RESUMO
B cell lymphoma therapy has been transformed by CD19-targeting cellular therapeutics that induce high clinical response rates and impressive remissions in relapsed and refractory patients. However, approximately half of all patients who respond to CD19-directed cell therapy relapse, the majority within 6 months. One characteristic of relapse is loss or reduction of CD19 expression on malignant B cells. We designed a unique therapeutic to prevent and reverse relapses due to lost or reduced CD19 expression. This novel biologic, a CAR T Engager, binds CD20 and displays the CD19 extracellular domain. This approach increases the apparent CD19 antigen density on CD19-positive/CD20-positive lymphoma cells, and prevents antigen-loss induced relapse, as CD19 bound to CD20 remains present on the cell surface. We demonstrate that this novel therapeutic prevents and reverses lymphoma relapse in vitro and prevents CD19-negative lymphoma growth and relapse in vivo.
Assuntos
Linfoma , Receptores de Antígenos Quiméricos , Antígenos CD19 , Antígenos CD20 , Humanos , Linfoma/terapia , Recidiva Local de Neoplasia , Receptores de Antígenos de Linfócitos T , Linfócitos TRESUMO
Refractory acute myeloid leukemia (AML) remains an incurable malignancy despite the clinical use of novel targeted therapies, new antibody-based therapies, and cellular therapeutics. Here, we describe the preclinical development of a novel cell therapy that targets the antigen CLEC12A with a biparatopic bridging protein. Bridging proteins are designed as "CAR-T cell engagers," with a CAR-targeted protein fused to antigen binding domains derived from antibodies. Here, we created a CD19-anti-CLEC12A bridging protein that binds to CAR19 T cells and to the antigen CLEC12A. Biparatopic targeting increases the potency of bridging protein-mediated cytotoxicity by CAR19 T cells. Using CAR19 T cells that secrete the bridging protein we demonstrate potent activity against aggressive leukemic cell lines in vivo This CAR-engager platform is facile and modular, as illustrated by activity of a dual-antigen bridging protein targeting CLEC12A and CD33, designed to counter tumor heterogeneity and antigen escape, and created without the need for extensive CAR T-cell genetic engineering. CAR19 T cells provide an optimal cell therapy platform with well-understood inherent persistence and fitness characteristics.
Assuntos
Antígenos CD19/imunologia , Imunoterapia Adotiva/métodos , Imunoterapia/métodos , Lectinas Tipo C/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Receptores Mitogênicos/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Linfócitos T/imunologia , Animais , Deriva e Deslocamento Antigênicos , Apoptose , Proliferação de Células , Citotoxicidade Imunológica/imunologia , Humanos , Técnicas In Vitro , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Successful CAR T cell therapy for the treatment of solid tumors requires exemplary CAR T cell expansion, persistence and fitness, and the ability to target tumor antigens safely. Here we address this constellation of critical attributes for successful cellular therapy by using integrated technologies that simplify development and derisk clinical translation. We have developed a CAR-CD19 T cell that secretes a CD19-anti-Her2 bridging protein. This cell therapy strategy exploits the ability of CD19-targeting CAR T cells to interact with CD19 on normal B cells to drive expansion, persistence and fitness. The secreted bridging protein potently binds to Her2-positive tumor cells, mediating CAR-CD19 T cell cytotoxicity in vitro and in vivo. Because of its short half-life, the secreted bridging protein will selectively accumulate at the site of highest antigen expression, ie. at the tumor. Bridging proteins that bind to multiple different tumor antigens have been created. Therefore, antigen-bridging CAR-CD19 T cells incorporate critical attributes for successful solid tumor cell therapy. This platform can be exploited to attack tumor antigens on any cancer.
