RESUMO
Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4ß7 in conventional T cells while preserving α4ß7 in regulatory T cells, with findings suggesting increased ß1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4ß7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Camundongos , Humanos , Animais , Transplante Homólogo , Receptores Notch/metabolismo , Transdução de Sinais , Doença Enxerto-Hospedeiro/metabolismo , PrimatasRESUMO
Memory CD8+ T cells populate non-lymphoid tissues (NLTs) following pathogen infection, but little is known about the establishment of endogenous tumor-specific tissue-resident memory T cells (TRM) during cancer immunotherapy. Using a transplantable mouse model of prostate carcinoma, here we report that tumor challenge leads to expansion of naïve neoantigen-specific CD8+ T cells and formation of a small population of non-recirculating TRM in several NLTs. Primary tumor destruction by irreversible electroporation (IRE), followed by anti-CTLA-4 immune checkpoint inhibitor (ICI), promotes robust expansion of tumor-specific CD8+ T cells in blood, tumor, and NLTs. Parabiosis studies confirm that TRM establishment following dual therapy is associated with tumor remission in a subset of cases and protection from subsequent tumor challenge. Addition of anti-PD-1 following dual IRE + anti-CTLA-4 treatment blocks tumor growth in non-responsive cases. This work indicates that focal tumor destruction using IRE combined with ICI is a potent in situ tumor vaccination strategy that generates protective tumor-specific TRM.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Eletroporação/métodos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoterapia/métodos , Neoplasias da Próstata/terapia , Animais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Memória Imunológica/imunologia , Estimativa de Kaplan-Meier , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias da Próstata/imunologia , Microambiente Tumoral/imunologiaRESUMO
Therapeutic activation of macrophage phagocytosis has the ability to restrain tumour growth through phagocytic clearance of tumour cells and activation of the adaptive immune response. Our objective for this study was to evaluate the effects of modulating pro- and anti-phagocytic pathways in malignant melanoma. In order to identify evolutionarily conserved mechanisms of resistance that may be important for melanoma cell survival, we utilized a multi-species approach and examined the phagocytosis of human, mouse, and dog melanoma cells. We observed that melanoma cells from all three species displayed unexpected resistance to phagocytosis that could not be fully mitigated by blockade of the 'don't eat me' signal CD47 or by chemotherapeutic enhancement of known 'eat me' signals. Additionally, CD47 blockade failed to promote anti-melanoma immune responses or tumour regression in vivo. This melanoma resistance to phagocytosis was not mediated by soluble factors, and it was unaffected by siRNA-mediated knockdown of 47 prospective 'don't eat me' signals or by CRISPR-Cas-mediated CD47 knockout. Unexpectedly, CD47 knockout also did not enhance phagocytosis of lymphoma cells, but it eliminated the pro-phagocytic effect of CD47 blockade, suggesting that the pro-phagocytic effects of CD47 blockade are due in part to Fc receptor engagement. From this study, we conclude that melanoma cells possess an evolutionarily conserved resistance to macrophage phagocytosis. Further investigation will be needed to overcome the mechanisms that mediate melanoma cell resistance to innate immunity.
Assuntos
Antígeno CD47/metabolismo , Melanoma/genética , Fagocitose/fisiologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Transdução de Sinais , Transfecção , Regulação para CimaRESUMO
Focal thermal therapy (Heat), cryosurgery (Cryo) and irreversible electroporation (IRE) are increasingly used to treat cancer. However, local recurrence and systemic spread are persistent negative outcomes. Nevertheless, emerging work with immunotherapies (i.e., checkpoint blockade or dendritic cell (DC) vaccination) in concert with focal therapies may improve outcomes. To understand the role of focal therapy in priming the immune system for immunotherapy, an in vitro model of T cell response after exposure to B16 melanoma cell lysates after lethal exposures was designed. Exposure included: Heat (50 °C, 30 min), Cryo (-80 °C, 30 min) and IRE (1250 V/cm, 99 pulses, 50 µs pulses with 1 Hz intervals). After viability assessment (CCK-8 assay), cell lysates were collected and assessed for protein release (BCA assay), protein denaturation (FTIR-spectroscopy), TRP-2 antigen release (western blot), and T cell activation (antigen-specific CD8 T cell proliferation). Results showed IRE released the most protein and antigen (TRP-2), followed by Cryo and Heat. In contrast, Cryo released the most native (not denatured) protein, compared to IRE and Heat. Finally, IRE dramatically outperformed both Cryo and Heat in T cell activation while Cryo modestly outperformed Heat. This study demonstrates that despite all focal therapies ability to destroy cells, the 'quantity' (i.e., amount) and 'quality' (i.e., molecular state) of tumor protein (including antigen) released can dramatically change the ensuing priming of the immune system. This suggests protein-based metrics whereby focal therapies can be designed to prime the immune system in concert with immunotherapies to eventually achieve improved and durable cancer treatment in vivo.
