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1.
Cell ; 144(4): 601-13, 2011 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-21295337

RESUMO

Understanding the factors that impede immune responses to persistent viruses is essential in designing therapies for HIV infection. Mice infected with LCMV clone-13 have persistent high-level viremia and a dysfunctional immune response. Interleukin-7, a cytokine that is critical for immune development and homeostasis, was used here to promote immunity toward clone-13, enabling elucidation of the inhibitory pathways underlying impaired antiviral immune response. Mechanistically, IL-7 downregulated a critical repressor of cytokine signaling, Socs3, resulting in amplified cytokine production, increased T cell effector function and numbers, and viral clearance. IL-7 enhanced thymic output to expand the naive T cell pool, including T cells that were not LCMV specific. Additionally, IL-7 promoted production of cytoprotective IL-22 that abrogated liver pathology. The IL-7-mediated effects were dependent on endogenous IL-6. These attributes of IL-7 have profound implications for its use as a therapeutic in the treatment of chronic viral diseases.


Assuntos
Interleucina-7/uso terapêutico , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Animais , Antígenos de Diferenciação/metabolismo , Regulação para Baixo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interleucina-6/imunologia , Interleucina-7/imunologia , Camundongos , Receptor de Morte Celular Programada 1 , Proteínas Recombinantes/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Linfócitos T/imunologia
2.
J Immunol ; 203(11): 2791-2806, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31659015

RESUMO

The paracaspase Malt1 is a key regulator of canonical NF-κB activation downstream of multiple receptors in both immune and nonimmune cells. Genetic disruption of Malt1 protease function in mice and MALT1 mutations in humans results in reduced regulatory T cells and a progressive multiorgan inflammatory pathology. In this study, we evaluated the altered immune homeostasis and autoimmune disease in Malt1 protease-deficient (Malt1PD) mice and the Ags driving disease manifestations. Our data indicate that B cell activation and IgG1/IgE production is triggered by microbial and dietary Ags preferentially in lymphoid organs draining mucosal barriers, likely as a result of dysregulated mucosal immune homeostasis. Conversely, the disease was driven by a polyclonal T cell population directed against self-antigens. Characterization of the Malt1PD T cell compartment revealed expansion of T effector memory cells and concomitant loss of a CD4+ T cell population that phenotypically resembles anergic T cells. Therefore, we propose that the compromised regulatory T cell compartment in Malt1PD animals prevents the efficient maintenance of anergy and supports the progressive expansion of pathogenic, IFN-γ-producing T cells. Overall, our data revealed a crucial role of the Malt1 protease for the maintenance of intestinal and systemic immune homeostasis, which might provide insights into the mechanisms underlying IPEX-related diseases associated with mutations in MALT1.


Assuntos
Autoimunidade/imunologia , Homeostase/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Linfócitos T Reguladores/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/deficiência , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética
3.
Immunol Cell Biol ; 96(1): 81-99, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29359407

RESUMO

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is essential for immune responses triggered by antigen receptors but the contribution of its paracaspase activity is not fully understood. Here, we studied how MALT1 proteolytic function regulates T-cell activation and fate after engagement of the T-cell receptor pathway. We show that MLT-827, a potent and selective MALT1 paracaspase inhibitor, does not prevent the initial phase of T-cell activation, in contrast to the pan-protein kinase C inhibitor AEB071. However, MLT-827 strongly impacted cell expansion after activation. We demonstrate this is the consequence of profound inhibition of IL-2 production as well as reduced expression of the IL-2 receptor alpha subunit (CD25), resulting from defective canonical NF-κB activation and accelerated mRNA turnover mechanisms. Accordingly, MLT-827 revealed a unique transcriptional fingerprint of MALT1 protease activity, providing evidence for broad control of T-cell signaling pathways. Altogether, this first report with a potent and selective inhibitor elucidates how MALT1 paracaspase activity integrates several T-cell activation pathways and indirectly controls gamma-chain receptor dependent survival, to impact on T-cell expansion.


