RESUMO
The immunological synapse (IS) serves a dual role for sustained T cell receptor (TCR) signaling and for TCR downregulation. TC21 (Rras2) is a RRas subfamily GTPase that constitutively associates with the TCR and is implicated in tonic TCR signaling by activating phosphatidylinositol 3-kinase. In this study, we demonstrate that TC21 both cotranslocates with the TCR to the IS and is necessary for TCR internalization from the IS through a mechanism dependent on RhoG, a small GTPase previously associated with phagocytosis. Indeed, we found that the TCR triggers T cells to phagocytose 1-6 µm beads through a TC21- and RhoG-dependent pathway. We further show that TC21 and RhoG are necessary for the TCR-promoted uptake of major histocompatibility complex (MHC) from antigen-presenting cells. Therefore, TC21 and RhoG dependence underlie the existence of a common phagocytic mechanism that drives TCR internalization from the IS together with its peptide-MHC ligand.
Assuntos
Sinapses Imunológicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fagocitose , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Apresentação de Antígeno , Antígenos/metabolismo , Comunicação Celular , Antígenos de Histocompatibilidade Classe II , Humanos , Sinapses Imunológicas/patologia , Células Jurkat , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Monoméricas de Ligação ao GTP/imunologia , Fragmentos de Peptídeos/imunologia , Fagocitose/imunologia , Transporte Proteico , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Proteínas rho de Ligação ao GTP/imunologiaRESUMO
The role of the center of the immunological synapse (the central supramolecular activation cluster or cSMAC) is controversial. One model suggests that the role of the cSMAC depends on antigen quality and can both enhance signaling and receptor downregulation, whereas a second model proposes that the sole function of the cSMAC is to downregulate signaling. An important distinction between the models is whether signaling occurs in the cSMAC. Here, we demonstrate that at early time points, signaling occurs outside the cSMAC, but occurs in the cSMAC at later time points. Additionally, we show that cSMAC formation enhances the stimulatory potency of weak agonists for the TCR. Combined with previous studies showing that cSMAC formation decreases the signaling by strong agonists, our data support a model proposing that signaling and receptor degradation both occur in the cSMAC and that the balance between signaling and degradation in the synapse is determined by antigen quality.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Junções Intercelulares/metabolismo , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteína-Tirosina Quinase ZAP-70/metabolismo , Animais , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Regulação para Baixo , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Peptídeos/metabolismo , Fosfatidilinositóis/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Receptores de Antígenos de Linfócitos T/agonistas , TransfecçãoRESUMO
Activator protein 1 (AP-1, also known as JUN) transcription factors are dimers of JUN, FOS, MAF and activating transcription factor (ATF) family proteins characterized by basic region and leucine zipper domains. Many AP-1 proteins contain defined transcriptional activation domains, but BATF and the closely related BATF3 (refs 2, 3) contain only a basic region and leucine zipper, and are considered to be inhibitors of AP-1 activity. Here we show that Batf is required for the differentiation of IL17-producing T helper (T(H)17) cells. T(H)17 cells comprise a CD4(+) T-cell subset that coordinates inflammatory responses in host defence but is pathogenic in autoimmunity. Batf(-/-) mice have normal T(H)1 and T(H)2 differentiation, but show a defect in T(H)17 differentiation, and are resistant to experimental autoimmune encephalomyelitis. Batf(-/-) T cells fail to induce known factors required for T(H)17 differentiation, such as RORgamma t (encoded by Rorc) and the cytokine IL21 (refs 14-17). Neither the addition of IL21 nor the overexpression of RORgamma t fully restores IL17 production in Batf(-/-) T cells. The Il17 promoter is BATF-responsive, and after T(H)17 differentiation, BATF binds conserved intergenic elements in the Il17a-Il17f locus and to the Il17, Il21 and Il22 (ref. 18) promoters. These results demonstrate that the AP-1 protein BATF has a critical role in T(H)17 differentiation.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular , Interleucina-17/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Fatores de Transcrição de Zíper de Leucina Básica/genética , Encefalomielite Autoimune Experimental/genética , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Interleucina-17/biossíntese , Interleucina-17/genética , Interleucinas/genética , Interleucinas/metabolismo , Interleucinas/farmacologia , Linfonodos/metabolismo , Masculino , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Regiões Promotoras Genéticas/genética , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Fator de Transcrição AP-1/deficiência , Fator de Transcrição AP-1/genéticaRESUMO
HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B-dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Antígenos CD/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Mieloma Múltiplo/terapia , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Degranulação Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Sinergismo Farmacológico , Feminino , Proteínas Ligadas por GPI/imunologia , Humanos , Células Matadoras Naturais/imunologia , Lenalidomida , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Depleção Linfocítica , Macaca fascicularis , Camundongos , Camundongos SCID , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Talidomida/administração & dosagem , Talidomida/análogos & derivados , Talidomida/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Engagement of the low-affinity Ab receptor FcγRIIb downregulates B cell activation, and its dysfunction is associated with autoimmunity in mice and humans. We engineered the Fc domain of an anti-human CD19 Ab to bind FcγRIIb with high affinity, promoting the coengagement of FcγRIIb with the BCR complex. This Ab (XmAb5871) stimulated phosphorylation of the ITIM of FcγRIIb and suppressed BCR-induced calcium mobilization, proliferation, and costimulatory molecule expression of human B cells from healthy volunteers and systemic lupus erythematosus (SLE) patients, as well as B cell proliferation induced by LPS, IL-4, or BAFF. XmAb5871 suppressed humoral immunity against tetanus toxoid and reduced serum IgM, IgG, and IgE levels in SCID mice engrafted with SLE or healthy human PBMC. XmAb5871 treatment also increased survival of mice engrafted with PBMC from a unique SLE patient. Unlike anti-CD20 Ab, coengagement of FcγRIIb and BCR complex did not promote B cell depletion in human PBMC cultures or in mice. Thus, amplification of the FcγRIIb inhibitory pathway in activated B cells may represent a novel B cell-targeted immunosuppressive therapeutic approach for SLE and other autoimmune diseases that should avoid the complications associated with B cell depletion.
Assuntos
Sítios de Ligação de Anticorpos , Comunicação Celular/imunologia , Imunidade Humoral , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de IgG/metabolismo , Animais , Antígenos CD19/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/patologia , Comunicação Celular/genética , Modelos Animais de Doenças , Feminino , Amplificação de Genes/imunologia , Células HEK293 , Humanos , Imunidade Humoral/genética , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/prevenção & controle , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/fisiologia , Receptores de IgG/deficiência , Receptores de IgG/fisiologiaRESUMO
Lymphocyte arrest and spreading on ICAM-1-expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca(2+). In contrast to LFA-1-activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1.
Assuntos
Quimiocinas/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Actinas/genética , Actinas/imunologia , Animais , Quimiocinas/farmacologia , Citoesqueleto/genética , Citoesqueleto/imunologia , Humanos , Molécula 1 de Adesão Intercelular/química , Molécula 1 de Adesão Intercelular/genética , Antígenos Comuns de Leucócito/química , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Transgênicos , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.
Assuntos
Imunidade Inata/imunologia , Imunoterapia/métodos , Interferons/metabolismo , Neoplasias/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , CamundongosRESUMO
Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.
Assuntos
Proteínas de Membrana , Neoplasias , Animais , Citocinas , Humanos , Imunoterapia/métodos , Interferons , Camundongos , Neoplasias/tratamento farmacológicoRESUMO
Immunological synapse formation between T cells and target cells can affect the functional outcome of TCR ligation by a given MHC-peptide complex. Although synapse formation is usually induced by TCR signaling, it is not clear whether other factors can affect the efficiency of synapse formation. Here, we tested whether cytokines could influence synapse formation between murine CTLs and target cells. We found that IL-12 enhanced synapse formation, whereas TGFbeta decreased synapse formation. The enhanced synapse formation induced by IL-12 appeared to be functional, given that IL-12-treated cells could respond to weak peptides, including self-peptides, to which the T cells were normally unresponsive. These responses correlated with expression of functionally higher avidity LFA-1 on IL-12-treated CTLs. These findings have implications for the function of IL-12 in T cell-mediated autoimmunity.
