , Animais , Antivirais/farmacologia , Proliferação de Células , Células Cultivadas , Quimioterapia Combinada , Feminino , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Nitrilas , Pirimidinas , Fator de Transcrição STAT1/genéticaRESUMO
Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a-/- Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer-driven inflammatory signaling.
Assuntos
Substituição de Aminoácidos , DNA (Citosina-5-)-Metiltransferases , Neoplasias Hematológicas , Células-Tronco Hematopoéticas , Mutação de Sentido Incorreto , Mielofibrose Primária , Transdução de Sinais/genética , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/patologia , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Camundongos Mutantes , Mielofibrose Primária/enzimologia , Mielofibrose Primária/genética , Mielofibrose Primária/patologiaRESUMO
Mucosal-associated invariant T (MAIT) cells are a unique innate-like T cell subset that responds to a wide array of bacteria and yeast through recognition of riboflavin metabolites presented by the MHC class I-like molecule MR1. Here, we demonstrate using MR1 tetramers that recipient MAIT cells are present in small but definable numbers in graft-versus-host disease (GVHD) target organs and protect from acute GVHD in the colon following bone marrow transplantation (BMT). Consistent with their preferential juxtaposition to microbial signals in the colon, recipient MAIT cells generate large amounts of IL-17A, promote gastrointestinal tract integrity, and limit the donor alloantigen presentation that in turn drives donor Th1 and Th17 expansion specifically in the colon after BMT. Allogeneic BMT recipients deficient in IL-17A also develop accelerated GVHD, suggesting MAIT cells likely regulate GVHD, at least in part, by the generation of this cytokine. Indeed, analysis of stool microbiota and colon tissue from IL-17A-/- and MR1-/- mice identified analogous shifts in microbiome operational taxonomic units (OTU) and mediators of barrier integrity that appear to represent pathways controlled by similar, IL-17A-dependent mechanisms. Thus, MAIT cells act to control barrier function to attenuate pathogenic T cell responses in the colon and, given their very high frequency in humans, likely represent an important population in clinical BMT.
Assuntos
Transplante de Medula Óssea , Colo/imunologia , Doenças do Colo/imunologia , Doença Enxerto-Hospedeiro/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Células Th17/imunologia , Aloenxertos , Animais , Colo/patologia , Doenças do Colo/genética , Doenças do Colo/patologia , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células T Invariantes Associadas à Mucosa/patologia , Células Th17/patologiaRESUMO
Eph receptors have emerged as targets for therapy in both neoplastic and non-neoplastic disease, however, particularly in non-neoplastic diseases, redundancy of function limits the effectiveness of targeting individual Eph proteins. We have shown previously that a soluble fusion protein, where the EphA4 ectodomain was fused to IgG Fc (EphA4 Fc), was an effective therapy in acute injuries and demonstrated that EphA4 Fc was a broad spectrum Eph/ephrin antagonist. However, a very short in vivo half-life effectively limited its therapeutic development. We report a unique glycoengineering approach to enhance the half-life of EphA4 Fc. Progressive deletion of three demonstrated N-linked sites in EphA4 progressively increased in vivo half-life such that the triple mutant protein showed dramatically improved pharmacokinetic characteristics. Importantly, protein stability, affinity for ephrin ligands and antagonism of cell expressed EphA4 was fully preserved, enabling it to be developed as a broad spectrum Eph/ephrin antagonist for use in both acute and chronic diseases.
Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Receptor EphA1/antagonistas & inibidores , Receptor EphA4/metabolismo , Receptor EphA4/farmacocinética , Análise Mutacional de DNA , Glicosilação , Meia-Vida , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptor EphA4/química , Receptor EphA4/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
Glioblastoma (GBM) is an essentially incurable and rapidly fatal cancer, with few markers predicting a favourable prognosis. Here we report that the transcription factor NFIB is associated with significantly improved survival in GBM. NFIB expression correlates inversely with astrocytoma grade and is lowest in mesenchymal GBM. Ectopic expression of NFIB in low-passage, patient-derived classical and mesenchymal subtype GBM cells inhibits tumourigenesis. Ectopic NFIB expression activated phospho-STAT3 signalling only in classical and mesenchymal GBM cells, suggesting a mechanism through which NFIB may exert its context-dependent tumour suppressor activity. Finally, NFIB expression can be induced in GBM cells by drug treatment with beneficial effects.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Fatores de Transcrição NFI/metabolismo , Animais , Linhagem Celular Tumoral , Genes Supressores de Tumor/fisiologia , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Supressoras de Tumor/metabolismoRESUMO
Significant endeavor has been applied to identify functional therapeutic targets in glioblastoma (GBM) to halt the growth of this aggressive cancer. We show that the receptor tyrosine kinase EphA3 is frequently overexpressed in GBM and, in particular, in the most aggressive mesenchymal subtype. Importantly, EphA3 is highly expressed on the tumor-initiating cell population in glioma and appears critically involved in maintaining tumor cells in a less differentiated state by modulating mitogen-activated protein kinase signaling. EphA3 knockdown or depletion of EphA3-positive tumor cells reduced tumorigenic potential to a degree comparable to treatment with a therapeutic radiolabelled EphA3-specific monoclonal antibody. These results identify EphA3 as a functional, targetable receptor in GBM.
Assuntos
Neoplasias Encefálicas/prevenção & controle , Glioblastoma/prevenção & controle , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Neoplásicas/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Apoptose , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Diferenciação Celular , Proliferação de Células , Citometria de Fluxo , Imunofluorescência , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptor EphA3 , Células Tumorais CultivadasRESUMO
Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.
Assuntos
Cavéolas/metabolismo , Cavéolas/ultraestrutura , Caveolinas/metabolismo , Escherichia coli , Mamíferos/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , HumanosRESUMO
Hsp90α/ß, the signal transduction chaperone, maintains intracellular communication in normal, stem, and cancer cells. The well characterised association of Hsp90α/ß with its client kinases form the framework of multiple signalling networks. GSK3ß, a known Hsp90α/ß client, mediates ß-catenin phosphorylation as part of a cytoplasmic destruction complex which targets phospho-ß-catenin to the 26S proteasome. The canonical Wnt/ß-catenin pathway promotes stem cell self-renewal as well as oncogenesis. The degree of Hsp90α/ß involvement in Wnt/ß-catenin signalling needs clarification. Here, we describe the association of Hsp90α/ß with GSK3ß, ß-catenin, phospho-ß-catenin and the molecular scaffold, axin1, in the human MCF-7 epithelial breast cancer cell model using selective inhibition of Hsp90α/ß, confocal laser scanning microscopy and immunoprecipitation. Our findings suggest that Hsp90α/ß modulates the phosphorylation of ß-catenin by interaction in common complex with GSK3ß/axin1/ß-catenin.
Assuntos
Proteína Axina/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , beta Catenina/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Feminino , Glicogênio Sintase Quinase 3 beta , Humanos , Imunoprecipitação , Modelos Biológicos , FosforilaçãoRESUMO
The ability of galantamine (Reminyl) to inhibit the aggregation and toxicity of the beta-amyloid peptide (Abeta) was investigated. Galantamine showed concentration-dependent inhibition of aggregation of both Abeta 1-40 and Abeta 1-42, as determined by an ELISA method. Electron microscope studies of Abeta 1-40 incubated in the presence of galantamine revealed fibrils that were disordered and clumped in appearance. MTT and lactate dehydrogenase assays, employing SH-SY5Y human neuroblastoma cells, showed that galantamine reduced the cytotoxicity induced by Abeta 1-40. Galantamine also dramatically reduced Abeta 1-40-induced cellular apoptosis in these cells. There is some evidence that galantamine may not be acting purely as a symptomatic treatment. Disease-modifying effects of the drug could be due to an additional effect on Abeta aggregation and/or toxicity.