Assuntos
Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , NF-kappa B/metabolismo , Linfócitos T/imunologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Imunomodulação , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Proteólise , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais
4.
J Immunol ; 194(8): 3723-34, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25762782

RESUMO

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.


Assuntos
Linfócitos B Reguladores/imunologia , Caspases/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Encefalomielite Autoimune Experimental/imunologia , Proteínas de Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B Reguladores/patologia , Caspases/genética , Diferenciação Celular/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Camundongos , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/genética , Linfócitos T Reguladores/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologia
5.
J Immunol ; 188(9): 4405-11, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22461693

RESUMO

Safety of human therapeutic Abs is generally assessed in nonhuman primates. Whereas IgG1 shows identical FcγR interaction and effector function profile in both species, fundamental differences in the IgG2 and IgG4 Ab subclasses were found between the two species. Granulocytes, the main effector cells against IgG2- and IgG4-opsonized bacteria and parasites, do not express FcγRIIIb, but show higher levels of FcγRII in cynomolgus monkey. In humans, IgG2 and IgG4 adapted a silent Fc region with weak binding to FcγR and effector functions, whereas, in contrast, cynomolgus monkey IgG2 and IgG4 display strong effector function as well as differences in IgG4 Fab arm exchange. To balance this shift toward activation, the cynomolgus inhibitory FcγRIIb shows strongly increased affinity for IgG2. In view of these findings, in vitro and in vivo results for human IgG2 and IgG4 obtained in the cynomolgus monkey have to be cautiously interpreted, whereas effector function-related effects of human IgG1 Abs are expected to be predictable for humans.


Assuntos
Granulócitos/imunologia , Imunoglobulina G/imunologia , Receptores de IgG/imunologia , Animais , Sequência de Bases , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Humanos , Imunoglobulina G/genética , Macaca fascicularis , Dados de Sequência Molecular , Receptores de IgG/genética , Especificidade da Espécie
6.
JCI Insight ; 7(13)2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35801586

RESUMO

IL-1 receptor-activated kinase 1 (IRAK1) is involved in signal transduction downstream of many TLRs and the IL-1R. Its potential as a drug target for chronic inflammatory diseases is underappreciated. To study its functional role in joint inflammation, we generated a mouse model expressing a functionally inactive IRAK1 (IRAK1 kinase deficient, IRAK1KD), which also displayed reduced IRAK1 protein expression and cell type-specific deficiencies of TLR signaling. The serum transfer model of arthritis revealed a potentially novel role of IRAK1 for disease development and neutrophil chemoattraction exclusively via its activity in nonhematopoietic cells. Consistently, IRAK1KD synovial fibroblasts showed reduced secretion of neutrophil chemoattractant chemokines following stimulation with IL-1ß or human synovial fluids from patients with rheumatoid arthritis (RA) and gout. Together with patients with RA showing prominent IRAK1 expression in fibroblasts of the synovial lining, these data suggest that targeting IRAK1 may be therapeutically beneficial. As pharmacological inhibition of IRAK1 kinase activity had only mild effects on synovial fibroblasts from mice and patients with RA, targeted degradation of IRAK1 may be the preferred pharmacologic modality. Collectively, these data position IRAK1 as a central regulator of the IL-1ß-dependent local inflammatory milieu of the joints and a potential therapeutic target for inflammatory arthritis.


Assuntos
Artrite Reumatoide , Quinases Associadas a Receptores de Interleucina-1 , Neutrófilos , Membrana Sinovial , Animais , Artrite Reumatoide/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-8/metabolismo , Camundongos , Neutrófilos/metabolismo , Membrana Sinovial/metabolismo
7.
Proc Natl Acad Sci U S A ; 105(8): 2999-3004, 2008 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-18287017

