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2.
Front Immunol ; 15: 1369780, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38868771

RESUMO

Although most follicular-derived thyroid cancers are well differentiated and have an overall excellent prognosis following treatment with surgery and radioiodine, management of advanced thyroid cancers, including iodine refractory disease and poorly differentiated/undifferentiated subtypes, is more challenging. Over the past decade, better understanding of the genetic drivers and immune milieu of advanced thyroid cancers has led to significant progress in the management of these patients. Numerous targeted kinase inhibitors are now approved by the U.S Food and Drug administration (FDA) for the treatment of advanced, radioiodine refractory differentiated thyroid cancers (DTC) as well as anaplastic thyroid cancer (ATC). Immunotherapy has also been thoroughly studied and has shown promise in selected cases. In this review, we summarize the progress in the understanding of the genetic landscape and the cellular and molecular basis of radioiodine refractory-DTC and ATC, as well as discuss the current treatment options and future therapeutic avenues.


Assuntos
Adenocarcinoma Folicular , Imunoterapia , Humanos , Imunoterapia/métodos , Adenocarcinoma Folicular/terapia , Adenocarcinoma Folicular/imunologia , Adenocarcinoma Folicular/genética , Neoplasias da Glândula Tireoide/terapia , Neoplasias da Glândula Tireoide/imunologia , Animais , Radioisótopos do Iodo/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico
3.
J Immunother Precis Oncol ; 6(2): 111-116, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-37214206

RESUMO

Pulmonary toxicity from immune checkpoint inhibitor therapy is typically a severe and potentially fatal complication, but these observations are driven by the most common toxicity, pneumonitis. Rarer pulmonary immune related adverse events, like airway disease and sarcoidosis, may have a more benign course. In this case report, we present a patient in whom therapy with the PD-1 inhibitor pembrolizumab resulted in severe eosinophilic asthma and sarcoidosis. This is the first case showing that anti-IL-5 inhibition may be safe in patients who develop eosinophilic asthma after ICI therapy. We further show that sarcoidosis does not necessarily require treatment cessation. This case highlights relevant nuances when clinicians face pulmonary toxicities other than pneumonitis.

4.
J Clin Invest ; 133(11)2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37053016

RESUMO

The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC); however, the complex intratumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single-cell transcriptomic and genetic alteration data from patients with different subtypes of thyroid cancer. The resulting spectrum of ATC transformation included stress-responsive DTC cells, inflammatory ATC cells (iATCs), and mitotic-defective ATC cells and extended all the way to mesenchymal ATC cells (mATCs). Furthermore, our analysis identified 2 important milestones: (a) a diploid stage, in which iATC cells were diploids with inflammatory phenotypes and (b) an aneuploid stage, in which mATCs gained aneuploid genomes and mesenchymal phenotypes, producing excessive amounts of collagen and collagen-interacting receptors. In parallel, cancer-associated fibroblasts showed strong interactions among mesenchymal cell types, macrophages shifted from M1 to M2 states, and T cells reprogrammed from cytotoxic to exhausted states, highlighting new therapeutic opportunities for the treatment of ATC.


Assuntos
Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Transcriptoma , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/genética , Perfilação da Expressão Gênica , Aneuploidia , Linhagem Celular Tumoral
5.
Front Cardiovasc Med ; 9: 988713, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36426217

RESUMO

Radiation therapy (RT) to the chest increases the patients' risk of cardiovascular disease (CVD). A complete understanding of the mechanisms by which RT induces CVD could lead to specific preventive, therapeutic approaches. It is becoming evident that both genotoxic chemotherapy agents and radiation induce mitochondrial dysfunction and cellular senescence. Notably, one of the common phenotypes observed in cancer survivors is accelerated senescence, and immunosenescence is closely related to both cancer risk and CVD development. Therefore, suppression of immunosenescence can be an ideal target to prevent cancer treatment-induced CVD. However, the mechanism(s) by which cancer treatments induce immunosenescence are incompletely characterized. We isolated peripheral blood mononuclear cells (PBMCs) before and 3 months after RT from 16 thoracic cancer patients. We characterized human immune cell lineages and markers of senescence, DNA damage response (DDR), efferocytosis, and determinants of clonal hematopoiesis of indeterminant potential (CHIP), using mass cytometry (CyTOF). We found that the frequency of the B cell subtype was decreased after RT. Unsupervised clustering of the CyTOF data identified 138 functional subsets of PBMCs. Compared with baseline, RT increased TBX21 (T-bet) expression in the largest B cell subset of Ki67-/DNMT3a+naïve B cells, and T-bet expression was correlated with phosphorylation of p90RSK expression. CD38 expression was also increased in naïve B cells (CD27-) and CD8+ effector memory CD45RA T cells (TEMRA). In vitro, we found the critical role of p90RSK activation in upregulating (1) CD38+/T-bet+ memory and naïve B, and myeloid cells, (2) senescence-associated ß-gal staining, and (3) mitochondrial reactive oxygen species (ROS) after ionizing radiation (IR). These data suggest the crucial role of p90RSK activation in immunosenescence. The critical role of p90RSK activation in immune cells and T-bet induction in upregulating atherosclerosis formation has been reported. Furthermore, T-bet directly binds to the CD38 promoter region and upregulates CD38 expression. Since both T-bet and CD38 play a significant role in the process of immunosenescence, our data provide a cellular and molecular mechanism that links RT-induced p90RSK activation and the immunosenescence with T-bet and CD38 induction observed in thoracic cancer patients treated by RT and suggests that targeting the p90RSK/T-bet/CD38 pathway could play a role in preventing the radiation-associated CVD and improving cancer prognosis by inhibiting immunosenescence.

