RESUMO
Bladder cancer invasion depends on mammalian target of rapamycin complex 2 (mTORC2) activity, although the downstream mTORC2 effectors that mediate this effect have not been fully defined. One potential downstream effector is the arginine derivative nitric oxide (NO). This study identified a stage-associated increase in the expression of the NO-generating enzymes endothelial NO synthase (eNOS) and inducible NOS (iNOS) in human bladder cancer. Reduction of NOS activity by pharmacologic inhibition or silencing of NOS enzymes reduced cancer cell invasion, with similar effects observed using the NO scavenger cobinamide. By contrast, enhanced invasion was seen with the NO donor Deta-NONOate and an analog of the downstream NO second messenger cGMP. Next, NOS expression was evaluated in invadopodia, which are cellular protrusions that form the invasive tips of cancer cells. Invadopodia were enriched in both iNOS protein and mTORC2 activity, and invadopodia formation was increased by Deta-NONOate and decreased by cobinamide and ablation of mTORC2 activity. Additionally, mTORC2 increased expression of iNOS. Using a zebrafish model, injection of iNOS- or rictor-silenced cells reduced the frequency of bladder cancer cell metastasis in zebrafish. These results indicate that mTORC2 can mediate bladder cancer cell invasion through increased iNOS expression, resulting in increased NO and cGMP production in invadopodia and further propagation of invadopodia formation.
Assuntos
Alvo Mecanístico do Complexo 2 de Rapamicina/fisiologia , Óxido Nítrico/metabolismo , Podossomos/metabolismo , Neoplasias da Bexiga Urinária/patologia , Animais , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Embrião não Mamífero , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Podossomos/genética , Podossomos/patologia , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Peixe-Zebra/embriologiaRESUMO
Treatment with partial (p)MHC class II-ß1α1 constructs (also referred to as recombinant T-cell receptor ligands - RTL) linked to antigenic peptides can induce T-cell tolerance, inhibit recruitment of inflammatory cells and reverse autoimmune diseases. Here we demonstrate a novel regulatory pathway that involves RTL binding to CD11b(+) mononuclear cells through a receptor comprised of MHC class II invariant chain (CD74), cell-surface histones and MHC class II itself for treatment of experimental autoimmune encephalomyelitis (EAE). Binding of RTL constructs with CD74 involved a previously unrecognized MHC class II-α1/CD74 interaction that inhibited CD74 expression, blocked activity of its ligand, macrophage migration inhibitory factor, and reduced EAE severity. These findings implicate binding of RTL constructs to CD74 as a key step in both antigen-driven and bystander T-cell tolerance important in treatment of inflammatory diseases.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Doenças Autoimunes/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Monócitos/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígeno CD11b/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica , Oxirredutases Intramoleculares , Fatores Inibidores da Migração de Macrófagos , Proteínas de Membrana , Camundongos , Receptores de Antígenos de Linfócitos T , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/imunologiaRESUMO
MHC class II-derived recombinant T cell receptor ligands (RTLs) modulate the behavior of pathogenic T cells and can reverse clinical and histological signs of autoimmune disease in experimental autoimmune encephalomyelitis (EAE), experimental autoimmune uveitis (EAU) and collagen-induced arthritis (CIA), and are currently in clinical trials for treatment of multiple sclerosis (MS). To expand the utility of these rationally-designed biologics and explore their mechanism(s) of activity in vivo, we have engineered RTL constructs bearing cysteine-tethered antigenic peptides and demonstrate that the appropriate cysteine-tethered RTLs effectively treat EAE. The data presented here suggests that the mechanism by which antigen-specific tolerance induction by RTLs bearing cysteine-tethered antigenic peptides in vivo involves delivery of RTL/antigen to endosomal compartments for processing and re-presentation by full-length MHC class II, with RTLs bearing cysteine-tethered antigenic peptides requiring gamma-interferon-inducible lysosomal thiol-reductase (GILT) for therapeutic activity.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Antígenos de Histocompatibilidade Classe II/uso terapêutico , Oxirredutases/metabolismo , Fragmentos de Peptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Endossomos/química , Endossomos/metabolismo , Genes MHC da Classe II/genética , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Mycobacterium tuberculosis/imunologia , Oxirredutases/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Engenharia de Proteínas , Proteínas Recombinantes/uso terapêutico , Linfócitos T/metabolismoRESUMO
The mammalian target of rapamycin (mTOR) pathway regulates important cellular functions. Aberrant activation of this pathway, either through upstream activation by growth factors, loss of inhibitory controls, or molecular alterations, can enhance cancer growth and progression. Bladder cancer shows high levels of mTOR activity in approximately 70% of urothelial carcinomas, suggesting a key role for this pathway in this cancer. mTOR signaling initiates through upstream activation of phosphatidylinositol 3 kinase (PI3K) and protein kinase B (AKT) and results in activation of either mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2). While these complexes share several key protein components, unique differences in their complex composition dramatically alter the function and downstream cellular targets of mTOR activity. While significant work has gone into analysis of molecular alterations of the mTOR pathway in bladder cancer, this has not yielded significant benefit in mTOR-targeted therapy approaches in urothelial carcinoma to date. New discoveries regarding signaling convergence onto mTOR complexes in bladder cancer could yield unique insights the biology and targeting of this aggressive disease. In this review, we highlight the functional significance of mTOR signaling in urothelial carcinoma and its potential impact on future therapy implications.