Assuntos
Autoantígenos/metabolismo , Sinapses Imunológicas/metabolismo , Interleucina-12/fisiologia , Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Sequência de Aminoácidos , Animais , Autoantígenos/imunologia , Células Cultivadas , Citotoxicidade Imunológica , Mediadores da Inflamação/fisiologia , Interleucina-12/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Transdução de Sinais/imunologiaRESUMO
Targeted pharmacologic activation of antigen-specific (AgS) T cells may bypass limitations inherent in current T cell-based cancer therapies. We describe two immunotherapeutics platforms for selective delivery of costimulatory ligands and peptide-HLA (pHLA) to AgS T cells. We engineered and deployed on these platforms an affinity-attenuated variant of interleukin-2, which selectively expands oligoclonal and polyfunctional AgS T cells in vitro and synergizes with CD80 signals for superior proliferation versus peptide stimulation.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Proteínas Recombinantes de Fusão/imunologia , Animais , Antígeno B7-1/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Antígenos HLA-A/genética , Antígenos HLA-A/imunologia , Humanos , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias/imunologia , Peptídeos/genética , Peptídeos/imunologia , Cultura Primária de Células , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genéticaRESUMO
Cancer immunotherapy has dramatically changed the approach to cancer treatment. The aim of targeting the immune system to recognize and destroy cancer cells has afforded many patients the prospect of achieving deep, long-term remission and potential cures. However, many challenges remain for achieving the goal of effective immunotherapy for all cancer patients. Checkpoint inhibitors have been able to achieve long-term responses in a minority of patients, yet improving response rates with combination therapies increases the possibility of toxicity. Chimeric antigen receptor T cells have demonstrated high response rates in hematological cancers, although most patients experience relapse. In addition, some cancers are notoriously immunologically "cold" and typically are not effective targets for immunotherapy. Overcoming these obstacles will require new strategies to improve upon the efficacy of current agents, identify biomarkers to select appropriate therapies, and discover new modalities to expand the accessibility of immunotherapy to additional tumor types and patient populations.
Assuntos
Imunoterapia Adotiva , Imunoterapia/tendências , Recidiva Local de Neoplasia/terapia , Neoplasias/terapia , Biomarcadores Tumorais/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Terapia Combinada , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Recidiva Local de Neoplasia/imunologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Microambiente Tumoral/imunologiaRESUMO
PURPOSE: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. EXPERIMENTAL DESIGN: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno HLA-A2/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Interleucina-2/imunologia , Neoplasias/terapia , Proteínas E7 de Papillomavirus/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Voluntários Saudáveis , Humanos , Leucócitos Mononucleares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/imunologia , Neoplasias/virologiaRESUMO
Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Membrana/metabolismo , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , HumanosRESUMO
The contact site between T cells and antigen-presenting cells or T cells and their targets--the immunological synapse--is a highly specialized structure potentially involved in T-cell activation and function. Although many insights have been obtained since the initial description of the immune synapse, recent advances have provided us with a better understanding of mechanisms involved in synapse formation and in the diversity of synapse morphologies. New potential roles for the synapse such as in polarized cytokine secretion or in adaptive control of T-cell activation have been proposed.