Assuntos
Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/imunologia , Neoplasias/imunologia , Animais , Humanos , Camundongos , Camundongos TransgênicosRESUMO
Vγ9Vδ2 T cells, the major subset of the human peripheral blood γδ T-cell, respond to microbial infection and stressed cells through the recognition of phosphoantigens. In contrast to the growing knowledge of antigen-mediated activation mechanisms, the antigen-independent and cytokine-mediated activation mechanisms of Vγ9Vδ2 T cells are poorly understood. Here, we show that interleukin (IL) -12 and IL-18 synergize to activate human ex vivo-expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells treated with IL-12 and IL-18 enhanced effector functions, including the expression of IFN-γ and granzyme B, and cytotoxicity. These enhanced effector responses following IL-12 and IL-18 treatment were associated with homotypic aggregation, enhanced expression of ICAM-1 and decreased expression of the B- and T-lymphocyte attenuator (BTLA), a co-inhibitory receptor. IL-12 and IL-18 also induced the antigen-independent proliferation of Vγ9Vδ2 T cells. Increased expression of IκBζ, IL-12Rß2 and IL-18Rα following IL-12 and IL-18 stimulation resulted in sustained activation of STAT4 and NF-κB. The enhanced production of IFN-γ and cytotoxic activity are critical for cancer immunotherapy using Vγ9Vδ2 T cells. Thus, the combined treatment of ex vivo-expanded Vγ9Vδ2 T cells with IL-12 and IL-18 may serve as a new strategy for the therapeutic activation of these cells.
Assuntos
Citocinas/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Proteínas I-kappa B/genética , Proteínas Nucleares/genéticaRESUMO
Hypertension often occurs in concurrence with obesity and diabetes mellitus, commonly referred to as metabolic syndrome. Renal denervation (RDNx) lowers arterial pressure (AP) and improves glucose metabolism in drug-resistant hypertensive patients with high body mass index. In addition, RDNx has been shown to reduce renal inflammation in the mouse model of angiotensin II hypertension. The present study tested the hypothesis that RDNx reduces AP and renal inflammation and improves glucose metabolism in obesity-induced hypertension. Eight-week-old C57BL/6J mice were fed either a low-fat diet (10 kcal%) or a high-fat diet (45 kcal%) for 10 weeks. Body weight, food intake, fasting blood glucose, and glucose metabolism (glucose tolerance test) were measured. In a parallel study, radiotelemeters were implanted in mice for AP measurement. High fat-fed C57BL/6J mice exhibited an inflammatory and metabolic syndrome phenotype, including increased fat mass, increased AP, and hyperglycemia compared with low-fat diet mice. RDNx, but not Sham surgery, normalized AP in high-fat diet mice (115.8±1.5 mm Hg in sham versus 96.6±6.7 mm Hg in RDNx). RDNx had no significant effect on AP in low-fat diet mice. Also, RDNx had no significant effect on glucose metabolism or renal inflammation as measured by the number of CD8, CD4, and T helper cells or levels of inflammatory cytokines in the kidneys. These results indicate that although renal nerves play a role in obesity-induced hypertension, they do not contribute to impaired glucose metabolism or renal inflammation in this model.