Assuntos
Antígenos CD19/genética , Imunoterapia Adotiva/métodos , Linfoma de Células B/terapia , Receptor ErbB-2/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Animais , Antígenos CD19/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Citotoxicidade Imunológica , Receptores ErbB/genética , Receptores ErbB/imunologia , Expressão Gênica , Vetores Genéticos/imunologia , Vetores Genéticos/metabolismo , Humanos , Lentivirus/genética , Lentivirus/imunologia , Ativação Linfocitária , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Camundongos , Camundongos SCID , Ligação Proteica , Receptor ErbB-2/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/citologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CD19-targeted chimeric antigen receptor (CAR) T-cells (CAR19s) show remarkable efficacy in the treatment of relapsed/refractory acute lymphocytic leukemia and Non-Hodgkin's lymphoma. However, the use of CAR T-cell therapy against CD19-negative hematological cancers and solid tumors has been challenging. We propose CD19-fusion proteins (CD19-FPs) to leverage the benefits of CAR19s while retargeting this validated cellular therapy to alternative tumor antigens. We demonstrate the ability of a fusion of CD19 extracellular domain (ECD) and a human epidermal growth factor receptor 2 (HER2) single-chain antibody fragment to retarget CAR19s to kill HER2+ CD19- tumor cells. To enhance the modularity of this technology, we engineered a more robust CD19 ECD via deep mutational scanning with yeast display and flow cytometric selections for improved protease resistance and anti-CD19 antibody binding. These enhanced CD19 ECDs significantly increase, and in some cases recover, fusion protein expression while maintaining target antigen affinity. Importantly, CD19-FPs retarget CAR19s to kill tumor cells expressing multiple distinct antigens, including HER2, CD20, EGFR, BCMA, and Clec12A as N- or C-terminal fusions and linked to both antibody fragments and fibronectin ligands. This study provides fundamental insights into CD19 sequence-function relationships and defines a flexible and modular platform to retarget CAR19s to any tumor antigen.
Assuntos
Antígenos CD19/metabolismo , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/metabolismo , Linfócitos T/imunologia , Antígenos CD19/genética , Antígenos CD19/imunologia , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Mutagênese , Neoplasias/imunologia , Neoplasias/patologia , Domínios Proteicos/genética , Engenharia de Proteínas , Receptor ErbB-2/antagonistas & inibidores , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplanteRESUMO
INTRODUCTION: CD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG1 antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab' anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8. METHODS: The potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE. RESULTS: CDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab' anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria. CONCLUSIONS: These findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.
Assuntos
Anticorpos Monoclonais/imunologia , Ligante de CD40/imunologia , Imunidade Humoral/imunologia , Trombose/imunologia , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Formação de Anticorpos/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/prevenção & controle , Modelos Animais de Doenças , Humanos , Imunidade Humoral/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/prevenção & controle , Macaca fascicularis , Macaca mulatta , Camundongos Endogâmicos NZB , Polietilenoglicóis/química , Toxoide Tetânico/imunologia , Trombose/induzido quimicamenteRESUMO
AIM/HYPOTHESIS: The adult mammalian pancreas has limited ability to regenerate in order to restore adequate insulin production from multipotent progenitors, the identity and function of which remain poorly understood. Here we test whether the TNF family member TWEAK (TNF-like weak inducer of apoptosis) promotes ß-cell neogenesis from proliferating pancreatic ductal epithelium in adult mice. METHODS: C57Bl/6J mice were treated with Fc-TWEAK and pancreas harvested at different time points for analysis by histology and immunohistochemistry. For lineage tracing, 4 week old double transgenic mice CAII-CreER(TM): R26R-eYFP were implanted with tamoxifen pellet, injected with Fc-TWEAK or control Ig twice weekly and analyzed at day 18 for TWEAK-induced duct cell progeny by costaining for insulin and YFP. The effect of TWEAK on pancreatic regeneration was determined by pancytokeratin immunostaining of paraffin embedded sections from wildtype and TWEAK receptor (Fn14) deficient mice after Px. RESULTS: TWEAK stimulates proliferation of ductal epithelial cells through its receptor Fn14, while it has no mitogenic effect on pancreatic α- or ß-cells or acinar cells. Importantly, TWEAK induces transient expression of endogenous Ngn3, a master regulator of endocrine cell development, and induces focal ductal structures with characteristics of regeneration foci. In addition, we identify by lineage tracing TWEAK-induced pancreatic ß-cells derived from pancreatic duct epithelial cells. Conversely, we show that Fn14 deficiency delays formation of regenerating foci after Px and limits their expansion. CONCLUSIONS/INTERPRETATION: We conclude that TWEAK is a novel factor mediating pancreatic ß-cell neogenesis from ductal epithelium in normal adult mice.