Assuntos
Antineoplásicos/uso terapêutico , Exossomos/patologia , Leucemia Mieloide Aguda/patologia , Células-Tronco Mesenquimais/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Adolescente , Adulto , Idoso , Criança , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/antagonistas & inibidores , Adulto JovemRESUMO
Chromosomal rearrangements of the NTRK genes generate kinase fusions that are targetable oncogenic drivers in diverse adult and pediatric malignancies. Despite robust clinical response to targeted NTRK inhibition, the emergence of therapeutic resistance poses a formidable clinical challenge. Here we report the characterization of an ETV6-NTRK3 fusion-driven pediatric glioma that progressed through NTRK-targeted treatments with entrectinib and selitrectinib. Genetic analysis of multifocal recurrent/resistant lesions identified a previously uncharacterized NTRK3 p.G623A and a known p.G623E resistance mutation, in addition to other alterations of potential pathogenic impact. Functional studies using heterologous reconstitution model systems and patient-derived tumor cell lines establish that NTRK3G623A and NTRK3G623E mutated kinases exhibit reduced sensitivity to entrectinib and selitrectinib, as well as other NTRK inhibitors tested herein. In summary, this genetic analysis of multifocal recurrent/resistant glioma driven by ETV6-NTRK3 fusion captured a cross section of resistance-associated alterations that, based on in vitro analysis, likely contributed to resistance to targeted therapy and disease progression.
Assuntos
Glioma , Proteínas de Fusão Oncogênica , Criança , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Recidiva Local de Neoplasia , Proteínas de Fusão Oncogênica/genética , Oncogenes , Receptores Proteína Tirosina QuinasesRESUMO
Recombinant T-cell receptor ligands (RTLs) can prevent and reverse clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner and are currently in clinical trials for treatment of subjects with multiple sclerosis (MS). To evaluate regulatory mechanisms, we designed and tested RTL551, containing the alpha1 and beta1 domains of the I-A(b) class II molecule covalently linked to the encephalitogenic MOG-35-55 peptide in C57BL/6 mice. Treatment of active or passive EAE with RTL551 after disease onset significantly reduced clinical signs and spinal cord lesions. Moreover, RTL551 treatment strongly and selectively reduced secretion of interleukin-17 and tumor necrosis factor alpha by transferred green fluorescent protein-positive (GFP+) MOG-35-55-reactive T-cells and almost completely abrogated existent GFP+ cellular infiltrates in affected spinal cord sections. Reduced inflammation in spinal cords of RTL551-treated mice was accompanied by a highly significant downregulation of chemokines and their receptors and inhibition of VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) expression by endothelial cells. Thus, RTL therapy cannot only inhibit systemic production of encephalitogenic cytokines by the targeted myelin oligodendrocyte glycoprotein-reactive T-cells but also impedes downstream local recruitment and retention of inflammatory cells in the CNS. These findings indicate that targeted immunotherapy of antigen-specific T-cells can result in a reversal of CNS lesion formation and lend strong support to the application of the RTL approach for therapy in MS.