Assuntos
Pressão Arterial/fisiologia , Denervação Autônoma/métodos , Hipertensão/fisiopatologia , Rim/cirurgia , Nefrite/patologia , Animais , Glicemia/metabolismo , Peso Corporal , Citocinas/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Citometria de Fluxo , Hipertensão/etiologia , Imuno-Histoquímica , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Nefrite/fisiopatologia , Obesidade/complicações , Distribuição Aleatória , Sensibilidade e EspecificidadeRESUMO
The inflammatory response regulates congestive heart failure (CHF) development. T cell activation plays an important role in tissue inflammation. We postulate that CD28 or B7 deficiency inhibits T cell activation and attenuates CHF development by reducing systemic, cardiac, and pulmonary inflammation. We demonstrated that chronic pressure overload-induced end-stage CHF in mice is characterized by profound accumulation of activated effector T cells (CD3(+)CD44(high) cells) in the lungs and a mild but significant increase of these cells in the heart. In knockout mice lacking either CD28 or B7, there was a dramatic reduction in the accumulation of activated effector T cells in both hearts and lungs of mice under control conditions and after transverse aortic constriction. CD28 or B7 knockout significantly attenuated transverse aortic constriction-induced CHF development, as indicated by less increase of heart and lung weight and less reduction of left ventricle contractility. CD28 or B7 knockout also significantly reduced transverse aortic constriction-induced CD45(+) leukocyte, T cell, and macrophage infiltration in hearts and lungs, lowered proinflammatory cytokine expression (such as tumor necrosis factor-α and interleukin-1ß) in lungs. Furthermore, CD28/B7 blockade by CTLA4-Ig treatment (250 µg/mouse every 3 days) attenuated transverse aortic constriction-induced T cell activation, left ventricle hypertrophy, and left ventricle dysfunction. Our data indicate that CD28/B7 deficiency inhibits activated effector T cell accumulation, reduces myocardial and pulmonary inflammation, and attenuates the development of CHF. Our findings suggest that strategies targeting T cell activation may be useful in treating CHF.
Assuntos
Abatacepte/farmacologia , Antígenos B7/metabolismo , Antígenos CD28/metabolismo , Insuficiência Cardíaca/fisiopatologia , Pneumonia/fisiopatologia , Análise de Variância , Animais , Antígenos B7/imunologia , Antígenos CD28/imunologia , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Insuficiência Cardíaca/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/imunologia , Distribuição Aleatória , Estatísticas não Paramétricas , Sístole/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Adhesion and degranulation promoting adapter protein (ADAP) is a multifunctional hematopoietic adapter protein that regulates TCR-dependent increases in both integrin function and activation of the NF-κB transcription factor. Activation of integrin function requires both ADAP and the ADAP-associated adapter Src kinase-associated phosphoprotein of 55 kDa (SKAP55). In contrast, ADAP-mediated regulation of NF-κB involves distinct binding sites in ADAP that promote the inducible association of ADAP, but not SKAP55, with the CARMA1 adapter and the TAK1 kinase. This suggests that the presence or absence of associated SKAP55 defines functionally distinct pools of ADAP. To test this hypothesis, we developed a novel SKAP-ADAP chimeric fusion protein and demonstrated that physical association of ADAP with SKAP55 is both sufficient and necessary for the rescue of integrin function in ADAP-deficient T cells. Similar to wild-type ADAP, the SKAP-ADAP chimera associated with the LFA-1 integrin after TCR stimulation. Although the SKAP-ADAP chimera contains the CARMA1 and TAK1 binding sequences from ADAP, expression of the chimera does not restore NF-κB signaling in ADAP(-/-) T cells. A single point mutation in the pleckstrin homology domain of SKAP55 (R131M) blocks the ability of the SKAP-ADAP chimera to restore integrin function and to associate with LFA-1. However, the R131M mutant was now able to restore NF-κB signaling in ADAP-deficient T cells. We conclude that integrin regulation by ADAP involves the recruitment of ADAP to LFA-1 integrin complexes by the pleckstrin homology domain of SKAP55, and this recruitment restricts the ability of ADAP to interact with the NF-κB signalosome and regulate NF-κB activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Integrinas/imunologia , NF-kappa B/imunologia , Fosfoproteínas/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação/genética , Proteínas Sanguíneas/metabolismo , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Integrinas/metabolismo , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Mutação , NF-kappa B/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologiaRESUMO
The long-term maintenance of memory CD4 T cells promotes protective immunity against future pathogen reinfection. As a site rich in survival cytokines, the bone marrow is proposed to be a critical niche for the survival of memory CD4 T cells. We demonstrate that endogenous, polyclonal Ag-specific CD4 T cells rapidly enter and are recovered long-term from the bone marrow following i.v. infection with Listeria monocytogenes. ß(1) integrin-deficient CD4 T cells also populate the bone marrow early following an infection, but their numbers in this site rapidly decline. This decline was not caused by increased death of T cells lacking ß(1) integrin but rather by reduced retention in the bone marrow after the primary immune response. The loss of memory CD4 T cells from the bone marrow does not lead to a loss of the predominant source of memory CD4 T cells in the spleen or the ability to mount a memory response. Thus, ß(1) integrin-dependent maintenance of memory CD4 T cells in the bone marrow is not required for long-term CD4 T cell memory.
Assuntos
Células da Medula Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Movimento Celular/imunologia , Epitopos de Linfócito T/imunologia , Memória Imunológica , Integrina beta1/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/microbiologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/microbiologia , Movimento Celular/genética , Epitopos de Linfócito T/genética , Memória Imunológica/genética , Cadeias beta de Integrinas/genética , Integrina beta1/genética , Integrina beta1/metabolismo , Listeriose/genética , Listeriose/imunologia , Listeriose/patologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fatores de TempoRESUMO
The alpha4beta7 integrin promotes homing of T cells to intestinal sites. The alpha4 integrin subunit that pairs with beta7 integrin can also pair with beta1 integrin. In this paper, we show that the preferential pairing of beta1 integrin with alpha4 integrin regulates the expression of alpha4beta7 on T cells. In the absence of beta1 integrin, naive mouse CD4 T cells have increased alpha4beta7 expression, resulting in increased adhesion to mucosal addressin cell adhesion molecule-1 and enhanced homing to Peyer's patches (PP). In a reciprocal manner, overexpression of beta1 integrin causes the loss of alpha4beta7 expression and decreased homing to PP. A similar upregulation of beta1 integrin and suppression of alpha4beta7 expression occurs rapidly after CD4 T cell activation. beta1 integrin thus dominates beta7 integrin for alpha4 integrin pairing, thereby controlling the abundance of unpaired alpha4 integrin. Increasing the abundance of alpha4 integrin relative to beta1 integrin is critical to retinoic acid-mediated expression of alpha4beta7 integrin during T cell activation. In the absence of beta1 integrin, endogenous Ag-specific CD4 T cells uniformly express high levels of alpha4beta7 after Listeria monocytogenes infection. The resulting beta1-deficient early memory T cells have decreased localization to the bone marrow and enhanced localization to PP after infection. Thus, the preferential association of beta1 integrin with alpha4 integrin suppresses alpha4beta7 integrin expression and regulates the localization of memory CD4 T cells.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Integrina beta1/metabolismo , Integrinas/metabolismo , Nódulos Linfáticos Agregados/metabolismo , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Integrina alfa4/genética , Integrina alfa4/metabolismo , Integrina beta1/genética , Integrinas/genética , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/metabolismo , Listeriose/microbiologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Nódulos Linfáticos Agregados/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tretinoína/farmacologiaRESUMO
WAVE2 regulates T cell receptor (TCR)-stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL-C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL-C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Integrinas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/fisiologia , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adesão Celular/fisiologia , Ativação Enzimática , Fibronectinas/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Humanos , Integrinas/genética , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-abl/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Following TCR stimulation, T cells utilize the hematopoietic specific adhesion and degranulation-promoting adapter protein (ADAP) to control both integrin adhesive function and NF-kappaB transcription