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1.
Genes Dev ; 33(23-24): 1657-1672, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31727774

RESUMO

In obesity, adipose tissue undergoes dynamic remodeling processes such as adipocyte hypertrophy, hypoxia, immune responses, and adipocyte death. However, whether and how invariant natural killer T (iNKT) cells contribute to adipose tissue remodeling are elusive. In this study, we demonstrate that iNKT cells remove unhealthy adipocytes and stimulate the differentiation of healthy adipocytes. In obese adipose tissue, iNKT cells were abundantly found nearby dead adipocytes. FasL-positive adipose iNKT cells exerted cytotoxic effects to eliminate hypertrophic and pro-inflammatory Fas-positive adipocytes. Furthermore, in vivo adipocyte-lineage tracing mice model showed that activation of iNKT cells by alpha-galactosylceramide promoted adipocyte turnover, eventually leading to potentiation of the insulin-dependent glucose uptake ability in adipose tissue. Collectively, our data propose a novel role of adipose iNKT cells in the regulation of adipocyte turnover in obesity.


Assuntos
Adipócitos/citologia , Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Morte Celular/fisiologia , Ativação Linfocitária/fisiologia , Células T Matadoras Naturais/fisiologia , Obesidade/fisiopatologia , Células 3T3 , Adipócitos/imunologia , Adipócitos/metabolismo , Animais , Proliferação de Células , Proteína Ligante Fas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor fas/metabolismo
2.
Presse Med ; 48(9): 919-930, 2019 Sep.
Artigo em Francês | MEDLINE | ID: mdl-31543394

RESUMO

Giant cell arteritis (GCA) is a large-vessel vasculitis involving the aorta and its main branches, especially supra aortic branches. Although much progress has been made, the pathophysiology remains incompletely understood. An initial trigger, suspected of infectious origin, lead to the maturation and recruitment of dendritic cells (DC). The lack of migration of these DC allows the local recruitment of T-lymphocytes (LT). These LT- CD4+ polarize in Type 1 helper (Th1), Th17 but also Th9. A qualitative and quantitative deficit in regulatory T cells (Treg) is observed under the influence of IL-21 overproduction. In addition, an imbalance in the Th17/Treg balance is favored by IL-6. The secretion of IFN-γ, IL-17, IL-6, IL-33 is responsible for a sustained local inflammatory reaction that is organized around tertiary lymphoid follicles. Locally recruited macrophages secrete reactive forms of oxygen together with VEGF and PDGF. These growth factors, together with neurotrophins and endothelin contribute to increase the proliferation of vascular smooth muscle cells (VSMCs). The imbalance between matrix metalloproteases (MMP)-2, MMP-9 and MMP-14 and tissue inhibitors of metalloproteases (TIMP)-1 and TIMP-2 also contribute to the remodeling process occurring in the vessel wall. Finally, arterial neovascularization contribute to the perpetuation of lymphocyte recruitment. This persistent remodeling is sometimes complicated by ischemic events responsible for the initial severity of the disease.


Assuntos
Arterite de Células Gigantes/fisiopatologia , Remodelação Vascular/fisiologia , Animais , Linfócitos B/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Movimento Celular , Proliferação de Células , Células Dendríticas/fisiologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Arterite de Células Gigantes/tratamento farmacológico , Arterite de Células Gigantes/etiologia , Arterite de Células Gigantes/patologia , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-33/metabolismo , Interleucina-6/fisiologia , Interleucinas/biossíntese , Ativação Linfocitária/fisiologia , Macrófagos/metabolismo , Metaloproteinases da Matriz/metabolismo , Músculo Liso Vascular/patologia , Neovascularização Patológica/fisiopatologia , Linfócitos T Reguladores/fisiologia , Células Th17/fisiologia
3.
DNA Cell Biol ; 38(11): 1402-1410, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31556705