RESUMO

There are many inhibitory mechanisms that function at the cellular and molecular levels to maintain tolerance. Despite this, self-reactive clones escape regulatory mechanisms and cause autoimmunity in certain circumstances. We hypothesized that the same mechanisms that permit T cells to expand during homeostatic proliferation may inadvertently promote autoimmunity under certain conditions. One major homeostatic cytokine is IL-7, and studies have linked it or its receptor to the development of multiple sclerosis and other autoimmune diseases. We show in a model of beta-islet cell self-reactivity that the transfer of activated autoreactive CD4 T cells can prime and expand endogenous autoreactive CD8 T cells in a CD28- and CD40-dependent manner through the licensing of dendritic cells. Despite this, mice do not develop diabetes. However, the provision of exogenous IL-7 or the physiological production of IL-7 associated with lymphopenia was able to profoundly promote the expansion of self-reactive clones even in the presence of regulatory T cells. Autoimmune diabetes rapidly ensued with CD4 help and the subsequent activation of CD8 T cells, which contributed to disease progression. With the advent of many biologicals targeting TNFalpha, IL-6, and IL-1 and their effective use in the treatment of autoimmune diseases, we propose that IL-7 and its receptor may be promising targets for biological agents in the treatment of autoimmunity.


Assuntos
Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Interleucina-7/imunologia , Linfopenia/imunologia , Modelos Biológicos , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/transplante , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos
8.
Oncogenesis ; 10(4): 32, 2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824280

RESUMO

CARD-CC complexes involving BCL10 and MALT1 are major cellular signaling hubs. They govern NF-κB activation through their scaffolding properties as well as MALT1 paracaspase function, which cleaves substrates involved in NF-κB regulation. In human lymphocytes, gain-of-function defects in this pathway lead to lymphoproliferative disorders. CARD10, the prototypical CARD-CC protein in non-hematopoietic cells, is overexpressed in several cancers and has been associated with poor prognosis. However, regulation of CARD10 remains poorly understood. Here, we identified CARD10 as the first MALT1 substrate in non-hematopoietic cells and showed that CARD10 cleavage by MALT1 at R587 dampens its capacity to activate NF-κB. Preventing CARD10 cleavage in the lung tumor A549 cell line increased basal levels of IL-6 and extracellular matrix components in vitro, and led to increased tumor growth in a mouse xenograft model, suggesting that CARD10 cleavage by MALT1 might be a built-in mechanism controlling tumorigenicity.

9.
J Clin Invest ; 117(12): 3833-45, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17992258

RESUMO

TNF-alpha antagonists are widely used in the treatment of inflammatory and autoimmune diseases, but their use is associated with reactivation of latent infections. This highlights the importance of TNF-alpha in immunity to certain pathogens and raises concerns that critical aspects of immune function are impaired in its absence. Unfortunately, the role of TNF-alpha in the regulation of T cell responses is clouded by a myriad of contradictory reports. Here, we show a role for TNF-alpha and its receptors, TNFR1 and TNFR2, specifically in antitumor immunity. TNF-alpha-deficient mice exhibited normal antiviral responses associated with strong inflammation. However, TNF-alpha/TNFR1-mediated signals on APCs and TNF-alpha/TNFR2 signals on T cells were critically required for effective priming, proliferation, and recruitment of tumor-specific T cells. Furthermore, in the absence of TNF-alpha signaling, tumor immune surveillance was severely abrogated. Finally, treatment with a CD40 agonist alone or in combination with TLR2 stimuli was able to rescue proliferation of TNF-alpha-deficient T cells. Therefore, TNF-alpha signaling may be required only for immune responses in conditions of limited immunostimulatory capacity, such as tumor surveillance. Importantly, these results suggest that prolonged continuous TNF-alpha blockade in patients may have long-term complications, including potential tumor development or progression.