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ductos Pancreáticos/efeitos dos fármacos , Fatores de Necrose Tumoral/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Citocina TWEAK , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Queratinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/metabolismo , Pâncreas/fisiologia , Pâncreas/cirurgia , Pancreatectomia , Ductos Pancreáticos/citologia , Ductos Pancreáticos/metabolismo , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Regeneração/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor de TWEAK , Fatores de TempoRESUMO
BACKGROUND: Inflammatory cytokines play an important role in the pathogenesis of heart failure. We have recently found the cytokine TWEAK (tumor necrosis factor (TNF)-like weak inducer of apoptosis), a member of the TNF superfamily, to be increased in patients with cardiomyopathy and result in the development of heart failure when overexpressed in mice. The molecular mechanisms underlying TWEAK-induced cardiac pathology, however, remain unknown. METHODOLOGY AND CRITICAL FINDING: Using mouse models of elevated circulating TWEAK levels, established through intravenous injection of adenovirus expressing TWEAK or recombinant TWEAK protein, we find that TWEAK induces a progressive dilated cardiomyopathy with impaired contractile function in mice. Moreover, TWEAK treatment is associated with decreased expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) and genes required for mitochondrial oxidative phosphorylation, which precede the onset of cardiac dysfunction. TWEAK-induced downregulation of PGC1α requires expression of its cell surface receptor, fibroblast growth factor-inducible 14 (Fn14). We further find that TWEAK downregulates PGC1α gene expression via the TNF receptor-associated factor 2 (TRAF2) and NFκB signaling pathways. Maintaining PGC1α levels through adenoviral-mediated gene expression is sufficient to protect against TWEAK-induced cardiomyocyte dysfunction. CONCLUSION: Collectively, our data suggest that TWEAK induces cardiac dysfunction via downregulation of PGC1α, through FN14-TRAF2-NFκB-dependent signaling. Selective targeting of the FN14-TRAF2-NFκB-dependent signaling pathway or augmenting PGC1α levels may serve as novel therapeutic strategies for cardiomyopathy and heart failure.
Assuntos
Fatores de Necrose Tumoral/metabolismo , Animais , Western Blotting , Células Cultivadas , Citocina TWEAK , Ecocardiografia , Fatores de Crescimento de Fibroblastos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator 2 Associado a Receptor de TNF/metabolismo , Transativadores/metabolismo , Fatores de Transcrição , Fatores de Necrose Tumoral/genéticaRESUMO
TWEAK, a TNF family ligand with pleiotropic cellular functions, was originally described as capable of inducing tumor cell death in vitro. TWEAK functions by binding its receptor, Fn14, which is up-regulated on many human solid tumors. Herein, we show that intratumoral administration of TWEAK, delivered either by an adenoviral vector or in an immunoglobulin Fc-fusion form, results in significant inhibition of tumor growth in a breast xenograft model. To exploit the TWEAK-Fn14 pathway as a therapeutic target in oncology, we developed an anti-Fn14 agonistic antibody, BIIB036. Studies described herein show that BIIB036 binds specifically to Fn14 but not other members of the TNF receptor family, induces Fn14 signaling, and promotes tumor cell apoptosis in vitro. In vivo, BIIB036 effectively inhibits growth of tumors in multiple xenograft models, including colon (WiDr), breast (MDA-MB-231), and gastric (NCI-N87) tumors, regardless of tumor cell growth inhibition response observed to BIIB036 in vitro. The anti-tumor activity in these cell lines is not TNF-dependent. Increasing the antigen-binding valency of BIB036 significantly enhances its anti-tumor effect, suggesting the contribution of higher order cross-linking of the Fn14 receptor. Full Fc effector function is required for maximal activity of BIIB036 in vivo, likely due to the cross-linking effect and/or ADCC mediated tumor killing activity. Taken together, the anti-tumor properties of BIIB036 validate Fn14 as a promising target in oncology and demonstrate its potential therapeutic utility in multiple solid tumor indications.