Assuntos
Movimento Celular/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Fatores Imunológicos/farmacologia , Interleucina-17/metabolismo , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/imunologia , Movimento Celular/imunologia , Quimiocinas/efeitos dos fármacos , Quimiocinas/imunologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Proteínas de Fluorescência Verde , Fatores Imunológicos/uso terapêutico , Imunoterapia/métodos , Interleucina-17/imunologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Esclerose Múltipla/fisiopatologia , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Therapeutic vaccination using T-cell receptor (TCR) peptides from V genes commonly expressed by potentially pathogenic T cells remains an approach of interest for treatment of multiple sclerosis (MS) and other autoimmune diseases. We developed a trivalent TCR vaccine containing complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 emulsified in incomplete Freund's adjuvant that reliably induced high frequencies of TCR-specific T cells. To evaluate induction of regulatory T-cell subtypes, immunological and clinical parameters were followed in 23 treatment-naïve subjects with relapsing-remitting or progressive MS who received 12 monthly injections of the trivalent peptide vaccine over 1 year in an open-label study design. Prior to vaccination, subjects had reduced expression of forkhead box (Fox) P3 message and protein, and reduced recognition of the expressed TCR repertoire by TCR-reactive cells compared with healthy control donors. After three or four injections, most vaccinated MS subjects developed high frequencies of circulating interleukin (IL)-10-secreting T cells specific for the injected TCR peptides and significantly enhanced expression of FoxP3 by regulatory T cells present in both 'native' CD4+ CD25+ and 'inducible' CD4+ CD25- peripheral blood mononuclear cells (PBMC). At the end of the trial, PBMC from vaccinated MS subjects retained or further increased FoxP3 expression levels, exhibited significantly enhanced recognition of the TCR V gene repertoire apparently generated by perturbation of the TCR network, and significantly suppressed neuroantigen but not recall antigen responses. These findings demonstrate that therapeutic vaccination using only three commonly expressed BV gene determinants can induce an expanded immunoregulatory network in vivo that may optimally control complex autoreactive responses that characterize the inflammatory phase of MS.
Assuntos
Fatores de Transcrição Forkhead/sangue , Esclerose Múltipla/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/uso terapêutico , Adulto , Idoso , Autoantígenos/imunologia , Autoimunidade/imunologia , Regiões Determinantes de Complementaridade/imunologia , Feminino , Genes Codificadores dos Receptores de Linfócitos T/imunologia , Humanos , Tolerância Imunológica/imunologia , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Proteínas do Tecido Nervoso/imunologia , Linfócitos T Reguladores/imunologia , Vacinação/métodosRESUMO
Exosomes are paracrine regulators of the tumor microenvironment and contain complex cargo. We previously reported that exosomes released from acute myeloid leukemia (AML) cells can suppress residual hematopoietic stem and progenitor cell (HSPC) function indirectly through stromal reprogramming of niche retention factors. We found that the systemic loss of hematopoietic function is also in part a consequence of AML exosome-directed microRNA (miRNA) trafficking to HSPCs. Exosomes isolated from cultured AML or the plasma from mice bearing AML xenografts exhibited enrichment of miR-150 and miR-155. HSPCs cocultured with either of these exosomes exhibited impaired clonogenicity, through the miR-150- and miR-155-mediated suppression of the translation of transcripts encoding c-MYB, a transcription factor involved in HSPC differentiation and proliferation. To discover additional miRNA targets, we captured miR-155 and its target transcripts by coimmunoprecipitation with an attenuated RNA-induced silencing complex (RISC)-trap, followed by high-throughput sequencing. This approach identified known and previously unknown miR-155 target transcripts. Integration of the miR-155 targets with information from the protein interaction database STRING revealed proteins indirectly affected by AML exosome-derived miRNA. Our findings indicate a direct effect of AML exosomes on HSPCs that, through a stroma-independent mechanism, compromises hematopoiesis. Furthermore, combining miRNA target data with protein-protein interaction data may be a broadly applicable strategy to define the effects of exosome-mediated trafficking of regulatory molecules within the tumor microenvironment.