Assuntos
Peptídeos beta-Amiloides/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Galantamina/farmacologia , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Análise de Variância , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores da Colinesterase/química , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Galantamina/química , Humanos , L-Lactato Desidrogenase/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Fármacos Neuroprotetores/química , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidadeRESUMO
PURPOSE: To review our attempt to devise a method of cultivated corneal endothelial transplantation using primates in which corneal endothelium, like that of humans, has low proliferative ability. METHODS: Monkey corneal endothelial cells (MCECs) were cultivated, with subcultures grown on collagen type I carriers. The corneal endothelia of 6 eyes of 6 monkeys were scraped intensively, after which cultivated MCEC sheets were inserted into the anterior chamber of 4 eyes. As controls, a collagen sheet without MCECs was transplanted in 1 eye of a monkey, and a suspension of cultivated MCECs was injected into the anterior chamber of 1 eye of another monkey. RESULTS: MCECs produced a confluent monolayer of closely attached hexagonal cells, which expressed both ZO-1 and Na-K adenosine triphosphatase. Early postoperative period MCEC sheets were attached to Descemet membrane, and corneal clarity was recovered. Six months after transplantation, MCEC-transplanted corneas remained clear, and closely attached hexagonal cells were observed. In 1 animal with longer observation, polygonal cells were observed by in vivo specular microscopy at a density of >2000 cells/mm2 and remained >1600 cells/mm2 for < or =2 years. Control eyes showed irreversible bullous keratopathy throughout the observation period. CONCLUSIONS: Cultivated MCECs become attached to the transplanted eye and maintain a clear cornea < or =2 years postoperatively, suggesting that corneal endothelial cells of primates might have proliferative ability in vivo once they have been cultured and proliferated in vitro. Our monkey model constitutes an important step forward for regenerative medicine with possible future application in patients with corneal endothelial dysfunction.
Assuntos
Doenças da Córnea/cirurgia , Endotélio Corneano/transplante , Engenharia Tecidual , Animais , Adesão Celular , Proliferação de Células , Células Cultivadas , Córnea/patologia , Doenças da Córnea/patologia , Lâmina Limitante Posterior/patologia , Endotélio Corneano/citologia , Endotélio Corneano/patologia , Endotélio Corneano/fisiopatologia , Macaca fascicularis , Microscopia Eletrônica , Medicina Regenerativa/tendências , Engenharia Tecidual/métodos , Transplante HomólogoRESUMO
Based on the life-time cost to the health care system, the Institute of Medicine has assigned the highest priority for a vaccine to control human cytomegalovirus (HCMV) disease in transplant patients and new born babies. In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive. Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide. Immunisation with this chimeric vaccine consistently generated strong HCMV-specific CD8(+) and CD4(+) T-cells which co-expressed IFN-gamma and TNF-alpha, while the humoral response induced by this vaccine showed strong virus neutralizing capacity. More importantly, immunization with adenoviral chimeric vaccine also afforded protection against challenge with recombinant vaccinia virus encoding HCMV antigens and this protection was associated with the induction of a pluripotent antigen-specific cellular and antibody response. Furthermore, in vitro stimulation with this adenoviral chimeric vaccine rapidly expanded multiple antigen-specific human CD8(+) and CD4(+) T-cells from healthy virus carriers. These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings.
Assuntos
Vacinas contra Citomegalovirus/uso terapêutico , Citomegalovirus/metabolismo , Vacinas/química , Animais , Formação de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/prevenção & controle , Genes MHC Classe I , Genes MHC da Classe II , Humanos , Sistema Imunitário/virologia , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/química , Vacinas/metabolismoRESUMO
Studies based on either MHC class II-knockout or CD4+ T cell-depleted murine models have demonstrated a critical role for CD4+ T cells in the generation of CD8+ T cell memory. However, it is difficult to extend these findings to immunocompromised humans where a complete loss of CD4+ T cells is rarely observed. Here, we have developed a model setting, which allows studies on the generation of CD8+ T cell memory responses in a transient CD4+ T cell-deficient setting similar to that seen in immunocompromised patients. Immunisation with an adenoviral vaccine under transient helpless or help-deficient conditions showed varying degrees of impact on the priming of CD8+ T cell responses. Antigen-specific T cells generated under normal CD4+ T cell help and transient help-deficient conditions showed similar effector phenotype and were capable of proliferation upon secondary antigen encounter. Most importantly, in spite of CD4+ T cell deficiency, the long-term CD8+ T cell memory response remained functionally stable and showed comparable cytotoxic effector function as seen in CD8+ T cells generated with normal CD4+ T cell numbers. These findings provide evidence that in spite of partially impaired activation of a primary CD8+ T cell response, a fully functional and stable memory CTL response can be induced under conditions of severe transient CD4+ T cell deficiency.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Herpesvirus Humano 4/imunologia , Hospedeiro Imunocomprometido , Memória Imunológica , Vacinas Virais/imunologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Ativação Linfocitária , Depleção Linfocítica , Camundongos , Camundongos Transgênicos , Vacinas Sintéticas/imunologia , Proteínas da Matriz Viral/imunologiaRESUMO