RESUMO

Tumor antigen-induced lymphocyte transformation (LT) represents the antitumor cellular immunity, which might correlate with the cancer treatment outcome. Currently, there is no LT assay (LTA) routinely used in clinic. To establish a sensitive and convenient procedure for LTA, the same samples were used to simultaneously perform three assays: 5-ethynyl-2'-deoxyuridine (EdU) assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and carboxyfluorescein succinimidyl ester (CFSE) assay, and then the three results were compared. Several conditions were optimized: the LT harvest time, sources of lymphocytes (blood, lymph nodes, or spleen), the added amount of stimulatory tumor antigen and in vivo immunization priming time for LTA. The results of side-by-side comparison showed that (1) the 72 h for coculture of lymphocytes with tumor antigens was optimal time to harvest cells for LTA; (2) 50 µg/mL of tumor antigens was the optimal concentration for activation LT from three sources; (3) EdU incorporation was the sensitive and convenient assay for LTA as compared with MTT and CFSE assays; (4) the day 21-28 after in vivo priming immunization was the testing time for LTA; and (5) peripheral blood LT could be a good representative of whole body's lymphocyte reaction and practically easy cell source for LTA. This comparison of the three LTA in mouse model suggests that the EdU incorporation assay might be useful to evaluate the antitumor immunity stimulated by specific tumor vaccine or different anticancer therapies.


Assuntos
Antígenos de Neoplasias/imunologia , Bioensaio/métodos , Ativação Linfocitária/fisiologia , Linfócitos/fisiologia , Animais , Antineoplásicos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Células Cultivadas , Técnicas de Cocultura , Monitoramento de Medicamentos/métodos , Feminino , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Humanos , Imunidade Celular/fisiologia , Imunoensaio/métodos , Camundongos , Camundongos Endogâmicos ICR
4.
Actas dermo-sifiliogr. (Ed. impr.) ; 110(5): 353-359, jun. 2019. graf, tab
Artigo em Espanhol | IBECS | ID: ibc-180923

RESUMO

La inmunoterapia en el cáncer emerge como un tratamiento novedoso y prometedor en una gran variedad de tumores, incluido el cáncer cutáneo no melanoma. Los anticuerpos inhibidores de proteínas de control inmunitario están dirigidos fundamentalmente a las moléculas de superficie CTLA-4 (antígeno citotóxico de los linfocitos T) y PD-1 (molécula de muerte programada 1). En el presente artículo se revisan las vías de CTLA-4 y PD-1/PD-L1 (PD-1/ligando de la PD-1) y las evidencias actuales de tratamiento con inhibidores de puntos de control inmunitario en los principales tipos de cáncer cutáneo no melanoma


Immunotherapy is emerging as a new and promising treatment for a great variety of tumors, including nonmelanoma skin cancer. Checkpoint inhibitors -antibodies that block proteins that regulate the immune system- mainly target the surface protein CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) and the PD-1/PD-L1 (programmed cell death protein 1/PD-ligand 1) axis. We review the CTLA-4 and PD-1/PD-L1 pathways and current evidence supporting checkpoint inhibitor therapy in the main types of nonmelanoma skin cancer


Assuntos
Humanos , Imunoterapia , Neoplasias Cutâneas/terapia , Antígeno CTLA-4/antagonistas & inibidores , Antígeno B7-H1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias Cutâneas/imunologia , Ativação Linfocitária/fisiologia , Carcinoma de Célula de Merkel/imunologia , Carcinoma de Célula de Merkel/terapia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/terapia , Carcinoma Basocelular/imunologia
5.
Nat Commun ; 10(1): 1507, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944315

RESUMO

Exhaustion of cytotoxic effector natural killer (NK) and CD8+ T cells have important functions in the establishment of persistent viral infections, but how exhaustion is induced during chronic hepatitis C virus (HCV) infection remains poorly defined. Here we show, using the humanized C/OTg mice permissive for persistent HCV infection, that NK and CD8+ T cells become sequentially exhausted shortly after their transient hepatic infiltration and activation in acute HCV infection. HCV infection upregulates Qa-1 expression in hepatocytes, which ligates NKG2A to induce NK cell exhaustion. Antibodies targeting NKG2A or Qa-1 prevents NK exhaustion and promotes NK-dependent HCV clearance. Moreover, reactivated NK cells provide sufficient IFN-γ that helps rejuvenate polyclonal HCV CD8+ T cell response and clearance of HCV. Our data thus show that NKG2A serves as a critical checkpoint for HCV-induced NK exhaustion, and that NKG2A blockade sequentially boosts interdependent NK and CD8+ T cell functions to prevent persistent HCV infection.