factor activation. We have investigated the molecular basis by which ADAP controls these events in primary murine ADAP(-/-) T cells. Naive DO11.10/ADAP(-/-) T cells show impaired adhesion to OVAp (OVA aa 323-339)-bearing APCs that is restored following reconstitution with wild-type ADAP. Mutational analysis demonstrates that the central proline-rich domain and the C-terminal domain of ADAP are required for rescue of T:APC conjugate formation. The ADAP proline-rich domain is sufficient to bind and stabilize the expression of SKAP55 (Src kinase-associated phosphoprotein of 55 kDa), which is otherwise absent from ADAP(-/-) T cells. Interestingly, forced expression of SKAP55 in the absence of ADAP is insufficient to drive T:APC conjugate formation, demonstrating that both ADAP and SKAP55 are required for optimal LFA-1 function. Additionally, the ADAP proline-rich domain is required for optimal Ag-induced activation of CD69, CD25, and Bcl-x(L), but is not required for assembly of the CARMA1/Bcl10/Malt1 (caspase-recruitment domain (CARD) membrane-associated guanylate kinase (MAGUK) protein 1/B-cell CLL-lymphoma 10/mucosa-associated lymphoid tissue lymphoma translocation protein 1) signaling complex and subsequent TCR-dependent NF-kappaB activity. Our results indicate that ADAP is used downstream of TCR engagement to delineate two distinct molecular programs in which the ADAP/SKAP55 module is required for control of T:APC conjugate formation and functions independently of ADAP/CARMA1-mediated NF-kappaB activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Apresentação de Antígeno , Integrinas/fisiologia , NF-kappa B/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Apresentação de Antígeno/genética , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Adesão Celular/genética , Adesão Celular/imunologia , Células Cultivadas , Humanos , Células Jurkat , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/genética , Ovalbumina/imunologia , Ovalbumina/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Domínios Proteicos Ricos em Prolina/fisiologia , Receptores Virais/biossíntese , Receptores Virais/genéticaRESUMO
The role of PI-3K in leukocyte function has been studied extensively. However, the specific role of the p110gamma isoform of PI- 3K in CD4 T lymphocyte function has yet to be defined explicitly. In this study, we report that although p110gamma does not regulate antigen-dependent CD4 T cell activation and proliferation, it plays a crucial role in regulating CD4 effector T cell migration. Naïve p110gamma(-/-) CD4 lymphocytes are phenotypically identical to their wild-type (WT) counterparts and do not exhibit any defects in TCR-mediated calcium mobilization or Erk activation. In addition, p110gamma-deficient CD4 OT.II T cells become activated and proliferate comparably with WT cells in response to antigen in vivo. Interestingly, however, antigen-experienced, p110gamma-deficient CD4 OT.II lymphocytes exhibit dramatic defects in their ability to traffic to peripheral inflammatory sites in vivo. Although antigen-activated, p110gamma-deficient CD4 T cells express P-selectin ligand, beta2 integrin, beta1 integrin, CCR4, CXCR5, and CCR7 comparably with WT cells, they exhibit impaired F-actin polarization and migration in response to stimulation ex vivo with the CCR4 ligand CCL22. These findings suggest that p110gamma regulates the migration of antigen-experienced effector CD4 T lymphocytes into inflammatory sites during adaptive immune responses in vivo.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Movimento Celular , Fosfatidilinositol 3-Quinases/fisiologia , Linfócitos T/imunologia , Actinas/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Cálcio/metabolismo , Adesão Celular/fisiologia , Quimiocinas/metabolismo , Classe I de Fosfatidilinositol 3-Quinases , Ativação Enzimática , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Quimiocinas/metabolismoRESUMO
A case with chondroblastoma arising from the right temporal bone was reported. A 52-year-old woman demonstrated residual tumor growth after surgical excision. The patient presented continuous right temporalgia and right facial twitch while opening her mouth. The tumor was an expansile mass (tumor volume: 12.8 cm3) and showed homogeneous hypo-intensity on T1 and T2-weighted images, but little contrast enhancement. The patient underwent gamma knife radiosurgery (GKR: marginal dose: 12 Gy, maximum dose: 24 Gy). One month later, her symptoms improved completely. The size of the tumor was reduced to 6.4 cm3 twenty months after GKR. The patient has been free of recurrence and side effects for four years since GKR. GKR may be useful to control residual chondroblastoma of the skull after surgery.