CD8(+) T-cell responses to persistent viral infections are characterized by the accumulation of an oligoclonal T-cell repertoire and a reduction in the naive T-cell pool. However, the precise mechanism for this phenomenon remains elusive. Here we show that human cytomegalovirus (HCMV)-specific CD8(+) T cells recognizing distinct epitopes from the pp65 protein and restricted through an identical HLA class I allele (HLA B*3508) exhibited either a highly conserved public T-cell repertoire or a private, diverse T-cell response, which was uniquely altered in each donor following in vitro antigen exposure. Selection of a public T-cell receptor (TCR) was coincident with an atypical major histocompatibility complex (MHC)-peptide structure, in that the epitope adopted a helical conformation that bulged from the peptide-binding groove, while a diverse TCR profile was observed in response to the epitope that formed a flatter, more "featureless" landscape. Clonotypes with biased TCR usage demonstrated more efficient recognition of virus-infected cells, a greater CD8 dependency, and were more terminally differentiated in their phenotype when compared with the T cells expressing diverse TCR. These findings provide new insights into our understanding on how the biology of antigen presentation in addition to the structural features of the pMHC-I might shape the T-cell repertoire and its phenotype.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Peptídeos/imunologia , Alelos , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Sequência de Bases , Linhagem Celular , Células Clonais , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Cristalografia por Raios X , Epitopos/imunologia , Antígenos HLA-B/imunologia , Humanos , Dados de Sequência Molecular , Peptídeos/química , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
PURPOSE: To examine the feasibility of cultivated corneal endothelial cell transplantation in a primate model. METHODS: Monkey corneal endothelial cells (MCECs) obtained from three cynomolgus monkeys were cultivated, with subcultures grown on collagen type I carriers for 4 weeks. The corneal endothelium of the right eye of six monkeys was mechanically scraped, after which a cultivated MCEC sheet was brought into the anterior chamber of four eyes and fixed to Descemet's membrane by air. As the control, a collagen sheet without MCECs was transplanted into one eye of one monkey, and a suspension of cultivated MCECs was injected into the anterior chamber in one eye. RESULTS: Cultivated MCECs produced a confluent monolayer of closely attached hexagonal cells that showed both ZO-1 and Na(+)-K(+) ATPase expression. In the early postoperative period MCEC sheets were attached to Descemet's membrane and corneal clarity was recovered. The recovered clarity was accompanied by a decrease in corneal thickness. Fluorescein DiI labeled donor corneal endothelial cells were detected on the host cornea on postoperative day 7. Six months after transplantation MCEC-transplanted corneas remained clear, and hexagonal cells were observed by in vivo specular microscopy with a density of 1992 to 2475 cells/mm(2). Control eyes showed irreversible bullous keratopathy that precluded pachymetry and specular microscopy. CONCLUSIONS: A model of cultivated corneal endothelial transplantation for corneal endothelial dysfunction was established in primates whose corneal endothelial cells have less proliferative capacity in vivo. Our results suggest that this is a useful model for long-term observation in advance of the future clinical application of cultivated corneal endothelial transplantation.
Assuntos
Câmara Anterior/cirurgia , Lâmina Limitante Posterior/cirurgia , Endotélio Corneano/transplante , Modelos Animais , Animais , Adesão Celular , Contagem de Células , Transplante de Células , Células Cultivadas , Endotélio Corneano/citologia , Endotélio Corneano/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes , Sobrevivência de Enxerto/fisiologia , Macaca fascicularis , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Fosfoproteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Transplante Homólogo , Proteína da Zônula de Oclusão-1RESUMO
Amyloid deposits derived from the amylin peptide accumulate within pancreatic islet beta-cells in most cases of type-2 diabetes mellitus (T2Dm). Human amylin 'oligomers' are toxic to these cells. Using two different experimental techniques, we found that H(2)O(2) was generated during the aggregation of human amylin into amyloid fibrils. This process was greatly stimulated by Cu(II) ions, and human amylin was retained on a copper affinity column. In contrast, rodent amylin, which is not toxic, failed to generate any H(2)O(2) and did not interact with copper. We conclude that the formation of H(2)O(2) from amylin could contribute to the progressive degeneration of islet cells in T2Dm.