Assuntos
Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Células Matadoras Naturais/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Hepatite C Crônica/virologia , Hepatócitos/virologia , Antígenos de Histocompatibilidade Classe I/imunologia , Interferon gama/imunologia , Ativação Linfocitária/fisiologia , Proteínas de Membrana/imunologia , Camundongos , Distribuição Aleatória
6.
J Vis Exp ; (144)2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30882785

RESUMO

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Ativação Linfocitária/fisiologia , Humanos , Transfecção
7.
Purinergic Signal ; 15(2): 127-137, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30919205

RESUMO

Previous studies have shown that T cell receptor (TCR) and CD28 coreceptor stimulation involves rapid ATP release, autocrine purinergic feedback via P2X receptors, and mitochondrial ATP synthesis that promote T cell activation. Here, we show that ADP formation and autocrine stimulation of P2Y1 receptors are also involved in these purinergic signaling mechanisms. Primary human CD4 T cells and the human Jurkat CD4 T cell line express P2Y1 receptors. The expression of this receptor increases following T cell stimulation. Inhibition of P2Y1 receptors impairs the activation of mitochondria, as assessed by mitochondrial Ca2+ uptake, and reduces cytosolic Ca2+ signaling in response to TCR/CD28 stimulation. We found that the addition of exogenous ADP or overexpression of P2Y1 receptors significantly increased IL-2 mRNA transcription in response to TCR/CD28 stimulation. Conversely, antagonists or silencing of P2Y1 receptors reduced IL-2 mRNA transcription and attenuated T cell functions. We conclude that P2Y1 and P2X receptors have non-redundant, synergistic functions in the regulation of T cell activation. P2Y1 receptors may represent potential therapeutic targets to modulate T cell function in inflammation and host defense.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Ativação Linfocitária/fisiologia , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Transdução de Sinais/imunologia , Comunicação Autócrina , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Humanos
8.
Croat Med J ; 60(1): 12-19, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30825273

RESUMO

AIM: To assess the immunomodulatory effect of tonsil-derived mesenchymal stem cells (MSCs) on T-lymphocyte proliferation and cytokine production. METHODS: Tonsils were obtained from children aged 3 to 12 years (n=15) who underwent tonsillectomy for obstructive sleep apnea from April 2012-October 2014 at the Merkur University Hospital, Zagreb. Tonsil-derived MSCs were co-cultured with peripheral blood mononuclear cells (PBMCs) and phytohemagglutinin as a mitogen. PBMCs were induced to differentiate into T helper 1 or T helper 2 cells in the presence or absence of tonsil-derived MSCs, after which the production of interferon-gamma in T helper 1 and interleukin-4 in T helper 2 cells was assessed. RESULTS: Tonsil-derived MSC suppressed phytohemagglutinin-induced proliferation of PBMCs. Compared with controls, tonsil-derived MSC co-culture significantly decreased interferon-gamma production (P<0.001) and increased interleukin-4 production (P<0.001). CONCLUSION: Tonsil-derived MSCs exert immunomodulatory effects on T lymphocyte proliferation and T helper 1- and T helper 2-specific cytokine production.