Assuntos
Antineoplásicos , Apoptose/efeitos dos fármacos , Neoplasias/terapia , Receptores do Fator de Necrose Tumoral/agonistas , Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/imunologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Citocina TWEAK , Células HT29 , Humanos , Ligantes , Camundongos , Neoplasias/imunologia , Ligação Proteica , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Receptor de TWEAK , Fatores de Necrose Tumoral/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, aka CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging. Therefore, to understand how BIO8898 achieves this feat, we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC(50) = 25 µM and inhibited CD40L-dependent apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the protein's 3-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.
Assuntos
Ligante de CD40/antagonistas & inibidores , Piridinas/farmacologia , Pirrolidinas/farmacologia , Animais , Antígenos CD40/imunologia , Antígenos CD40/isolamento & purificação , Ligante de CD40/imunologia , Ligante de CD40/isolamento & purificação , Linhagem Celular , Cricetinae , Cristalografia por Raios X , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Ligação Proteica/efeitos dos fármacos , Piridinas/síntese química , Piridinas/química , Pirrolidinas/síntese química , Pirrolidinas/químicaRESUMO
INTRODUCTION: TNF-like weak inducer of apoptosis (TWEAK) has been implicated as a mediator of chronic inflammatory processes via prolonged activation of the NF-kappaB pathway in several tissues, including the kidney. Evidence for the importance of TWEAK in the pathogenesis of lupus nephritis (LN) has been recently introduced. Thus, TWEAK levels may serve as an indication of LN presence and activity. METHODS: Multicenter cohorts of systemic lupus erythematosus (SLE) patients and controls were recruited for cross-sectional and longitudinal analysis of urinary TWEAK (uTWEAK) and/or serum TWEAK (sTWEAK) levels as potential biomarkers of LN. The performance of TWEAK as a biomarker for nephritis was compared with routinely used laboratory tests in lupus patients, including anti-double stranded DNA antibodies and levels of C3 and C4. RESULTS: uTWEAK levels were significantly higher in LN patients than in non-LN SLE patients and other disease control groups (P = 0.039). Furthermore, uTWEAK was better at distinguishing between LN and non-LN SLE patients than anti-DNA antibodies and complement levels, while high uTWEAK levels predicted LN in SLE patients with an odds ratio of 7.36 (95% confidence interval = 2.25 to 24.07; P = 0.001). uTWEAK levels peaked during LN flares, and were significantly higher during the flare than at 4 and 6 months prior to or following the flare event. A linear mixed-effects model showed a significant association between uTWEAK levels in SLE patients and their disease activity over time (P = 0.008). sTWEAK levels, however, were not found to correlate with the presence of LN or the degree of nephritis activity. CONCLUSIONS: High uTWEAK levels are indicative of LN, as opposed to non-LN SLE and other healthy and disease control populations, and reflect renal disease activity in longitudinal follow-up. Thus, our study further supports a role for TWEAK in the pathogenesis of LN, and provides strong evidence for uTWEAK as a candidate clinical biomarker for LN.
Assuntos
Nefrite Lúpica/urina , Fatores de Necrose Tumoral/urina , Adulto , Idoso , Biomarcadores/sangue , Biomarcadores/urina , Estudos de Coortes , Estudos Transversais , Citocina TWEAK , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Nefrite Lúpica/sangue , Masculino , Pessoa de Meia-Idade , Fatores de Necrose Tumoral/sangueRESUMO
Misincorporation of amino acids in proteins expressed in Escherichia coli has been well documented but not in proteins expressed in mammalian cells under normal recombinant protein production conditions. Here we report for the first time that Ser can be incorporated at Asn positions in proteins expressed in Chinese hamster ovary cells. This misincorporation was discovered as a result of intact mass measurement, peptide mapping analysis, and tandem mass spectroscopy sequencing. Our analyses showed that the substitution was not related to specific protein molecules or DNA codons and was not site-specific. We believe that the incorporation of Ser at sites coded for Asn was due to mischarging of tRNA(Asn) rather than to codon misreading. The rationale for substitution of Asn by Ser and not by other amino acids is also discussed. Further investigation indicated that the substitution was due to the starvation for Asn in the cell culture medium and that the substitution could be limited by using the Asn-rich feed. These observations demonstrate that the quality of expressed proteins should be closely monitored when altering cell culture conditions.