Assuntos
Vigilância Imunológica , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Ligante de CD40/genética , Ligante de CD40/imunologia , Proliferação de Células , Células Cultivadas , Humanos , Vigilância Imunológica/genética , Inflamação/genética , Inflamação/imunologia , Coriomeningite Linfocítica/genética , Camundongos , Camundongos Knockout , Neoplasias/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética
10.
Arthritis Rheumatol ; 72(6): 919-930, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31943941

RESUMO

OBJECTIVE: Fcγ receptors (FcγR) play important roles in both protective and pathogenic immune responses. The assembly of the CBM signalosome encompassing caspase recruitment domain-containing protein 9, B cell CLL/lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) is required for optimal FcγR-induced canonical NF-κB activation and proinflammatory cytokine release. This study was undertaken to clarify the relevance of MALT-1 protease activity in FcγR-driven events and evaluate the therapeutic potential of selective MALT-1 protease inhibitors in FcγR-mediated diseases. METHODS: Using genetic and pharmacologic disruption of MALT-1 scaffolding and enzymatic activity, we assessed the relevance of MALT-1 function in murine and human primary myeloid cells upon stimulation with immune complexes (ICs) and in murine models of autoantibody-driven arthritis and immune thrombocytopenic purpura (ITP). RESULTS: MALT-1 protease function is essential for optimal FcγR-induced production of proinflammatory cytokines by various murine and human myeloid cells stimulated with ICs. In contrast, MALT-1 protease inhibition did not affect the Syk-dependent, FcγR-mediated production of reactive oxygen species or leukotriene B4 . Notably, pharmacologic MALT-1 protease inhibition in vivo reduced joint inflammation in the murine K/BxN serum-induced arthritis model (mean area under the curve for paw swelling of 45.42% versus 100% in control mice; P = 0.0007) but did not affect platelet depletion in a passive model of ITP. CONCLUSION: Our findings indicate a specific contribution of MALT-1 protease activity to FcγR-mediated events and suggest that MALT-1 protease inhibitors have therapeutic potential in a subset of FcγR-driven inflammatory disorders.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Receptores de IgG/imunologia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Plaquetas/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Células Mieloides/metabolismo
11.
J Med Chem ; 63(23): 14594-14608, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33216547

RESUMO

The paracaspase MALT1 has gained increasing interest as a target for the treatment of subsets of lymphomas as well as autoimmune diseases, and there is a need for suitable compounds to explore the therapeutic potential of this target. Here, we report the optimization of the in vivo potency of pyrazolopyrimidines, a class of highly selective allosteric MALT1 inhibitors. High doses of the initial lead compound led to tumor stasis in an activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) xenograft model, but this compound suffered from a short in vivo half-life and suboptimal potency in whole blood. Guided by metabolism studies, we identified compounds with reduced metabolic clearance and increased in vivo half-life. In the second optimization step, masking one of the hydrogen-bond donors of the central urea moiety through an intramolecular interaction led to improved potency in whole blood. This was associated with improved in vivo potency in a mechanistic model of B cell activation. The optimized compound led to tumor regression in a CARD11 mutant ABC-DLBCL lymphoma xenograft model.


Assuntos
Sangue/metabolismo , Inibidores de Caspase/uso terapêutico , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Ureia/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Inibidores de Caspase/síntese química , Inibidores de Caspase/metabolismo , Inibidores de Caspase/farmacocinética , Linhagem Celular Tumoral , Feminino , Meia-Vida , Humanos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microssomos Hepáticos/metabolismo , Neoplasias/tratamento farmacológico , Pirazóis/síntese química , Pirazóis/metabolismo , Pirazóis/farmacocinética , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratos Sprague-Dawley , Ovinos , Ureia/síntese química , Ureia/metabolismo , Ureia/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Front Immunol ; 11: 745, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32425939

RESUMO

Genetic disruption or short-term pharmacological inhibition of MALT1 protease is effective in several preclinical models of autoimmunity and B cell malignancies. Despite these protective effects, the severe reduction in regulatory T cells (Tregs) and the associated IPEX-like pathology occurring upon congenital disruption of the MALT1 protease in mice has raised concerns about the long-term safety of MALT1 inhibition. Here we describe the results of a series of toxicology studies in rat and dog species using MLT-943, a novel potent and selective MALT1 protease inhibitor. While MLT-943 effectively prevented T cell-dependent B cell immune responses and reduced joint inflammation in the collagen-induced arthritis rat pharmacology model, in both preclinical species, pharmacological inhibition of MALT1 was associated with a rapid and dose-dependent reduction in Tregs and resulted in the progressive appearance of immune abnormalities and clinical signs of an IPEX-like pathology. At the 13-week time point, rats displayed severe intestinal inflammation associated with mast cell activation, high serum IgE levels, systemic T cell activation and mononuclear cell infiltration in multiple tissues. Importantly, using thymectomized rats we demonstrated that MALT1 protease inhibition affects peripheral Treg frequency independently of effects on thymic Treg output and development. Our data confirm the therapeutic potential of MALT1 protease inhibitors but highlight the safety risks and challenges to consider before potential application of such inhibitors into the clinic.


Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/etiologia , Doenças Genéticas Ligadas ao Cromossomo X/etiologia , Doenças do Sistema Imunitário/congênito , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 1/etiologia , Cães , Feminino , Humanos , Doenças do Sistema Imunitário/etiologia , Inflamação/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Linfócitos T Reguladores/imunologia
13.
Mol Cancer Ther ; 19(10): 2089-2104, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32847974

RESUMO

The sole inhibitory Fcγ receptor CD32b (FcγRIIb) is expressed throughout B and plasma cell development and on their malignant counterparts. CD32b expression on malignant B cells is known to provide a mechanism of resistance to rituximab that can be ameliorated with a CD32b-blocking antibody. CD32b, therefore, represents an attractive tumor antigen for targeting with a monoclonal antibody (mAb). To this end, two anti-CD32b mAbs, NVS32b1 and NVS32b2, were developed. Their complementarity-determining regions (CDR) bind the CD32b Fc binding domain with high specificity and affinity while the Fc region is afucosylated to enhance activation of FcγRIIIa on immune effector cells. The NVS32b mAbs selectively target CD32b+ malignant cells and healthy B cells but not myeloid cells. They mediate potent killing of opsonized CD32b+ cells via antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) as well as complement-dependent cytotoxicity (CDC). In addition, NVS32b CDRs block the CD32b Fc-binding domain, thereby minimizing CD32b-mediated resistance to therapeutic mAbs including rituximab, obinutuzumab, and daratumumab. NVS32b mAbs demonstrate robust antitumor activity against CD32b+ xenografts in vivo and immunomodulatory activity including recruitment of macrophages to the tumor and enhancement of dendritic cell maturation in response to immune complexes. Finally, the activity of NVS32b mAbs on CD32b+ primary malignant B and plasma cells was confirmed using samples from patients with B-cell chronic lymphocytic leukemia (CLL) and multiple myeloma. The findings indicate the promising potential of NVS32b mAbs as a single agent or in combination with other mAb therapeutics for patients with CD32b+ malignant cells.


Assuntos
Linfoma de Células B/genética , Neoplasias de Plasmócitos/genética , Receptores de IgG/imunologia , Animais , Células CHO , Cricetulus , Humanos
14.
J Vis Exp ; (143)2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30663686

RESUMO

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-κB activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-κB activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor -signaling by contrast to TLR-signaling.


Assuntos
Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Animais , Humanos , Camundongos , Transdução de Sinais
15.
Glia ; 56(15): 1625-36, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18551629

RESUMO

T cell mediated immunotherapies are proposed for many cancers including malignant astrocytoma. As such therapies become more potent, but not necessarily more tumor-specific, the risk of collateral autoimmune damage to normal tissue increases. Tumors of the brain present significant challenges in this respect, as autoimmune destruction of brain tissue could have severe consequences. To investigate local immune reactivity toward a tumor-associated antigen in the brain, transgenic mice were generated that express a defined antigen (CW3 170-179) in astroglial cells. The resulting six transgenic mouse lines expressed the transgenic self-antigen in cells of the gastrointestinal tract and CNS compartments, or in the CNS alone. By challenging transgenic mice with tumor cells that express CW3, self/tumor-specific immune responses were visualized within a normal polyclonal T cell repertoire. A large expansion of the endogenous CW3 170-179-specific CD8 T cell population was observed in nontransgenic mice after both subcutaneous and intracerebral implantation of tumor cells. In contrast, CW3 170-179-specific immune responses were not observed in transgenic mice that exhibited extracerebral transgene expression. Importantly, in certain groups of mice in which transgene expression was restricted to the CNS, antigen-specific immune responses occurred when tumor was implanted subcutaneously, but not intracerebrally. This local immune tolerance in the brain was induced via peripheral (extrathymic) rather than central (thymic) tolerance mechanisms. Thus, this study highlights the role of regional immune regulation in the prevention of autoimmunity in the brain, and the potential impact of these mechanisms for brain tumor immunotherapy.