Assuntos
Exossomos/metabolismo , Hematopoese , Leucemia Mieloide Aguda/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myb/biossíntese , RNA Neoplásico/metabolismo , Animais , Exossomos/genética , Exossomos/patologia , Células HL-60 , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , MicroRNAs/genética , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-myb/genética , RNA Neoplásico/genéticaRESUMO
Estrogen (E2) upregulates the FoxP3 gene that marks regulatory CD4+CD25+ T cells (Treg cells). However, E2 also inhibits the ability of antigen presenting cells (APC) to activate T cells. It is possible that these opposing functions might affect the degree of overt suppression during pregnancy and autoimmunity. To evaluate E2 effects on Treg cell function, we quantified FoxP3 levels and Treg suppression in CD4+CD25+ T cells from pregnant and E2-treated mice, and overt Treg suppression in E2- vs. placebo-pretreated mice with autoimmune encephalomyelitis. The data clearly demonstrate that enhanced expression of FoxP3, which occurs in pregnant mice and in mice treated exogenously with E2 pellets, results in a concomitant increase in functional suppression within the CD4+CD25(bright) Treg fraction of splenocytes. The similarities in FoxP3 expression and Treg cell function in E2-treated and pregnant mice implicate E2 as a major contributor for increasing Treg function during pregnancy. Surprisingly, suppression was not enhanced when Treg cells from E2-treated mice were activated with APC and CD4+CD25- responder T cells from the same E2-treated mice, a result consistent with impaired APC activation of Treg cells. In contrast, Treg suppression was strikingly enhanced in combined cell cultures from E2-pretreated mice that were protected from EAE induced with neuroantigen in complete Freund's adjuvant. These results suggest that E2 treatment may have opposing effects on Treg cells vs. APC that both contribute to overt suppression, but such effects are overcome and focused towards enhanced suppression in inflammatory environments produced during pregnancy and EAE.
Assuntos
Estradiol/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Prenhez/fisiologia , Linfócitos T Reguladores/fisiologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/prevenção & controle , Feminino , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Prenhez/metabolismo , Receptores de Interleucina-2/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Células Th2/metabolismoRESUMO
Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the ß-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the `empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure.
RESUMO
Relapse remains the major cause of mortality for patients with Acute Myeloid Leukemia (AML). Improved tracking of minimal residual disease (MRD) holds the promise of timely treatment adjustments to preempt relapse. Current surveillance techniques detect circulating blasts that coincide with advanced disease and poorly reflect MRD during early relapse. Here, we investigate exosomes as a minimally invasive platform for a microRNA (miRNA) biomarker. We identify a set of miRNA enriched in AML exosomes and track levels of circulating exosome miRNA that distinguish leukemic xenografts from both non-engrafted and human CD34+ controls. We develop biostatistical models that reveal circulating exosomal miRNA at low marrow tumor burden and before circulating blasts can be detected. Remarkably, both leukemic blasts and marrow stroma contribute to serum exosome miRNA. We propose development of serum exosome miRNA as a platform for a novel, sensitive compartment biomarker for prospective tracking and early detection of AML recurrence.
Assuntos
Biomarcadores Tumorais/sangue , Exossomos/metabolismo , Leucemia Mieloide Aguda/sangue , MicroRNAs/sangue , Neoplasias Experimentais/sangue , RNA Neoplásico/sangue , Animais , Células HL-60 , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Experimentais/patologia , Células U937RESUMO
Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Ensaios Clínicos como Assunto , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinase/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-6 , Transdução de Sinais , Quinase Syk , Regulação para Cima , Quinases da Família src/metabolismoRESUMO
PURPOSE: To evaluate the immunotherapeutic efficacy of recombinant T cell receptor ligands (RTLs) specific for arrestin immunity in treatment of experimental autoimmune uveitis (EAU) in humanized leukocyte antigen (HLA-DR3) transgenic (Tg) mice. METHODS: We generated de novo recombinant human DR3-derived RTLs bearing covalently tethered arrestin peptides 291-310 (RTL351) or 305-324 (RTL352). EAU was induced by immunization of HLA-DR3 mice with arrestin or arrestin peptide and treated with RTLs by subcutaneous delivery. T cell proliferation and cytokine expression was measured in RTL-treated and control mice. RESULTS: RTL351 prevented the migration of cells outside of the spleen and the recruitment of inflammatory cells into the eye, and provided full protection against inflammation from EAU induced with arrestin or arrestin peptides. RTL351 significantly inhibited T cell proliferation and secretion of inflammatory cytokines interleukin 2 (IL-2), interferon γ (IFN-γ), IL-6, and IL-17 and chemokines (macrophage inflammatory proteins [MIP-1a] and regulated and normal T cell expressed and secreted [RANTES]), which is in agreement with the suppression of intraocular inflammation. RTL350 ("empty," no peptide) and RTL352 were not effective. CONCLUSIONS: Immunotherapy with a single RTL351 successfully prevented and treated arrestin-induced EAU in HLA-DR3 mice and provided proof of concept for therapy of autoimmune uveitis in human patients. The beneficial effects of RTL351 should be attributed to a significant decrease in Th1/Th17 mediated inflammation. TRANSLATIONAL RELEVANCE: Successful therapies for autoimmune uveitis must specifically inhibit pathogenic inflammation without inducing generalized immunosuppression. RTLs can offer such an option. The single retina-specific RTLs may have a value as potential immunotherapeutic drug for human autoimmune uveitis because they effectively prevent disease induced by multiple T cell specificities.