6.
Radiother Oncol ; 176: 149-156, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36209942

RESUMO

PURPOSE: We compared treatment-related pulmonary adverse events (TRPAE), progression-free survival (PFS), and overall survival (OS) among locally advanced non-small cell lung cancer (NSCLC) patients who received concurrent chemoradiotherapy (CRT) versus CRT followed by immune check point inhibitor (ICI) immunotherapy (CRTI). MATERIALS AND METHODS: TRPAE was defined as any pulmonary events as defined in CTCAE v.5 occurring within 12 months after completion of radiotherapy. Outcomes were compared between CRT and CTRI by Cox proportional hazard regression and Kaplan-Meier analyses. We also assessed if TRPAE-induced discontinuation of ICI affected survival. RESULTS: We analyzed 326 patients treated between July 2010 and November 2019; 195 patients received CRT and 131 received CRTI. The incidences of severe grade ≥ 3 TRPAE were similar between the two groups, however, symptomatic TRPAE was almost doubled in CRTI group (65.7 % CTRI vs 35.9 % CRT, P < 0.0001). The rates of 4-year OS and PFS were 54.5 % vs 36.7 % (P = 0.0003) and 43.8 % vs 35.8 % (P = 0.038) in CRT + Durvalumab and CRT group, respectively. Receipt of ICI Durvalumab was associated with better 4-year OS (HR 0.53, 95 % CI 0.36-0.78, P = 0.001) and PFS (HR 0.55, 95 % CI 0.38-0.80, P = 0.002). Patients who discontinued ICI because of TRPAE had worse 4-year OS (P = 0.001) and higher rates of distant metastasis (P = 0.003) than those who completed planned ICI after developing TRPAE. CONCLUSION: CRT followed by adjuvant ICI led to improved 4-year OS and PFS consistent with published data. CRTI was associated with higher incidence of grade ≥ 2 TRPAE in both high and low mean lung dose groups without significant difference in grade ≥ 3 TRPAE. Discontinuation of ICI due to TRPAE was associated with poorer OS and distant disease control than completing ICI as planned after developing TRPAE.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/patologia , Quimiorradioterapia/efeitos adversos , Pulmão/patologia
7.
Cancer Cell ; 40(5): 509-523.e6, 2022 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-35537412

RESUMO

Immune checkpoint blockade (ICB) therapy frequently induces immune-related adverse events. To elucidate the underlying immunobiology, we performed a deep immune analysis of intestinal, colitis, and tumor tissue from ICB-treated patients with parallel studies in preclinical models. Expression of interleukin-6 (IL-6), neutrophil, and chemotactic markers was higher in colitis than in normal intestinal tissue; T helper 17 (Th17) cells were more prevalent in immune-related enterocolitis (irEC) than T helper 1 (Th1). Anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) induced stronger Th17 memory in colitis than anti-program death 1 (anti-PD-1). In murine models, IL-6 blockade associated with improved tumor control and a higher density of CD4+/CD8+ effector T cells, with reduced Th17, macrophages, and myeloid cells. In an experimental autoimmune encephalomyelitis (EAE) model with tumors, combined IL-6 blockade and ICB enhanced tumor rejection while simultaneously mitigating EAE symptoms versus ICB alone. IL-6 blockade with ICB could de-couple autoimmunity from antitumor immunity.