Assuntos
Asparagina/química , Proteínas Recombinantes/química , Serina/química , Animais , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Escherichia coli/metabolismo , Glicosilação , Espectrometria de Massas/métodos , Modelos Biológicos , Mapeamento de Peptídeos , Peptídeos/química , RNA de Transferência/metabolismoRESUMO
BACKGROUND: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, is a multifunctional cytokine known to regulate cellular functions in contexts of injury and disease through its receptor, fibroblast growth factor-inducible molecule 14 (Fn14). Although many of the processes and downstream signals regulated by the TWEAK/Fn14 pathway have been implicated in the development of cardiac dysfunction, the role of TWEAK in the cardiovascular system is completely unknown. METHODS AND RESULTS: Herein, we demonstrate that mouse and human cardiomyocytes express the TWEAK receptor Fn14. Furthermore, we determine that elevated circulating levels of TWEAK, induced via transgenic or adenoviral-mediated gene expression in mice, result in dilated cardiomyopathy with subsequent severe cardiac dysfunction. This phenotype was mediated exclusively by the Fn14 receptor, independent of tumor necrosis factor-alpha, and was associated with cardiomyocyte elongation and cardiac fibrosis but not cardiomyocyte apoptosis. Moreover, we find that circulating TWEAK levels were differentially upregulated in patients with idiopathic dilated cardiomyopathy compared with other forms of heart disease and normal control subjects. CONCLUSIONS: Our data suggest that TWEAK/Fn14 may be important in regulating myocardial structural remodeling and function and may play a role in the pathogenesis of dilated cardiomyopathy.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Fatores de Necrose Tumoral/fisiologia , Animais , Apoptose , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatia Dilatada/complicações , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Tamanho Celular , Doença das Coronárias/metabolismo , Doença das Coronárias/patologia , Citocina TWEAK , Feminino , Fibrose , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Humanos , Hipertensão/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Pessoa de Meia-Idade , Fenótipo , Proteínas Recombinantes de Fusão/fisiologia , Receptor de TWEAK , Transdução Genética , Fatores de Necrose Tumoral/sangue , Fatores de Necrose Tumoral/genéticaRESUMO
T helper type 17 (Th17) cells play an important pathogenic function in autoimmune diseases; their regulation, however, is not well understood. We show that the expression of a tumor necrosis factor receptor family member, death receptor 3 (DR3; also known as TNFRSF25), is selectively elevated in Th17 cells, and that TL1A, its cognate ligand, can promote the proliferation of effector Th17 cells. To further investigate the role of the TL1A-DR3 pathway in Th17 regulation, we generated a TL1A-deficient mouse and found that TL1A(-/-) dendritic cells exhibited a reduced capacity in supporting Th17 differentiation and proliferation. Consistent with these data, TL1A(-/-) animals displayed decreased clinical severity in experimental autoimmune encephalomyelitis (EAE). Finally, we demonstrated that during EAE disease progression, TL1A was required for the optimal differentiation as well as effector function of Th17 cells. These observations thus establish an important role of the TL1A-DR3 pathway in promoting Th17 cell function and Th17-mediated autoimmune disease.