Assuntos
Células-Tronco Mesenquimais/imunologia , Tonsila Palatina/citologia , Linfócitos T/imunologia , Proliferação de Células/fisiologia , Células Cultivadas , Criança , Pré-Escolar , Técnicas de Cocultura , Citocinas/imunologia , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/fisiologia , Masculino , Fito-Hemaglutininas/farmacologia
9.
Proc Natl Acad Sci U S A ; 116(13): 5914-5919, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850527

RESUMO

An essential feature of the adaptive immune system is the proliferation of antigen-specific lymphocytes during an immune reaction to form a large pool of effector cells. This proliferation must be regulated to ensure an effective response to infection while avoiding immunopathology. Recent experiments in mice have demonstrated that the expansion of a specific clone of T cells in response to cognate antigen obeys a striking inverse power law with respect to the initial number of T cells. Here, we show that such a relationship arises naturally from a model in which T cell expansion is limited by decaying levels of presented antigen. The same model also accounts for the observed dependence of T cell expansion on affinity for antigen and on the kinetics of antigen administration. Extending the model to address expansion of multiple T cell clones competing for antigen, we find that higher-affinity clones can suppress the proliferation of lower-affinity clones, thereby promoting the specificity of the response. Using the model to derive optimal vaccination protocols, we find that exponentially increasing antigen doses can achieve a nearly optimized response. We thus conclude that the dynamics of presented antigen is a key regulator of both the size and specificity of the adaptive immune response.


Assuntos
Apresentação do Antígeno/fisiologia , Linfócitos T/fisiologia , Animais , Imunidade Celular , Ativação Linfocitária/imunologia , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Imunológicos , Modelos Teóricos , Vacinação/métodos
10.
J Bone Joint Surg Am ; 101(3): 257-264, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30730485

RESUMO

BACKGROUND: The utilization of lymphocyte transformation testing (LTT) has increased for diagnosing metal sensitivity associated with total knee arthroplasty (TKA), but its validity for the diagnosis of TKA failure due to an immune reaction has not been established. In this study, we sought to characterize the relationship of a positive LTT result to histopathologic findings and clinical and functional outcomes. METHODS: This was a retrospective study of 27 well-fixed, aseptic, primary TKA cases in which the patient had persistent pain and/or stiffness and underwent revision due to a suspected metal allergy to nickel, as determined on the basis of positive LTT. Revision procedures were performed by a single experienced arthroplasty surgeon. Periprosthetic tissue samples obtained at the time of revision surgery were scored using the aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL) scoring system. RESULTS: Eight patients were categorized as mildly reactive; 8 patients, moderately reactive; and 11 patients, highly reactive to nickel by LTT. The predominant findings on routine histopathologic analysis were fibrosis and varying degrees of lymphocytic infiltration in 17 (63%) of the 27 cases. The average ALVAL score of the cohort was 3.1 ± 1.9, of a maximum score of 10. Average Knee Society Score (KSS) values improved post-revision, as did range of motion (all p < 0.01). Neither LTT stimulation index as a continuous variable nor as a categorical variable (mildly reactive, moderately reactive, highly reactive) was correlated with ALVAL score, pre-revision function (as assessed by KSS-clinical, KSS-functional, and range of motion), or change in function at the most recent follow-up (0.015 < r < 0.30, 0.13 < p < 0.95). In addition, the ALVAL score did not correlate significantly with either pre-revision or post-revision KSS or range of motion (0.061 < r < 0.365, 0.09 < p < 0.88). CONCLUSIONS: On the basis of this analysis, including histopathologic assessment, LTT results alone were insufficient for the diagnosis of TKA failure due to an immune reaction. A positive LTT may not indicate that an immune reaction is the cause of pain and stiffness post-TKA. The role of LTT in assessing TKA failure from an immune reaction needs further investigation. Diagnostic criteria for such TKA failure need to be established. LEVEL OF EVIDENCE: Diagnostic Level IV. See Instructions for Authors for a complete description of levels of evidence.