Assuntos
Comunicação Celular/imunologia , Junções Intercelulares/imunologia , Linfócitos T/imunologia , Animais , Humanos , Modelos Imunológicos , Transdução de Sinais/imunologiaRESUMO
Understanding the physiology of complex relationships between components of signaling pathways and the actin cytoskeleton is an important challenge. CD2AP is a membrane scaffold protein implicated in a variety of physiological and disease processes. The physiological function of CD2AP is unclear, but its biochemical interactions suggest that it has a role in dynamic actin assembly. Here, we report that CD2AP functions to facilitate the recruitment of actin capping protein (CP) to the Src kinase substrate, cortactin, at the cell periphery, and that this is necessary for formation of the short branched filaments that characterize lamellipodium formation and are required for cell migration. Superresolution fluorescence microscopy demonstrated that the efficient colocalization of CP and cortactin at the cell periphery required CD2AP. As both cortactin and CP function to enhance branched actin filament formation, CD2AP functions synergistically to enhance the function of both proteins. Our data demonstrate how the interplay between specialized actin regulatory molecules shapes the actin cytoskeleton.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cortactina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Podócitos/citologia , Pseudópodes/metabolismo , Proteínas de Capeamento de Actina/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Movimento Celular , Cortactina/genética , Proteínas do Citoesqueleto/genética , Camundongos , Camundongos Mutantes , Podócitos/metabolismo , Domínios de Homologia de src , Quinases da Família src/metabolismoRESUMO
Mast cells and basophils play a central role in allergy, asthma, and anaphylaxis, as well as in non-allergic inflammatory, neurological and autoimmune diseases. Allergen-mediated cross-linking of IgE bound to FcεRI leads to cellular activation, and the low-affinity Fc receptor FcγRIIb is a key inhibitor of subsequent degranulation. FcγRIIb, when coengaged with FcεRI via allergen bound to IgE, stimulates ITIM domain-mediated inhibitory signaling that efficiently suppresses mast cell and basophil activation. To assess the therapeutic potential of directed coengagement of FcεRI and FcγRIIb in the absence of FcεRI crosslinking, we developed a fusion protein comprising the coupled Fc domains of murine IgE and human IgG1. As a key functional component of this tandem Fcε-Fcγ biologic, we engineered its IgG1 Fc domain to bind to human FcγRIIb with 100-fold enhanced affinity relative to native IgG1 Fc. Using mast cells from mice transgenic for human FcγRIIb, we show that this tandem Fc binds with high affinity to murine FcεRI and human FcγRIIb on mast cells, triggers phosphorylation of FcγRIIb, and inhibits FcεRI-dependent calcium mobilization. Control tandem Fc biologics containing a native IgG1 Fc domain or lacking binding to Fcγ receptors were markedly less active, demonstrating that the affinity-optimized tandem Fc can inhibit degranulation through stimulation of FcγRIIb signaling as well as through competition with allergen-IgE immune complex for FcεRI binding. We propose that in the context of a fully human tandem Fc biologic, high-affinity coengagement of FcεRI and FcγRIIb has potential as a novel therapy for allergy and other mast cell and basophil-mediated pathologies.
Assuntos
Degranulação Celular , Imunoglobulina E/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Mastócitos/fisiologia , Receptores de IgG/imunologia , Animais , Cálcio/metabolismo , Diferenciação Celular , Humanos , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Fosforilação , Proteínas Recombinantes/imunologiaRESUMO
BACKGROUND: T cell receptor (TCR) engagement leads to formation of signaling microclusters and induction of rapid and dynamic changes in the actin cytoskeleton, although the exact mechanism by which the TCR initiates actin polymerization is incompletely understood. The Vav family of guanine nucleotide exchange factors (GEF) has been implicated in generation of TCR signals and immune synapse formation, however, it is currently not known if Vav's GEF activity is required in T cell activation by the TCR in general, and in actin polymerization downstream of the TCR in particular. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that Vav1 assembles into signaling microclusters at TCR contact sites and is critical for TCR-initiated actin polymerization. Surprisingly, Vav1 functions in TCR signaling and Ca(++) mobilization via a mechanism that does not appear to strictly depend on the intrinsic GEF activity. CONCLUSIONS/SIGNIFICANCE: We propose here a model in which Vav functions primarily as a tyrosine phosphorylated linker-protein for TCR activation of T cells. Our results indicate that, contrary to expectations based on previously published studies including from our own laboratory, pharmacological inhibition of Vav1's intrinsic GEF activity may not be an effective strategy for T cell-directed immunosuppressive therapy.
Assuntos
Actinas/fisiologia , Citoesqueleto/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Ativação Linfocitária , Proteínas Proto-Oncogênicas c-vav/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/imunologia , Animais , Biopolímeros/fisiologia , Camundongos , Camundongos KnockoutRESUMO
T cell activation is predicated on the interaction between the T cell receptor and peptide-major histocompatibility (pMHC) ligands. The factors that determine the stimulatory potency of a pMHC molecule remain unclear. We describe results showing that a peptide exhibiting many hallmarks of a weak agonist stimulates T cells to proliferate more than the wild-type agonist ligand. An in silico approach suggested that the inability to form the central supramolecular activation cluster (cSMAC) could underlie the increased proliferation. This conclusion was supported by experiments that showed that enhancing cSMAC formation reduced stimulatory capacity of the weak peptide. Our studies highlight the fact that a complex interplay of factors determines the quality of a T cell antigen.