RESUMO
Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet, it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study, we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment, investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore, their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together, our findings revealed that AML exosome trafficking alters the proliferative, angiogenic, and migratory responses of cocultured stromal and hematopoietic progenitor cell lines, helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML.
Assuntos
Exossomos/fisiologia , Leucemia Mieloide Aguda/genética , RNA Neoplásico/metabolismo , Medula Óssea/patologia , Linhagem Celular , Movimento Celular , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Transdução de Sinais , Microambiente TumoralRESUMO
Recombinant T cell ligands (RTLs) ameliorate experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner. We evaluated effects of RTL401 (I-A(s) alpha1beta1+PLP-139-151) on splenocytes from SJL/J mice with EAE to study RTL-T cell tolerance-inducing mechanisms. RTLs bound to B, macrophages and DCs, through RTL-MHC-alpha1beta1 moiety. RTL binding reduced CD11b expression on splenic macrophages/DC, and RTL401-conditioned macrophages/DC, not B cells, inhibited T cell activation. Reduced ability of RTL- incubated splenocytes to transfer EAE was likely mediated through macrophages/DC, since B cells were unnecessary for RTL treatment of EAE. These results demonstrate a novel pathway of T cell regulation by RTL-bound APCs.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Antígeno CD11b/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Ligantes , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteína Proteolipídica de Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Disease modifying effects of interferon (IFN)-beta therapy in patients with multiple sclerosis (MS) may be mediated in part through enhanced immunoregulation by the CD56(bright) subpopulation of natural killer (NK) cells and by Foxp3+ (not italicized) CD4+CD25+ regulatory T cells (Treg). We found that IFN-beta-1a(IM) treatment of relapsing-remitting (RR)MS subjects over 12 months significantly increased both percentage of CD56(bright) NK cells and Foxp3 mRNA expression compared to baseline values, untreated RRMS subjects and healthy controls (HC). This striking enhancement of two prominent immunoregulatory pathways lends support to the idea that beneficial effects of IFN-beta-1a in MS include control of pernicious autoimmunity.
Assuntos
Antígeno CD56/biossíntese , Fatores de Transcrição Forkhead/biossíntese , Interferon beta/uso terapêutico , Células Matadoras Naturais/imunologia , Esclerose Múltipla/imunologia , Linfócitos T Reguladores/imunologia , Regulação para Cima/imunologia , Adulto , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Interferon beta-1a , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Projetos Piloto , Linfócitos T Reguladores/metabolismo , Resultado do Tratamento , Adulto JovemRESUMO
We previously demonstrated the therapeutic effects of MHC class II derived recombinant T cell receptor ligands (RTL), single-chain two domain complexes of the alpha1 and beta1 domains of MHC class II molecules genetically linked with an immunodominant peptide, in experimental autoimmune encephalomyelitis. In the current study, we produced a monomeric murine I-Aq-derived RTL construct covalently linked with bovine collagen type II peptide (bCII257-270) suitable for use in DBA/1LacJ mice that develop collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis, after immunization with bCII protein in CFA. In this study, we demonstrate that the I-Aq-derived RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term modulation of T cells specific for arthritogenic Ags. Our results showed that the I-Aq/bCII257-270 molecule could systemically reduce proinflammatory IL-17 and IFN-gamma production and significantly increase anti-inflammatory IL-10, IL-13, and FoxP3 gene expression in splenocytes. Moreover, I-Aq/bCII257-270 molecule could also selectively inhibit IL-1beta, IL-6, and IL-23 expression in local joint tissue. This is the first report demonstrating effective prevention of joint inflammation and clinical signs of CIA with an I-Aq-derived RTL, thus supporting the possible clinical use of this approach for treating rheumatoid arthritis in humans.