Assuntos
Artroplastia do Joelho/efeitos adversos , Ativação Linfocitária/fisiologia , Metais/efeitos adversos , Dor Pós-Operatória/imunologia , Feminino , Humanos , Hipersensibilidade/imunologia , Prótese do Joelho/efeitos adversos , Masculino , Pessoa de Meia-Idade , Fraturas Periprotéticas/diagnóstico por imagem , Fraturas Periprotéticas/cirurgia , Radiografia , Reoperação/estatística & dados numéricos , Estudos Retrospectivos
11.
Nat Commun ; 10(1): 687, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737409

RESUMO

How innate T cells (ITC), including invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells, maintain a poised effector state has been unclear. Here we address this question using low-input and single-cell RNA-seq of human lymphocyte populations. Unbiased transcriptomic analyses uncover a continuous 'innateness gradient', with adaptive T cells at one end, followed by MAIT, iNKT, γδ T and natural killer cells at the other end. Single-cell RNA-seq reveals four broad states of innateness, and heterogeneity within canonical innate and adaptive populations. Transcriptional and functional data show that innateness is characterized by pre-formed mRNA encoding effector functions, but impaired proliferation marked by decreased baseline expression of ribosomal genes. Together, our data shed new light on the poised state of ITC, in which innateness is defined by a transcriptionally-orchestrated trade-off between rapid cell growth and rapid effector function.


Assuntos
Proliferação de Células/fisiologia , Linfócitos/metabolismo , Feminino , Ontologia Genética , Humanos , Imunidade Inata/fisiologia , Imunofenotipagem , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/fisiologia , Masculino , Células T Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/metabolismo
12.
J Immunol ; 202(6): 1735-1746, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30728213

RESUMO

Long-term survival of adoptively transferred chimeric Ag receptor (CAR) T cells is often limited. Transplantation of hematopoietic stem cells (HSCs) transduced to express CARs could help to overcome this problem as CAR-armed HSCs can continuously deliver CAR+ multicell lineages (e.g., T cells, NK cells). In dependence on the CAR construct, a variable extent of tonic signaling in CAR T cells was reported; thus, effects of CAR-mediated tonic signaling on the hematopoiesis of CAR-armed HSCs is unclear. To assess the effects of tonic signaling, two CAR constructs were established and analyzed 1) a signaling CAR inducing a solid Ag-independent tonic signaling termed CAR-28/ζ and 2) a nonstimulating control CAR construct lacking intracellular signaling domains termed CAR-Stop. Bone marrow cells from immunocompetent mice were isolated, purified for HSC-containing Lin-cKit+ cells or the Lin-cKit+ Sca-1+ subpopulation (Lin-Sca-1+cKit+), and transduced with both CAR constructs. Subsequently, modified bone marrow cells were transferred into irradiated mice, in which they successfully engrafted and differentiated into hematopoietic progenitors. HSCs expressing the CAR-Stop sustained normal hematopoiesis. In contrast, expression of the CAR-28/ζ led to elimination of mature CAR+ T and B cells, suggesting that the CAR-mediated tonic signaling mimics autorecognition via the newly recombined immune receptors in the developing lymphocytes.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Ativação Linfocitária/fisiologia , Linfopoese/fisiologia , Receptores de Antígenos Quiméricos/metabolismo , Transdução de Sinais/fisiologia , Transferência Adotiva , Animais , Diferenciação Celular/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Camundongos , Camundongos Endogâmicos C57BL
13.
Proc Natl Acad Sci U S A ; 116(5): 1692-1697, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30635425

RESUMO

Immune checkpoint therapy (ICT) has transformed cancer treatment in recent years; however, treatment response is not uniform across tumor types. The tumor immune microenvironment plays a critical role in determining response to ICT; therefore, understanding the differential immune infiltration between ICT-sensitive and ICT-resistant tumor types will help to develop effective treatment strategies. We performed a comprehensive analysis of the immune tumor microenvironment of an ICT-sensitive tumor (melanoma, n = 44) and an ICT-resistant tumor (pancreatic cancer, n = 67). We found that a pancreatic tumor has minimal to moderate infiltration of CD3, CD4, and CD8 T cells; however, the immune infiltrates are predominantly present in the stromal area of the tumor and are excluded from tumoral area compared with melanoma, where the immune infiltrates are primarily present in the tumoral area. Metastatic pancreatic ductal adenocarcinomas (PDACs) had a lower infiltration of total T cells compared with resectable primary PDACs, suggesting that metastatic PDACs have poor immunogenicity. Further, a significantly higher number of CD68+ macrophages and VISTA+ cells (also known as V-domain immunoglobulin suppressor of T cell activation) were found in the pancreatic stromal area compared with melanoma. We identified VISTA as a potent inhibitory checkpoint that is predominantly expressed on CD68+ macrophages on PDACs. These data suggest that VISTA may be a relevant immunotherapy target for effective treatment of patients with pancreatic cancer.