Assuntos
Colite , Neoplasias , Animais , Colite/induzido quimicamente , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Interleucina-6 , Camundongos , Células Mieloides , Neoplasias/tratamento farmacológico
8.
Nat Commun ; 13(1): 1970, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35413951

RESUMO

Immune checkpoint inhibitors are associated with immune-related adverse events (irAEs), including arthritis (arthritis-irAE). Management of arthritis-irAE is challenging because immunomodulatory therapy for arthritis should not impede antitumor immunity. Understanding of the mechanisms of arthritis-irAE is critical to overcome this challenge, but the pathophysiology remains unknown. Here, we comprehensively analyze peripheral blood and/or synovial fluid samples from 20 patients with arthritis-irAE, and unmask a prominent Th1-CD8+ T cell axis in both blood and inflamed joints. CX3CR1hi CD8+ T cells in blood and CXCR3hi CD8+ T cells in synovial fluid, the most clonally expanded T cells, significantly share TCR repertoires. The migration of blood CX3CR1hi CD8+ T cells into joints is possibly mediated by CXCL9/10/11/16 expressed by myeloid cells. Furthermore, arthritis after combined CTLA-4 and PD-1 inhibitor therapy preferentially has enhanced Th17 and transient Th1/Th17 cell signatures. Our data provide insights into the mechanisms, predictive biomarkers, and therapeutic targets for arthritis-irAE.


Assuntos
Artrite , Neoplasias , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Linfócitos T CD8-Positivos , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Imunoterapia/efeitos adversos , Neoplasias/tratamento farmacológico , Neoplasias/etiologia
10.
Nat Immunol ; 21(11): 1467, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32884131

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

11.
Front Immunol ; 11: 590494, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33552049

RESUMO

Patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) treated with immune checkpoint inhibitors (ICIs) are at risk of pneumonitis as well as pneumonia (combined henceforth as ICI-related pulmonary complications). Little is known about the cellular and molecular mechanisms underlying ICI-related pulmonary complications. We characterized lymphocytes from bronchoalveolar lavage (BAL) fluid and peripheral blood from seven AML/MDS patients with pulmonary symptoms after ICI-based therapy (ICI group) and four ICI-naïve AML/MDS patients with extracellular bacterial or fungal pneumonias (controls). BAL T cells in the ICI group were clonally expanded, and BAL IFNγ+ IL-17- CD8+ T and CXCR3+ CCR6+ Th17/Th1 cells were enriched in the ICI group. Our data suggest that these cells may play a critical role in the pathophysiology of ICI-related pulmonary complications. Understanding of these cell populations may also provide predictive and diagnostic biomarkers of ICI-related pulmonary complications, eventually enabling differentiation of pneumonitis from pneumonia in AML/MDS patients receiving ICI-based therapies.


Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Leucemia Mieloide Aguda/imunologia , Síndromes Mielodisplásicas/imunologia , Pneumonia/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/efeitos adversos , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Imunofenotipagem , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/tratamento farmacológico , Pneumonia/induzido quimicamente
12.
J Immunother Cancer ; 7(1): 126, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088575

RESUMO

BACKGROUND: Despite ground-breaking clinical success in the treatment of different cancers, immune checkpoint inhibitors can cause profound inflammatory and immune-related adverse events. Autoimmune inflammatory arthritis following immune checkpoint inhibitor treatment has been reported; however, to date, no cases of crystal arthritis following immune checkpoint inhibitors have been identified. CASE PRESENTATION: We report the first case of recurrent pseudogout, an inflammatory crystal arthritis, in a patient treated with nivolumab, a PD-1 inhibitor, for renal cell carcinoma. The patient had recurrent pseudogout flares about week to 10 days after each nivolumab infusion. After treatment with prophylactic colchicine, the patient well tolerated additional nivolumab infusions without adverse events. In parallel, we characterized immune cells of synovial fluid at each flare. Immunoprofiling of synovial fluid showed that the proportion of inflammatory IL-17-producing CD4+ T cells and amount of IL-17 were notably increased in synovial fluid with every recurrent flair, and correlated with the increase in number of synovial neutrophils, suggesting a potential role of T helper 17 (Th17) cells in neutrophil-driven inflammation during pseudogout arthritis. CONCLUSIONS: This case suggests a potential influence of Th17 cells on the neutrophil recruitment and neutrophil-driven inflammatory events leading to pseudogout induced by immune checkpoint inhibitor therapy.