Assuntos
Antígenos de Neoplasias/imunologia , Astrócitos/imunologia , Astrocitoma/imunologia , Neoplasias Encefálicas/imunologia , Encéfalo/imunologia , Linfócitos T CD8-Positivos/imunologia , Tolerância Imunológica/imunologia , Animais , Antígenos de Neoplasias/genética , Astrócitos/patologia , Astrocitoma/fisiopatologia , Autoantígenos/genética , Autoantígenos/imunologia , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/prevenção & controle , Autoimunidade/genética , Autoimunidade/imunologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Neoplasias Encefálicas/fisiopatologia , Transplante de Tecido Encefálico , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Camundongos , Camundongos Transgênicos , Células Tumorais Cultivadas
16.
PLoS One ; 12(3): e0173176, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28257518

RESUMO

Although the role of T cells in autoimmunity has been explored for many years, the mechanisms leading to the initial priming of an autoimmune T cell response remain enigmatic. The 'hit and run' model suggests that self-antigens released upon cell death can provide the initial signal for a self-sustaining autoimmune response. Using a novel transgenic mouse model where we could induce the release of self-antigens via caspase-dependent apoptosis. We tracked the fate of CD8+ T cells specific for the self-antigen. Our studies demonstrated that antigens released from apoptotic cells were cross-presented by CD11c+ cells in the draining lymph node. This cross-presentation led to proliferation of self-antigen specific T cells, followed by a transient ability to produce IFN-γ, but did not lead to the development of autoimmune diabetes. Using this model we examined the consequences on T cell immunity when apoptosis was combined with dendritic cell maturation signals, an autoimmune susceptible genetic background, and the deletion of Tregs. The results of our study demonstrate that autoimmune diabetes cannot be initiated by the presentation of antigens released from apoptotic cells in vivo even in the presence of factors known to promote autoimmunity.


Assuntos
Autoantígenos/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Linfócitos T Reguladores/imunologia , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Apoptose/genética , Autoantígenos/imunologia , Autoimunidade/genética , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Humanos , Tolerância Imunológica , Interferon gama/genética , Camundongos , Camundongos Transgênicos/imunologia
17.
PLoS One ; 12(1): e0169026, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28052131

RESUMO

The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A.


Assuntos
Caspases/metabolismo , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/metabolismo , Linfócitos T/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 10 de Linfoma CCL de Células B , Western Blotting , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspases/genética , Linhagem Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Immunoblotting , Células Jurkat , Linfócitos/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Mutagênese , Proteínas de Neoplasias/genética , Isoformas de Proteínas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/genética , Ubiquitinação/genética , Ubiquitinação/fisiologia
18.
Sci Transl Med ; 8(367): 367ra166, 2016 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-27903862