Assuntos
Antígenos B7/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Humanos , Imunoterapia/métodos , Ativação Linfocitária/fisiologia , Microambiente Tumoral/fisiologia
14.
Oncogene ; 38(17): 3316-3324, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30635655

RESUMO

The immune microenvironment of pancreatic ductal adenocarcinoma (PDA) is comprised of a heterogeneous population of cells that are critical for disease evolution. Prominent among these are the specialized CD1dhiCD5+ regulatory B (Breg) cells that exert a pro-tumorigenic role by promoting tumor cell proliferation. Dissecting the molecular pathways regulating this immune sub-population can thus be valuable for uncovering potential therapeutic targets. Here, we investigate Bruton's tyrosine kinase (BTK), a key B-cell kinase, as a potential regulator of CD1dhiCD5+ Breg differentiation in the pancreatic tumor microenvironment. Treatment of cytokine-induced B cells in vitro with the high specificity BTK inhibitor Tirabrutinib inhibited CD1dhiCD5+ Breg differentiation and production of IL-10 and IL-35, essential mediators of Breg immunosuppressive functions. The BTK signaling pathway was also found to be active in vivo in PanIN-associated regulatory B cells. Tirabrutinib treatment of mice bearing orthotopic KrasG12D-pancreatic lesions severely compromised stromal accumulation of the CD1dhiCD5+ Breg population. This was accompanied by an increase in stromal CD8+IFNγ+ cytotoxic T cells and significant attenuation of tumor cell proliferation and PanIN growth. Our results uncover a novel role for BTK in regulating CD1dhiCD5+ Breg differentiation and emphasize its potential as a therapeutic target for pancreatic cancer.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Antígenos CD1d/metabolismo , Linfócitos B Reguladores/metabolismo , Antígenos CD5/metabolismo , Carcinogênese/metabolismo , Diferenciação Celular/fisiologia , Neoplasias Pancreáticas/metabolismo , Animais , Linfócitos B Reguladores/patologia , Carcinogênese/patologia , Proliferação de Células/fisiologia , Interleucina-10/metabolismo , Interleucinas/metabolismo , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/patologia , Transdução de Sinais/fisiologia
15.
Invest Ophthalmol Vis Sci ; 60(1): 192-201, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30654385

RESUMO

Purpose: To examine how circulating immune mediators in vivo may affect gene and protein expression at the RPE/choroid interface. Methods: Young mice were systemically infected with lymphocytic choriomeningitis virus (LCMV) or continuously infused with IFN-γ. RPE/choroid was isolated and analyzed with whole-transcriptome gene expression microarrays. Selected gene expression findings were validated at the protein level. Results: Both the systemic immune activation from virus infection and the sterile systemically increased level of IFN-γ resulted in increased expression of chemokine ligands, chemokine receptors, and early complement components in isolates of RPE/choroid. These findings were largely absent from LCMV-infected mice deficient in either the interferon α/ß receptor or IFN-γ. Conclusions: Together, these findings demonstrate that acute systemic immune activation results in a local response at the RPE/choroid interface that may include chemokine-dependent recruitment of inflammatory cells and engagement of the complement system. This may represent a link between the systemic low-grade inflammation and the retinal pathology observed in several multifactorial entities such as aging, AMD, and diabetes.