Assuntos
Artrite Experimental/prevenção & controle , Receptores de Antígenos de Linfócitos T/agonistas , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Formação de Anticorpos , Antígenos/imunologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Bovinos , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Fator 3-gama Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/uso terapêutico , Humanos , Articulações/patologia , Ligantes , Camundongos , Camundongos Endogâmicos DBA , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Baço/imunologia , Líquido Sinovial/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Regulação para CimaRESUMO
Chronic beryllium disease is a lung disorder caused by beryllium exposure in the workplace and is characterized by granulomatous inflammation and the accumulation of beryllium-specific, HLA-DP2-restricted CD4+ T lymphocytes in the lung that proliferate and secrete Th1-type cytokines. To characterize the interaction among HLA-DP2, beryllium, and CD4+ T cells, we constructed rHLA-DP2 and rHLA-DP4 molecules consisting of the alpha-1 and beta-1 domains of the HLA-DP molecules genetically linked into single polypeptide chains. Peptide binding to rHLA-DP2 and rHLA-DP4 was consistent with previously published peptide-binding motifs for these MHC class II molecules, with peptide binding dominated by aromatic residues in the P1 pocket. 9Be nuclear magnetic resonance spectroscopy showed that beryllium binds to the HLA-DP2-derived molecule, with no binding to the HLA-DP4 molecule that differs from DP2 by four amino acid residues. Using beryllium-specific CD4+ T cell lines derived from the lungs of chronic beryllium disease patients, beryllium presentation to those cells was independent of Ag processing because fixed APCs were capable of presenting BeSO4 and inducing T cell proliferation. Exposure of beryllium-specific CD4+ T cells to BeSO4 -pulsed, plate-bound rHLA-DP2 molecules induced IFN-gamma secretion. In addition, pretreatment of beryllium-specific CD4+ T cells with BeSO4-pulsed, plate-bound HLA-DP2 blocked proliferation and IL-2 secretion upon re-exposure to beryllium presented by APCs. Thus, the rHLA-DP2 molecules described herein provide a template for engineering variants that retain the ability to tolerize pathogenic CD4+ T cells, but do so in the absence of the beryllium Ag.
Assuntos
Beriliose/imunologia , Berílio/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos HLA-DP/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Beriliose/terapia , Berílio/imunologia , Linhagem Celular , Doença Crônica , Antígenos HLA-DP/genética , Cadeias beta de HLA-DP , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Ratos , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/genéticaRESUMO
The major goal of this study was to evaluate the efficacy and mechanism of a rTCR ligand (RTL) construct (I-A(s)/proteolipid protein (PLP)-139-151 peptide = RTL401) for treatment of SJL/J mice developing passive experimental autoimmune encephalomyelitis (EAE) that did not involve coimmunization with the highly inflammatory CFA. Our results demonstrated clearly that RTL401 was highly effective in treating passive EAE, with kinetics of recovery from disease very similar to treatment of actively induced EAE. The potent RTL401 treatment effect was reflected by a partial reduction of infiltrating mononuclear cells into CNS, minimal inflammatory lesions in spinal cord, and preservation of axons injured in vehicle-treated mice during the progression of EAE. Interestingly, in the absence of CFA, RTL401 treatment strongly enhanced production of the Th2 cytokine, IL-13, in spleen, blood, and spinal cord tissue, with variable effects on other Th1 and Th2 cytokines, and no significant effect on the Th3 cytokine, TGF-beta1, or on FoxP3 that is expressed by regulatory T cells. Moreover, pretreatment of PLP-139-151-specific T cells with RTL401 in vitro induced high levels of secreted IL-13, with lesser induction of other pro- and anti-inflammatory cytokines. Given the importance of IL-13 for protection against EAE, these data strongly implicate IL-13 as a dominant regulatory cytokine induced by RTL therapy. Pronounced IL-13 levels coupled with marked reduction in IL-6 levels secreted by PLP-specific T cells from blood after treatment of mice with RTL401 indicate that IL-13 and IL-6 may be useful markers for following effects of RTL therapy in future clinical trials in multiple sclerosis.