Assuntos
Condroblastoma/cirurgia , Radiocirurgia , Neoplasias Cranianas/cirurgia , Osso Temporal , Condroblastoma/diagnóstico , Feminino , Humanos , Angiografia por Ressonância Magnética , Pessoa de Meia-Idade , Neoplasias Cranianas/diagnóstico , Resultado do TratamentoRESUMO
T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin.
Assuntos
Integrina beta1/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Talina/metabolismo , Vinculina/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Antígenos CD18/genética , Antígenos CD18/metabolismo , Cálcio/metabolismo , Linhagem Celular , Citoesqueleto/metabolismo , Humanos , Sistema Imunitário/fisiologia , Integrina beta1/genética , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Complexos Multiproteicos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/fisiologia , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The adapter protein ADAP regulates T lymphocyte adhesion and activation. We present evidence for a previously unrecognized function for ADAP in regulating T cell receptor (TCR)-mediated activation of the transcription factor NF-kappaB. Stimulation of ADAP-deficient mouse T cells with antibodies to CD3 and CD28 resulted in impaired nuclear translocation of NF-kappaB, a reduced DNA binding, and delayed degradation and decreased phosphorylation of IkappaB (inhibitor of NF-kappaB). TCR-stimulated assembly of the CARMA1-BCL-10-MALT1 complex was substantially impaired in the absence of ADAP. We further identified a region of ADAP that is required for association with the CARMA1 adapter and NF-kappaB activation but is not required for ADAP-dependent regulation of adhesion. These findings provide new insights into ADAP function and the mechanism by which CARMA1 regulates NF-kappaB activation in T cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína 10 de Linfoma CCL de Células B , Antígenos CD28/imunologia , Complexo CD3/imunologia , Caspases/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Isoenzimas/metabolismo , Células Jurkat , Ativação Linfocitária , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Mutação , Proteínas de Neoplasias/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Natural killer (NK) cells play a key role in inflammation and tumor regression through their ability to migrate into tissues. CXCL12 is a chemokine that promotes lymphocyte invasion and migration into tissues; however, the mechanism for this process remains incompletely understood. In this study, we show that CXCL12 significantly enhanced CD16(+)CD56(+) human peripheral NK-cell invasion into type I collagen by the catalytic activity of matrix metalloproteinase-1 (MMP-1). Confocal immunofluorescence and co-immunoprecipitation studies suggest that MMP-1 colocalized with alpha(2)beta(1) integrin on CXCL-12-stimulated NK-cell surface. The binding of pro-MMP-1 with alpha(2)beta(1) integrin required activation of G(i)-coupled pathway. However, the production of MMP-1 from CXCL12-stimulated NK cells was mediated by p38 and mitogen-activated or extracellular signal-regulation protein kinase kinase 1/2 in a manner independent of the G(i)-coupled pathway. These results suggest that CXCL12/CXCR4 interaction transduces the two signaling pathways to promote NK-cell invasion, which stimulates pericellular degradation of extracellular matrix proteins by membrane-associated MMP-1. The mechanisms would thus play a role in facilitating lymphocyte trafficking and accumulation in tissues during physiological and pathological processes.