Assuntos
Quimiocinas/genética , Corioide/metabolismo , Regulação da Expressão Gênica/fisiologia , Interferon gama/sangue , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Sistema Imunitário/fisiologia , Ativação Linfocitária/fisiologia , Coriomeningite Linfocítica/genética , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Sequenciamento Completo do Exoma
16.
Nat Commun ; 10(1): 277, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30655520

RESUMO

Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation. Whereas microclusters have been studied extensively using confocal microscopy, the spatial and kinetic relationships of their signaling components have not been well characterized due to limits in image resolution and acquisition speed. Here we show, using TIRF-SIM to examine the organization of microclusters at sub-diffraction resolution, the presence of two spatially distinct domains composed of ZAP70-bound TCR and LAT-associated signaling complex. Kinetic analysis of microcluster assembly reveal surprising delays between the stepwise recruitment of ZAP70 and signaling proteins to the TCR, as well as distinct patterns in their disassociation. These delays are regulated by intracellular calcium flux downstream of T cell activation. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation.


Assuntos
Cálcio/metabolismo , Ativação Linfocitária/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cálcio/imunologia , Retroalimentação Fisiológica , Técnicas de Inativação de Genes , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Intravital/métodos , Células Jurkat , Cinética , Leucócitos Mononucleares , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Cultura Primária de Células , Domínios Proteicos/fisiologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/imunologia
17.
Brain Behav Immun ; 75: 48-59, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30218784

RESUMO

Maternal immune activation (mIA) in rodents is rapidly emerging as a key model for neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. Here, we optimise a mIA model in rats, aiming to address certain limitations of current work in this field. Specifically, the lack of clear evidence for methodology chosen, identification of successful induction of mIA in the dams and investigation of male offspring only. We focus on gestational and early juvenile changes in offspring following mIA, as detailed information on these critical early developmental time points is sparse. Following strain (Wistar, Lister Hooded, Sprague Dawley) comparison and selection, and polyriboinosinic-polyribocytidylic acid (poly I:C) dose selection (2.5-15 mg/kg single or once daily for 5 days), mIA was induced in pregnant Wistar rats with 10 mg/kg poly I:C i.p. on gestational day (GD) 15. Early morphometric analysis was conducted in male and female offspring at GD21 and postnatal day (PD) 21, eight dams for each treatment at each time point were used, 32 in total. Subsequent microglia analysis was conducted at PD21 in a small group of offspring. Poly I:C at 10 mg/kg i.p. induced a robust, but variable, plasma IL-6 response 3 h post-injection and reduced body weight at 6 h and 24 h post-injection in two separate cohorts of Wistar rats at GD15. Plasma IL-6 was not elevated at PD21 in offspring or dams. Poly I:C-induced mIA did not affect litter numbers, but resulted in PD21 pup, and GD21 placenta growth restriction. Poly I:C significantly increased microglial activation at PD21 in male hippocampi. We have identified 10 mg/kg poly I:C i.p on GD15 as a robust experimental approach for inducing mIA in Wistar rats and used this to identify early neurodevelopmental changes. This work provides a framework to study the developmental trajectory of disease-relevant, sex-specific phenotypic changes in rats.


Assuntos
Imunidade Ativa/fisiologia , Ativação Linfocitária/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Animais , Comportamento Animal/fisiologia , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Hipocampo/efeitos dos fármacos , Imunidade Ativa/imunologia , Interleucina-6/metabolismo , Ativação Linfocitária/fisiologia , Masculino , Modelos Animais , Atividade Motora/efeitos dos fármacos , Transtornos do Neurodesenvolvimento , Placenta/metabolismo , Poli I-C/farmacologia , Gravidez , Ratos , Ratos Wistar , Esquizofrenia/imunologia , Linfócitos T/imunologia
18.
Leukemia ; 33(3): 625-637, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30267008