RESUMO

Interleukin-2 (IL-2) immunotherapy is an attractive approach in treating advanced cancer. However, by binding to its IL-2 receptor α (CD25) subunit, IL-2 exerts unwanted effects, including stimulation of immunosuppressive regulatory T cells (Tregs) and contribution to vascular leak syndrome. We used a rational approach to develop a monoclonal antibody to human IL-2, termed NARA1, which acts as a high-affinity CD25 mimic, thereby minimizing association of IL-2 with CD25. The structure of the IL-2-NARA1 complex revealed that NARA1 occupies the CD25 epitope of IL-2 and precisely overlaps with CD25. Association of NARA1 with IL-2 occurs with 10-fold higher affinity compared to CD25 and forms IL-2/NARA1 complexes, which, in vivo, preferentially stimulate CD8+ T cells while disfavoring CD25+ Tregs and improving the benefit-to-adverse effect ratio of IL-2. In two transplantable and one spontaneous metastatic melanoma model, IL-2/NARA1 complex immunotherapy resulted in efficient expansion of tumor-specific and polyclonal CD8+ T cells. These CD8+ T cells showed robust interferon-γ production and expressed low levels of exhaustion markers programmed cell death protein-1, lymphocyte activation gene-3, and T cell immunoglobulin and mucin domain-3. These effects resulted in potent anticancer immune responses and prolonged survival in the tumor models. Collectively, our data demonstrate that NARA1 acts as a CD25-mimobody that confers selectivity and increased potency to IL-2 and warrant further assessment of NARA1 as a therapeutic.


Assuntos
Anticorpos Monoclonais/química , Imunoterapia/métodos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Interleucina-2/antagonistas & inibidores , Neoplasias/terapia , Animais , Sítios de Ligação , Linfócitos T CD8-Positivos/citologia , Proliferação de Células , Epitopos/química , Inativação Gênica , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/imunologia , Conformação Proteica , Recombinação Genética , Linfócitos T Citotóxicos/citologia , Linfócitos T Reguladores/imunologia
19.
J Invest Dermatol ; 121(4): 745-50, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14632191

RESUMO

Dendritic cells are excellent targets for antigen-specific immune intervention. Here we attempted to introduce a CD8 T cell-dependent epitope into dendritic cells for presentation on major histocompatibility complex class I and induction of immunity. Murine bone-marrow-derived dendritic cells were subjected to electroporation with mRNA transcribed in vitro from a plasmid encoding lymphocytic choriomeningitis virus glycoprotein or enhanced green fluorescent protein under the control of a T7 promotor. The transfection efficiency of dendritic cells was 22 to 40%. Maturation was not inhibited by the electroporation. Dendritic cells electroporated with the appropriate antigen induced cell number-dependent in vitro proliferation in CD8 T cells expressing a transgenic receptor recognizing the 33 to 41 sequence of lymphocytic choriomeningitis virus glycoprotein in association with H-2Kb/Db, indicating correct synthesis, processing, and presentation of the epitope. Naive C57BL/6 mice vaccinated with electroporated dendritic cells and challenged with lymphocytic choriomeningitis virus were protected. Vaccination induced epitope-specific T cells as assessed by tetramer staining in blood and spleen. These results indicate that targeting dendritic cells with antigen-encoding mRNA can induce antigen-specific CD8 T cell responses as well as protective anti-viral immunity in vivo. Targeting dendritic cells with antigen-encoding mRNA may find wider application for immune intervention in disorders such as autoimmunity and cancer.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/transplante , Coriomeningite Linfocítica/prevenção & controle , Vírus da Coriomeningite Linfocítica/imunologia , Vacinação , Animais , Apresentação de Antígeno , Células Dendríticas/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Camundongos Transgênicos , RNA Mensageiro , Transfecção , Transgenes/genética , Transgenes/imunologia
20.
PLoS One ; 8(3): e59630, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23544080

RESUMO

BACKGROUND: Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span. METHODOLOGY: We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation. PRINCIPAL FINDINGS: The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS. SIGNIFICANCE: The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.


Assuntos
Aldeído Liases/deficiência , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/prevenção & controle , Aldeído Liases/metabolismo , Animais , Encéfalo/metabolismo , Linfócitos T CD4-Positivos/imunologia , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/complicações , Fatores de Transcrição Forkhead/metabolismo , Hipersensibilidade Tardia/sangue , Hipersensibilidade Tardia/complicações , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/patologia , Memória Imunológica/imunologia , Integrases/metabolismo , Linfonodos/imunologia , Linfonodos/patologia , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Ovinos , Esfingolipídeos/metabolismo , Baço/imunologia , Baço/patologia , Análise de Sobrevida , Timo/imunologia , Timo/patologia
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