Assuntos
Antineoplásicos Imunológicos/efeitos adversos , Condrocalcinose/diagnóstico , Condrocalcinose/etiologia , Antineoplásicos Imunológicos/uso terapêutico , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Carcinoma de Células Renais/complicações , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/tratamento farmacológico , Condrocalcinose/metabolismo , Citocinas/metabolismo , Progressão da Doença , Humanos , Masculino , Pessoa de Meia-Idade , Nivolumabe/efeitos adversos , Nivolumabe/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Recidiva , Líquido Sinovial/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
13.
Nature ; 567(7749): 525-529, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30814730

RESUMO

T cells become dysfunctional when they encounter self antigens or are exposed to chronic infection or to the tumour microenvironment1. The function of T cells is tightly regulated by a combinational co-stimulatory signal, and dominance of negative co-stimulation results in T cell dysfunction2. However, the molecular mechanisms that underlie this dysfunction remain unclear. Here, using an in vitro T cell tolerance induction system in mice, we characterize genome-wide epigenetic and gene expression features in tolerant T cells, and show that they are distinct from effector and regulatory T cells. Notably, the transcription factor NR4A1 is stably expressed at high levels in tolerant T cells. Overexpression of NR4A1 inhibits effector T cell differentiation, whereas deletion of NR4A1 overcomes T cell tolerance and exaggerates effector function, as well as enhancing immunity against tumour and chronic virus. Mechanistically, NR4A1 is preferentially recruited to binding sites of the transcription factor AP-1, where it represses effector-gene expression by inhibiting AP-1 function. NR4A1 binding also promotes acetylation of histone 3 at lysine 27 (H3K27ac), leading to activation of tolerance-related genes. This study thus identifies NR4A1 as a key general regulator in the induction of T cell dysfunction, and a potential target for tumour immunotherapy.


Assuntos
Regulação da Expressão Gênica/genética , Genoma , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologia , Acetilação , Animais , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Linhagem Celular Tumoral , Colite/imunologia , Colite/patologia , Colite/terapia , Epigênese Genética , Feminino , Histonas/química , Histonas/metabolismo , Tolerância Imunológica/genética , Imunoterapia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Linfócitos T/imunologia , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica
14.
Front Immunol ; 9: 1884, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30158933

RESUMO

T follicular helper (Tfh) cells play key role in providing help to B cells during germinal center (GC) reactions. Generation of protective antibodies against various infections is an important aspect of Tfh-mediated immune responses and the dysregulation of Tfh cell responses has been implicated in various autoimmune disorders, inflammation, and malignancy. Thus, their differentiation and maintenance must be closely regulated to ensure appropriate help to B cells. The generation and function of Tfh cells is regulated by multiple checkpoints including their early priming stage in T zones and throughout the effector stage of differentiation in GCs. Signaling pathways activated downstream of cytokine and costimulatory receptors as well as consequent activation of subset-specific transcriptional factors are essential steps for Tfh cell generation. Thus, understanding the mechanisms underlying Tfh cell-mediated immunity and pathology will bring into spotlight potential targets for novel therapies. In this review, we discuss the recent findings related to the molecular mechanisms of Tfh cell differentiation and their role in normal immune responses and antibody-mediated diseases.


Assuntos
Suscetibilidade a Doenças , Imunidade Celular , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Autoimunidade , Biomarcadores , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Citocinas/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Linfócitos T Auxiliares-Indutores/citologia
15.
Cancer Immunol Res ; 6(7): 788-797, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29764837

RESUMO

Somatic KRAS mutations are the most common oncogenic variants in lung cancer and are associated with poor prognosis. Using a Kras-induced lung cancer mouse model, CC-LR, we previously showed a role for inflammation in lung tumorigenesis through activation of the NF-κB pathway, along with induction of interleukin 6 (IL6) and an IL17-producing CD4+ T-helper cell response. IL22 is an effector molecule secreted by CD4+ and γδ T cells that we previously found to be expressed in CC-LR mice. IL22 mostly signals through the STAT3 pathway and is thought to act exclusively on nonhematopoietic cells with basal IL22 receptor (IL22R) expression on epithelial cells. Here, we found that higher expression of IL22R1 in patients with KRAS-mutant lung adenocarcinoma was an independent indicator of poor recurrence-free survival. We then showed that genetic ablation of Il22 in CC-LR mice (CC-LR/IL22KO mice) caused a significant reduction in tumor number and size. This was accompanied by significantly lower tumor cell proliferation, angiogenesis, and STAT3 activation. Il22 ablation was also associated with significant reduction in lung-infiltrating inflammatory cells and expression of protumor inflammatory cytokines. Conversely, this was accompanied with increased antitumor Th1 and cytotoxic CD8+ T-cell responses, while suppressing the protumor immunosuppressive T regulatory cell response. In CC-LR/IL22KO mice, we found significantly reduced expression of core stemness genes and the number of prototypical SPC+CCSP+ stem cells. Thus, we conclude that IL22 promotes Kras-mutant lung tumorigenesis by driving a protumor inflammatory microenvironment with proliferative, angiogenic, and stemness contextual cues in epithelial/tumor cells. Cancer Immunol Res; 6(7); 788-97. ©2018 AACR.