Assuntos
Quimiocinas CXC/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/enzimologia , Metaloproteinase 1 da Matriz/biossíntese , Metaloproteinase 1 da Matriz/metabolismo , Antígeno CD56/imunologia , Quimiocina CXCL12 , Colágeno Tipo I/metabolismo , Meios de Cultivo Condicionados , Humanos , Integrina alfa2beta1/metabolismo , Células Matadoras Naturais/citologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Toxina Pertussis/farmacologia , Receptores de IgG/imunologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
NK cells are key mediators of the innate immune response and anti-tumor surveillance. Adhesion and degranulation-promoting adapter protein (ADAP, formerly known as SLAP-130 or Fyb) is a hematopoietic-specific adapter that is required for efficient TCR signaling and T cell activation. Herein, we examine a potential role for ADAP in NK development and function. ADAP is expressed in primary NK cells and in IL-2 stimulated lymphokine-activated killers. However, ADAP-deficient mice show no defects in NK development. Further, ADAP is dispensable for key NK functions, including cytotoxicity in response to engagement of activating receptors, cytokine production, conjugate formation and tumor suppression in vivo. These results indicate that, unlike events stimulated by TCR engagement, signaling events engaged by immunoreceptor tyrosine-based activation motif-associated and cytokine receptors on NK cells can occur independently of ADAP.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Células Matadoras Naturais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos Ly/imunologia , Antígenos Ly/metabolismo , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-2/imunologia , Células Matadoras Ativadas por Linfocina/citologia , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Naturais/citologia , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores Semelhantes a Lectina de Células NKRESUMO
Dendritic cells (DCs) are a heterogeneous population of APCs with critical roles in T cell activation and immune regulation. We report in this study the identification and characterization of a novel subset of DCs resident in skin-draining peripheral lymph nodes of normal mice. This subset of CD11c(high)CD40(high)CD8alpha(intermediate (int)) DCs expresses the collagen-binding integrin, alpha1beta1, and the E-cadherin-binding integrin, alphaEbeta7. Although alpha1beta1 and alphaEbeta7 are also expressed on CD11c(high)CD40(int)CD8alpha(high) lymphoid DCs, CD11c(high)CD40(high)CD8alpha(int) DCs demonstrate preferential integrin-mediated adhesion to collagen and fibronectin. This DC subset most likely acquires expression of these integrins in peripheral lymph node, as this subset is not found in the spleen or mesenteric lymph node, and recent DC migrants from the skin lack expression of alpha1beta1 and alphaEbeta7 integrins. Resident CD40(high) DCs express alpha1beta1 integrin and colocalize with collagen in lymph nodes. When compared with CD11c(high)CD40(high)CD8alpha(int) DCs lacking expression of these integrins, the alpha1beta1+alphaEbeta7+DC subset exhibits more efficient formation of Ag-independent conjugates with T cells, and a decreased ability to acquire soluble Ag. Thus, the alpha1beta1 and alphaEbeta7 integrins define a unique population of peripheral lymph node-derived DCs with altered functional properties and adhesive potential that localizes these cells to sites in lymph nodes where Ag presentation to T cells occurs.
Assuntos
Antígenos/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Integrina alfa1beta1/biossíntese , Integrinas/biossíntese , Linfonodos/citologia , Linfonodos/imunologia , Animais , Antígeno CD11c/biossíntese , Antígenos CD40/biossíntese , Antígenos CD8/biossíntese , Caderinas/metabolismo , Adesão Celular/imunologia , Movimento Celular/imunologia , Colágeno/metabolismo , Células Dendríticas/metabolismo , Epitopos de Linfócito T/metabolismo , Fibronectinas/metabolismo , Imunofenotipagem , Integrina alfa1beta1/metabolismo , Integrina alfa1beta1/fisiologia , Integrina alfa2/metabolismo , Integrinas/metabolismo , Integrinas/fisiologia , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica/imunologia , Pele/citologia , Pele/imunologia , Pele/metabolismo , SolubilidadeRESUMO
Among the myriad receptors expressed by T cells, the sine qua non is the CD3/T cell receptor (CD3/TCR) complex, because it is uniquely capable of translating the presence of a specific antigen into intracellular signals necessary to trigger an immune response against a pathogen or tumor. Much work over the past 2 decades has attempted to define the signaling pathways leading from the CD3/TCR complex that culminate ultimately in the functions necessary for effective T cell immune responses, such as cytokine production. Here, we summarize recent advances in our understanding of the mechanisms by which the CD3/TCR complex controls integrin-mediated T cell adhesion, and discuss new information that suggests that there may be unexpected facets to this pathway that distinguish it from those previously defined.