Assuntos
Doenças Autoimunes/imunologia , Encefalomielite Autoimune Experimental/imunologia , Antígeno HLA-DR3/imunologia , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/deficiência , Animais , Encéfalo/imunologia , Encéfalo/patologia , Diferenciação Celular , Divisão Celular , Citocinas/metabolismo , Primers do DNA , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Medula Espinal/imunologia , Medula Espinal/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/fisiologiaRESUMO
TNF-like weak inducer of apoptosis (TWEAK) is a TNF family member with pleiotropic effects on a variety of cell types, one of which is the induction of proinflammatory cytokines by synovial fibroblasts derived from rheumatoid arthritis (RA) patients. In this study, we report that the serum TWEAK level was dramatically elevated during mouse collagen-induced arthritis (CIA) and blocking TWEAK by a neutralizing mAb significantly reduced the clinical severity of CIA. Histological analyses also revealed that TWEAK inhibition diminished joint inflammation, synovial angiogenesis, as well as cartilage and bone erosion. Anti-TWEAK treatment proved efficacious when administered just before the disease onset but not during the priming phase of CIA. Consistent with this, TWEAK inhibition did not affect either cellular or humoral responses to collagen. In contrast, TWEAK inhibition significantly reduced serum levels of a panel of arthritogenic mediators, including chemokines such as MIP-1beta (CCL-4), lymphotactin (XCL-1), IFN-gamma-inducible protein 10 (IP-10) (CXCL-10), MCP-1 (CCL-2), and RANTES (CCL-5), as well as the matrix metalloprotease-9. Exploring the possible role of the TWEAK/Fn14 pathway in human RA pathogenesis, we showed that TWEAK can target human primary chondrocytes and osteoblast-like cells, in addition to synovial fibroblasts. We further demonstrated that TWEAK induced the production of matrix metalloproteases in human chondrocytes and potently inhibited chondrogenesis and osteogenesis using in vitro models. These results provide evidence for a novel cytokine pathway that contributes to joint tissue inflammation, angiogenesis, and damage, as well as may inhibit endogenous repair, suggesting that TWEAK may be a new therapeutic target for human RA.
Assuntos
Artrite Experimental/metabolismo , Artrite Experimental/patologia , Mediadores da Inflamação/fisiologia , Fatores de Necrose Tumoral/fisiologia , Animais , Apoptose/imunologia , Artrite Experimental/sangue , Células Cultivadas , Colágeno Tipo II/administração & dosagem , Citocina TWEAK , Adjuvante de Freund/administração & dosagem , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/sangue , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Receptor de TWEAK , Inibidores do Fator de Necrose Tumoral , Fatores de Necrose Tumoral/biossíntese , Fatores de Necrose Tumoral/sangueRESUMO
TNF-like weak inducer of apoptosis, or TWEAK, is a relatively new member of the TNF-ligand superfamily. Ligation of the TWEAK receptor Fn14 by TWEAK has proinflammatory effects on fibroblasts, synoviocytes, and endothelial cells. Several of the TWEAK-inducible cytokines are important in the pathogenesis of kidney diseases; however, whether TWEAK can induce a proinflammatory effect on kidney cells is not known. We found that murine mesangial cells express cell surface TWEAK receptor. TWEAK stimulation of mesangial cells led to a dose-dependent increase in CCL2/MCP-1, CCL5/RANTES, CXCL10/IFN-gamma-induced protein 10 kDa, and CXCL1/KC. The induced levels of chemokines were comparable to those found following mesangial cell exposure to potent proinflammatory stimuli such as TNF-alpha + IL-1beta. CXCL11/interferon-inducible T cell alpha chemoattractant, CXCR5, mucosal addressin cell adhesion molecule-1, and VCAM-1 were up-regulated by TWEAK as well. TWEAK stimulation of mesangial cells resulted in an increase in phosphorylated Ikappa-B, while pretreatment with an Ikappa-B phosphorylation inhibitor significantly blocked chemokine induction, implicating activation of the NF-kappaB signaling pathway in TWEAK-induced chemokine secretion. Importantly, the Fn14-mediated proinflammatory effects of TWEAK on kidney cells were confirmed using mesangial cells derived from Fn14-deficient mice and by injection in vivo of TWEAK into wild-type vs Fn14-deficient mice. Finally, TWEAK-induced chemokine secretion was prevented by treatment with novel murine anti-TWEAK Abs. We conclude that TWEAK induces mesangial cells to secrete proinflammatory chemokines, suggesting a prominent role for TWEAK in the pathogenesis of renal injury. Our results support Ab inhibition of TWEAK as a potential new approach for the treatment of chemokine-dependent inflammatory kidney diseases.