RESUMO

Chronic lymphocytic leukemia (CLL) is associated with substantial alterations in T-cell composition and function. However, the role of T-cells in CLL remains largely controversial. Here, we utilized the Eµ-TCL1 mouse model of CLL as well as blood and lymph node samples of CLL patients to investigate the existence of anti-tumoral immune responses in CLL, and to characterize involved immune cell populations. Thereby, we identified an oligoclonal CD8+ effector T-cell population that expands along with CLL progression and controls disease development. We further show that a higher percentage of CD8+ effector T-cells produces IFNγ, and demonstrate that neutralization of IFNγ results in faster CLL progression in mice. Phenotypical and functional analyses of expanded CD8+ effector T-cells show significant differences in disease-affected tissues in mice, with cells in secondary lymphoid organs harboring hallmarks of activation-induced T-cell exhaustion. Notably, we further describe a respective population of exhausted CD8+ T-cells that specifically accumulate in lymph nodes, but not in peripheral blood of CLL patients. Collectively, these data emphasize the non-redundant role of CD8+ T-cells in suppressing CLL progression and highlight their dysfunction that can be exploited as target of immunotherapy in this malignancy.


Assuntos
Linfócitos T CD8-Positivos/patologia , Leucemia Linfocítica Crônica de Células B/patologia , Tecido Linfoide/patologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Humanos , Imunoterapia/métodos , Interferon gama/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfonodos/patologia , Ativação Linfocitária/fisiologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
19.
Mov Disord ; 34(1): 138-141, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30485547

RESUMO

OBJECTIVE: Lymphocyte activation gene-3 (LAG-3) could mediate pathological α-synuclein transmission in neurodegeneration and may be involved in the pathogenesis of Parkinson's disease (PD). The aim of the present study was to explore soluble LAG-3 (sLAG-3) as a potential diagnostic biomarker for PD. METHODS: Serum sLAG-3 concentrations were measured by a quantitative ELISA for patients with PD, essential tremor (ET) and age- and sex-matched controls. The relationships between sLAG-3 and clinical phenotype were assessed via correlation analysis and logistic regression. RESULTS: Serum sLAG-3 levels in patients with PD were significantly higher than those in ET patients and age- and sex-matched controls. The area under the curve of serum sLAG-3 in differentiating PD from age- and sex-matched controls was 0.82. Serum sLAG-3 was associated with non-motor symptoms and excessive daytime sleep. CONCLUSION: sLAG-3 is a candidate novel biomarker for PD. © 2018 International Parkinson and Movement Disorder Society.


Assuntos
Antígenos CD/sangue , Tremor Essencial/sangue , Ativação Linfocitária/fisiologia , Doença de Parkinson/sangue , Biomarcadores/sangue , Tremor Essencial/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/complicações , Fenótipo
20.
Oncogene ; 38(15): 2830-2843, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30546089

RESUMO

γδ T cells are a unique lymphocyte population that have been reported to have either anti- or pro-tumoral functions in several cancer types, but the mechanisms are underinvestigated. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. Hypoxia is a common feature of solid tumors and is believed to alter tumor-derived exosomes (TEXs), which mediate the hypoxic evolution of the tumor microenvironment in return. This study sought to investigate whether TEXs mediate the anti- and pro-tumoral equilibrium of γδ T cells under different oxygen pressures in the tumor microenvironment. We show that TEXs can alter the expansion and cytotoxicity of γδ T cells in an HSP70-dependent but dendritic cell-independent manner. The stimulating effects of normoxic TEXs on γδ T-cell activity were absent from hypoxic TEXs, which enhanced the suppressive effect of myeloid-derived suppressor cells (MDSCs) on γδ T cells through a miR-21/PTEN/PD-L1 regulation axis. Finally, the therapeutic outcome benefited from combined miR-21 and PD-L1 targeting in oral squamous cell carcinoma (OSCC)-bearing immunocompetent mice. We conclude that oxygen pressure in the tumor microenvironment orchestrates an anti- and pro-tumoral γδ T-cell equilibrium by altering TEX content, which subsequently regulates MDSC function in a miR-21/PTEN/PD-L1-axis-dependent manner. Our results should prompt further investigation into integrated exosomal miRNA inhibition and immune checkpoint inhibitor therapeutic modalities for patients with OSCC.


Assuntos
Exossomos/metabolismo , Oxigênio/metabolismo , Linfócitos T/metabolismo , Microambiente Tumoral/fisiologia , Animais , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/metabolismo , Humanos , Hipóxia/metabolismo , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Células Supressoras Mieloides/metabolismo
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