RESUMO
Platelets protect vascular integrity during inflammation. Recent evidence suggests that this action is independent of thrombus formation and requires the engagement of glycoprotein VI (GPVI), but it remains unclear how platelets prevent inflammatory bleeding. We investigated whether platelets and GPVI act primarily by preventing detrimental effects of neutrophils using models of immune complex (IC)-mediated inflammation in mice immunodepleted in platelets and/or neutrophils or deficient in GPVI. Depletion of neutrophils prevented bleeding in thrombocytopenic and GPVI(-/-) mice during IC-mediated dermatitis. GPVI deficiency did not modify neutrophil recruitment, which was reduced by thrombocytopenia. Neutrophil cytotoxic activities were reduced in thrombocytopenic and GPVI(-/-) mice during IC-mediated inflammation. Intravital microscopy revealed that in this setting, intravascular binding sites for platelets were exposed by neutrophils, and GPVI supported the recruitment of individual platelets to these spots. Furthermore, the platelet secretory response accompanying IC-mediated inflammation was partly mediated by GPVI, and blocking of GPVI signaling impaired the vasculoprotective action of platelets. Together, our results show that GPVI plays a dual role in inflammation by enhancing neutrophil-damaging activities while supporting the activation and hemostatic adhesion of single platelets to neutrophil-induced vascular breaches.
Assuntos
Plaquetas/metabolismo , Doenças do Complexo Imune/patologia , Inflamação/patologia , Neutrófilos/patologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Modelos Animais de Doenças , Doenças do Complexo Imune/complicações , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Thrombosis is common in patients suffering from myeloproliferative neoplasm (MPN), whereas bleeding is less frequent. JAK2(V617F), the main mutation involved in MPN, is considered as a risk factor for thrombosis, although the direct link between the mutation and hemostatic disorders is not strictly established. We investigated this question using conditional JAK2(V617F) knock-in mice with constitutive and inducible expression of JAK2(V617F) in hematopoietic cells, which develop a polycythemia vera (PV)-like disorder evolving into myelofibrosis. In vitro, thrombosis was markedly impaired with an 80% decrease in platelet-covered surface, when JAK2(V617F) blood was perfused at arterial shear over collagen. JAK2(V617F) platelets presented only a moderate glycoprotein (GP) VI deficiency not responsible for the defective platelet accumulation. In contrast, a decreased proportion of high-molecular-weight von Willebrand factor multimers could reduce platelet adhesion. Accordingly, the tail bleeding time was prolonged. In the FeCl3-induced thrombosis model, platelet aggregates formed rapidly but were highly unstable. Interestingly, vessels were considerably dilated. Thus, mice developing PV secondary to constitutive JAK2(V617F) expression exhibit a bleeding tendency combined with the accelerated formation of unstable clots, reminiscent of observations made in patients. Hemostatic defects were not concomitant with the induction of JAK2(V617F) expression, suggesting they were not directly caused by the mutation but were rather the consequence of perturbations in blood and vessel homeostasis.
Assuntos
Modelos Animais de Doenças , Transtornos Hemostáticos/genética , Janus Quinase 2/genética , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/genética , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Tempo de Sangramento , Plaquetas/metabolismo , Citometria de Fluxo , Técnicas de Introdução de Genes , Humanos , Immunoblotting , Camundongos Transgênicos , Transtornos Mieloproliferativos/sangue , Ativação Plaquetária/genética , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Policitemia Vera/sangue , Policitemia Vera/genética , Mielofibrose Primária/sangue , Mielofibrose Primária/genética , Trombose/sangue , Trombose/genética , Vasodilatação/genética , Fator de von Willebrand/metabolismoRESUMO
The immune system plays a critical role in the control and eradication of tumors. A better understanding of the anti-tumor immune mechanisms over the last decade has led to the development of immunotherapies, including cellular therapies such as those using CAR-T cells. These therapies have been remarkably effective in hematological malignancies. However, their application to solid tumors requires some optimization. Many efforts are being made in this regard, both to increase the efficacy of CAR-T cells, and to make them more secure. For the former goal, there is a need for the identification of new targets, better activation strategies, or arming T cells in a way that makes them able to overcome intra-tumoral barriers. For the latter goal, dose adjustment, locoregional administration or use of suicide genes are currently investigated as ways to mitigate the risks of this therapy. Together, these adjustments will permit larger applicability of CAR-T cells, in anti-tumor immunity, but also in the context of auto-immune diseases or fibrolytic therapies.