Assuntos
Mesângio Glomerular/metabolismo , Mediadores da Inflamação/fisiologia , Células Mesangiais/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Fatores de Necrose Tumoral/fisiologia , Animais , Anticorpos Monoclonais , Apoptose/imunologia , Linhagem Celular Transformada , Células Cultivadas , Quimiocinas/biossíntese , Citocina TWEAK , Proteínas I-kappa B/metabolismo , Mediadores da Inflamação/metabolismo , Interferon gama/fisiologia , Interleucina-1/fisiologia , Interleucina-6/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Inibidor de NF-kappaB alfa , Fosforilação , Receptores do Fator de Necrose Tumoral/genética , Receptor de TWEAK , Inibidores do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/fisiologia , Fatores de Necrose Tumoral/imunologia , Fatores de Necrose Tumoral/metabolismo , Regulação para Cima/fisiologiaRESUMO
The TNF superfamily cytokine TWEAK induces mesangial cells, podocytes, and endothelial cells to secrete pro-inflammatory chemokines including MCP-1, IP-10 and RANTES, which are crucial in the pathogenesis of lupus nephritis (LN). As TWEAK regulates the secretion of these inflammatory mediators, we studied whether urinary TWEAK (uTWEAK) levels might be predictive and/or diagnostic in LN. In a cross-sectional study of a large, multi-center cohort of systemic lupus erythematosus (SLE) patients, uTWEAK levels were higher in patients with active as compared to never or non-active nephritis (median (IQR): 16.3 (9.9-23.0) versus 5.5 (2.3-16.8) pg/mg creatinine, p=0.001), and levels of uTWEAK correlated with the renal SLE disease activity index (rSLEDAI) score (r=0.405, p<0.001). uTWEAK levels were higher in patients undergoing a flare as compared to patients with chronic stable disease (11.1 (8.1-18.2) and 5.2 (2.3-15.3) pg/mg creatinine, respectively; p=0.036). Moreover, uTWEAK levels were significantly higher in patients undergoing a renal flare, as opposed to a non-renal flare (12.4 (9.1-18.2) and 5.2 (3.0-11.9) pg/mg creatinine, respectively; p=0.029). An accurate, non-invasive method to repeatedly assess kidney disease in lupus would be very helpful in managing these often challenging patients. Our study indicates that urinary TWEAK levels may be useful as a novel biomarker in LN.
Assuntos
Lúpus Eritematoso Sistêmico/urina , Nefrite Lúpica/urina , Fatores de Necrose Tumoral/urina , Adulto , Biomarcadores/urina , Quimiocina CCL2/biossíntese , Quimiocina CCL2/imunologia , Quimiocina CCL2/urina , Estudos Transversais , Citocina TWEAK , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/imunologia , Masculino , Pessoa de Meia-Idade , Fatores de Necrose Tumoral/imunologia , Urina/químicaRESUMO
Herein we demonstrate that B cell-activating factor of the TNF family (BAFF), a B cell survival factor, also regulates CD21/35 and CD23 expression. BAFF blockade in wild-type mice down-modulates CD21/35 and CD23 on B cells while survival remains intact, and BAFF exposure causes elevated CD21/35 and CD23 expression. Similar down-modulation is observed in bcl-2-transgenic mice treated with a BAFF inhibitor. This is the first evidence that BAFF has a function independent of B cell survival. Reports using CD21/35 and CD23 expression to assess splenic B cell subsets in BAFF-null mice concluded a lack of B cells beyond the immature stage. Since CD21/35 and CD23 are inadequate for delineating B cell subpopulations in BAFF-null mice, we used expression of BAFF-R and several B cell markers to identify more mature splenic B cells in these mice. These data broaden our understanding of BAFF function and correct the view that BAFF-null mice lack mature B cells.