Assuntos
Antígenos/imunologia , Antígenos de Histocompatibilidade/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Proliferação de Células , Regulação para Baixo , Antígenos de Histocompatibilidade/genética , Cinética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Fosforilação , Receptores de Antígenos de Linfócitos T/agonistas , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores Imunológicos/imunologia , Receptores de Células Matadoras NaturaisRESUMO
In several human pathologies (e.g. cancer, rheumatoid arthritis, AIDS and leprosy) oxidative stress induces T cell hyporesponsiveness. Hyporesponsive T cells often appear to display impaired expression of some (e.g. TCR-zeta, p56(lck) and LAT) but not all (e.g. TCR-alphabeta and CD3-epsilon) crucial TCR-proximal signaling molecules but the underlying mechanisms have as yet not been identified. Using an in vitro system for oxidative-stress-induced T cell hyporesponsiveness we here report two sequential effects of oxidative stress on TCR signaling molecules: protein alterations and proteasomal degradation. We have identified the C-terminal part of TCR-zeta and the membrane-proximal domain of p56(lck) as potential targets for modifications induced by reactive oxygen species. Oxidative-stress-exposed proteins were differentially susceptible to proteasomal degradation: whereas modified TCR-zeta was relatively resistant, reactive oxygen species (ROS)-altered LAT and p56(lck) were much more susceptible. Importantly, we found that T cell hyporesponsiveness best correlated with ROS-dependent protein alteration since inhibition of proteasomal degradation did not restore function. Finally, our data provide an explanation for the paradox of reduced TCR-zeta signals combined with unaltered TCR-alphabeta and CD3-epsilon expression levels: the TCR-zeta chain in hyporesponsive T cells is still expressed but no longer detectable by certain mAb recognizing ROS-sensitive epitopes.
Assuntos
Acetilcisteína/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Membrana , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Acetilcisteína/farmacologia , Proteínas de Transporte/metabolismo , Técnicas de Cocultura , Cisteína Endopeptidases/metabolismo , Humanos , Tolerância Imunológica , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Neutrófilos/imunologia , Estresse Oxidativo , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma , Receptores de Antígenos de Linfócitos T gama-delta/química , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Transdução de Sinais , Linfócitos T/efeitos dos fármacosRESUMO
Oxidative stress plays an important role in the induction of T lymphocyte hyporesponsiveness observed in several human pathologies including cancer, rheumatoid arthritis, leprosy, and AIDS. To investigate the molecular basis of oxidative stress-induced T cell hyporesponsiveness, we have developed an in vitro system in which T lymphocytes are rendered hyporesponsive by co-culture with oxygen radical-producing activated neutrophils. We have observed a direct correlation between the level of T cell hyporesponsiveness induced and the concentration of reactive oxygen species produced. Moreover, induction of T cell hyporesponsiveness is blocked by addition of N-acetyl cysteine, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, and catalase, confirming the critical role of oxidative stress in this system. The pattern of tyrosine-phosphorylated proteins was profoundly altered in hyporesponsive as compared with normal T cells. In hyporesponsive T cells, T cell receptor (TCR) ligation no longer induced phospholipase C-gamma1 activation and caused reduced Ca(2+) flux. In contrast, despite increased levels of ERK1/2 phosphorylation, TCR-dependent activation of mitogen-activated protein kinase ERK1/2 was unaltered in hyporesponsive T lymphocytes. A late TCR-signaling event such as caspase 3 activation was as well unaffected in hyporesponsive T lymphocytes. Our data indicate that TCR-signaling pathways are differentially affected by physiological levels of oxidative stress and would suggest that although "hyporesponsive" T cells have lost certain effector functions, they may have maintained or gained others.