Title: Optimisation de l'efficacité et de la sécurité d'utilisation des lymphocytes CAR-T. Abstract: Le système immunitaire joue un rôle déterminant dans le contrôle et l'éradication des tumeurs. Une meilleure compréhension des mécanismes en jeu a permis le développement des immunothérapies, et notamment des thérapies par lymphocytes CAR-T. Ces thérapies ont montré une grande efficacité dans les maladies hématologiques, mais leur application aux tumeurs solides nécessite des optimisations pour améliorer leur efficacité et leur sécurité. Ces ajustements permettront une plus grande applicabilité des lymphocytes CAR-T, non seulement pour les traitements anti-tumoraux mais aussi pour le traitement de maladies auto-immunes ou fibreuses.
Assuntos
Imunoterapia Adotiva , Neoplasias , Receptores de Antígenos Quiméricos , Linfócitos T , Humanos , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/efeitos adversos , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Monitorização Imunológica/métodos , Linfócitos T/imunologia , Resultado do Tratamento , AnimaisRESUMO
We previously showed that chimeric antigen receptor (CAR) T-cell therapy targeting epidermal growth factor receptor variant III (EGFRvIII) produces upregulation of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Here we conducted a phase 1 trial (NCT03726515) of CAR T-EGFRvIII cells administered concomitantly with the anti-PD1 (aPD1) monoclonal antibody pembrolizumab in patients with newly diagnosed, EGFRvIII+ glioblastoma (GBM) (n = 7). The primary outcome was safety, and no dose-limiting toxicity was observed. Secondary outcomes included median progression-free survival (5.2 months; 90% confidence interval (CI), 2.9-6.0 months) and median overall survival (11.8 months; 90% CI, 9.2-14.2 months). In exploratory analyses, comparison of the TME in tumors harvested before versus after CAR + aPD1 administration demonstrated substantial evolution of the infiltrating myeloid and T cells, with more exhausted, regulatory, and interferon (IFN)-stimulated T cells at relapse. Our study suggests that the combination of CAR T cells and PD-1 inhibition in GBM is safe and biologically active but, given the lack of efficacy, also indicates a need to consider alternative strategies.
Assuntos
Anticorpos Monoclonais Humanizados , Glioblastoma , Humanos , Glioblastoma/terapia , Receptores ErbB , Recidiva Local de Neoplasia/metabolismo , Linfócitos T , Microambiente TumoralRESUMO
Background: Filamin (FLN) regulates many cell functions through its scaffolding activity cross-linking cytoskeleton and integrins. FLN was shown to inhibit integrin activity, but the exact mechanism remains unclear. Objectives: The aim of this study was to evaluate the role of filamin A (FLNa) subdomains on the regulation of integrin αIIbß3 signaling. Methods: Three FLNa deletion mutants were overexpressed in the erythro-megakaryocytic leukemic cell line HEL: Del1, which lacks the N-terminal CH1-CH2 domains mediating the FLNa-actin interaction; Del2, lacking the Ig-like repeat 21, which mediates the FLNa-ß3 interaction; and Del3, lacking the C-terminal Ig repeat 24, responsible for FLNa dimerization and interaction with the small Rho guanosine triphosphatase involved in actin cytoskeleton reorganisation. Fibrinogen binding to HEL cells in suspension and talin-ß3 proximity in cells adherent to immobilized fibrinogen were assessed before and after αIIbß3 activation by the protein kinase C agonist phorbol 12-myristate 13-acetate. Results: Our results show that FLNa-actin and FLNa-ß3 interactions negatively regulate αIIbß3 activation. Moreover, FLNa-actin interaction represses Rac activation, contributing to the negative regulation of αIIbß3 activation. In contrast, the FLNa dimerization domain, which maintains Rho inactive, was found to negatively regulate αIIbß3 outside-in signaling. Conclusion: We conclude that FLNa negatively controls αIIbß3 activation by regulating actin polymerization and restraining activation of Rac, as well as outside-in signaling by repressing Rho.
RESUMO
BACKGROUND: Filaminopathies A are rare disorders affecting the brain, intestine, or skeleton, characterized by dominant X-linked filamin A (FLNA) gene mutations. Macrothrombocytopenia with functionally defective platelets is frequent. We have described a filaminopathy A male patient, exhibiting a C-terminal frame-shift FLNa mutation (Berrou et al., Arterioscler Thromb Vasc Biol. 2017;37:1087-1097). Contrasting with female patients, this male patient exhibited gain of platelet functions, including increased platelet aggregation, integrin αIIbß3 activation, and secretion at low agonist concentration, raising the issue of thrombosis risk. OBJECTIVES: Our goal is to assess the thrombotic potential of the patient FLNa mutation in an in vivo model. METHODS: We have established a mutant FlnA knock-in mouse model. RESULTS: The mutant FlnA mouse platelets phenocopied patient platelets, showing normal platelet count, lower expression level of mutant FlnA, and gain of platelet functions: increased platelet aggregation, secretion, and αIIbß3 activation, as well as increased spreading and clot retraction. Surprisingly, mutant FlnA mice exhibited a normal bleeding time, but with increased re-bleeding (77%) compared to wild type (WT) FlnA mice (27%), reflecting hemostatic plug instability. Again, in an in vivo thrombosis model, the occlusion time was not altered by the FlnA mutation, but arteriolar embolies were increased (7-fold more frequent in mutant FlnA mice versus WT mice), confirming thrombus instability. CONCLUSIONS: This study shows that the FlnA mutation found in the male patient induced gain of platelet functions in vitro, but thrombus instability in vivo. Implications for the role of FLNa in physiology of thrombus formation are discussed.
Assuntos
Hemostáticos , Trombose , Masculino , Feminino , Camundongos , Animais , Filaminas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Mutação com Ganho de Função , Trombose/genética , Trombose/metabolismo , MutaçãoRESUMO
Myeloproliferative neoplasms (MPN) are associated with an increased risk of arterial and venous thrombosis. Pegylated-interferon alpha (IFN) and hydroxyurea (HU) are commonly used to treat MPN, but their effect on hemostasis has not yet been studied. The aim of our study was to determine whether IFN and HU impact the biological hemostatic profile of MPN patients by studying markers of endothelial, platelet, and coagulation activation. A total of 85 patients (50 polycythemia vera and 35 essential thrombocythemia) were included: 28 treated with IFN, 35 with HU, and 22 with no cytoreductive drug (non-treated, NT). Von Willebrand factor, shear-induced platelet aggregation, factor VIII coagulant activity (FVIII:C), fibrinogen, and thrombin generation with and without exogenous thrombomodulin were significantly higher in IFN-treated patients compared to NT patients, while protein S anticoagulant activity was lower. In 10 patients in whom IFN therapy was discontinued, these hemostatic biomarkers returned to the values observed in NT patients, strongly suggesting an impact of IFN therapy on endothelial and coagulation activation. Overall, our study shows that treatment with IFN is associated with significant and reversible effects on the biological hemostatic profile of MPN patients. Whether they could be associated with an increased thrombotic risk remains to be determined in further randomized clinical studies.
RESUMO
Although intravital microscopy models of thrombosis in mice have contributed to dissect the mechanisms of thrombus formation and stability, they have not been well adapted to study long-term evolution of occlusive thrombi. Here, we assessed the suitability of the dorsal skinfold chamber (DSC) for the study of thrombolysis and testing of thrombolytic agents by intravital microscopy. We show that induction of FeCl3-induced occlusive thrombosis is achievable in microvessels of DSCs, and that thrombi formed in DSCs can be visualised by intravital microscopy using brightfield transmitted light, or fluorescent staining of thrombus components such as fibrinogen, platelets, leukocytes, and von Willebrand factor. Direct application of control saline or recombinant tissue-plasminogen activator (rtPA) to FeCl3-produced thrombi in DSCs did not affect thrombus size or induce recanalisation. However, in the presence of hirudin, rtPA treatment caused a rapid dose-dependent lysis of occlusive thrombi, resulting in recanalisation within 1 hour after treatment. Skin haemorrhage originating from vessels located inside and outside the FeCl3-injured area was also observed in DSCs of rtPA-treated mice. We further show that rtPA-induced thrombolysis was enhanced in plasminogen activator inhibitor-1-deficient (PAI-1-/-) mice, and dropped considerably as the time between occlusion and treatment application increased. Together, our results show that by allowing visualization and measurement of thrombus lysis and potential bleeding complications of thrombolytic treatments, the DSC provides a model for studying endogenous fibrinolysis and for first-line screening of thrombolytic agents. Furthermore, using this system, we found that thrombin and clot aging impair the thrombolytic action of rtPA towards FeCl3-produced thrombi.