Assuntos
Interleucinas/metabolismo , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Mutação , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Animais Geneticamente Modificados , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Humanos , Imuno-Histoquímica , Interleucinas/genética , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Microambiente Tumoral , Interleucina 22
16.
J Allergy Clin Immunol ; 141(6): 2061-2073.e5, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-28943467

RESUMO

BACKGROUND: Allergic asthma is a heterogeneous chronic inflammatory disease of the airways with a massive infiltration of eosinophils or neutrophils mediated by allergen-specific TH2 and TH17 cells, respectively. Therefore successful treatment of allergic asthma will require suppression of both TH2 and TH17 cells. OBJECTIVE: We sought to investigate the role of the TH17 cell pathway in regulating TH2 cell responses in allergic asthma. METHODS: Allergic asthma was induced by intranasal challenge with proteinase allergens in C57BL/6, Il17a-/-Il17f-/-, and retinoic acid receptor-related orphan receptor γt (RORγt)gfp/gfp mice. A pharmacologic RORγt inhibitor was used to evaluate its preventive and therapeutic effects in allergic asthma. Characteristics of allergic airway inflammation were analyzed by using flow cytometry, histology, quantitative real-time PCR, and ELISA. Mixed bone marrow chimeric mice, fate mapping analysis, short hairpin RNA transduction, and in vitro T-cell differentiation were used for mechanistic studies. RESULTS: Mice deficient in IL-17A and IL-17F, as well as RORγt, exhibited a significant reduction not only in TH17 cell responses but also in TH2 cell responses in an animal model of allergic asthma. Similarly, mice treated with an RORγt inhibitor had significantly diminished TH17 and TH2 cell responses, leading to reduced neutrophil and eosinophil numbers in the airway. RORγt-deficient T cells were intrinsically defective in differentiating into TH2 cells and expressed increased levels of B-cell lymphoma 6 (Bcl6). Bcl6 knockdown resulted in a remarkable restoration of TH2 cell differentiation in RORγt-deficient T cells. Blockade of RORγt also significantly hampered the differentiation of human TH2 and TH17 cells from naive CD4+ T cells. CONCLUSION: RORγt in T cells is required for optimal TH2 cell differentiation by suppressing Bcl6 expression; this finding suggests that targeting RORγt might be a promising approach for the treatment of allergic asthma by concomitantly suppressing TH17 and TH2 cell responses in the airway.


Assuntos
Asma/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Células Th17/imunologia , Células Th2/imunologia , Animais , Diferenciação Celular/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores
17.
J Vis Exp ; (129)2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-29286361

RESUMO

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.


Assuntos
Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Linfócitos T/imunologia , Vacinação/métodos , Citometria de Fluxo/métodos , Humanos
18.
Cell Rep ; 21(1): 195-207, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28978473

RESUMO

Th17 cells are potent mediators in autoimmune diseases, and RORγt is required for their development. Recent studies have shown that RORγt+ Treg cells in the gut regulate intestinal inflammation by inhibiting effector T cell function. In the current study, we report that RORγt+ Treg cells were also found in lymph nodes following immunization. Not only distinct from intestinal RORγt+ Treg cells in their transcriptomes, peripheral RORγt+ Treg cells were derived from Foxp3+ thymic Treg cells in an antigen-specific manner. Development of these RORγt+ Treg cells, coined T regulatory 17 (Tr17) cells, depended on IL-6/Stat3 signaling. Tr17 cells showed suppressive activity against antigen-specific effector T cells in vitro. In addition, Tr17 cells efficiently inhibited myelin-specific Th17-cell-mediated CNS auto-inflammation in a passive EAE model. Collectively, our study demonstrates that Tr17 cells are effector Treg cells that potentially restrict autoimmunity.


Assuntos
Autoimunidade/genética , Encefalomielite Autoimune Experimental/genética , Fatores de Transcrição Forkhead/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Transferência Adotiva , Animais , Diferenciação Celular , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Fragmentos de Peptídeos/administração & dosagem , Receptores CCR6/genética , Receptores CCR6/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/transplante , Células Th17/patologia
19.
J Immunol ; 199(10): 3453-3465, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29055004

RESUMO

The metabolic syndrome and diabetic conditions support atherosclerosis, but the exact mechanisms for accelerated atherogenesis remain unclear. Although the proinflammatory role of STAT4 in atherosclerosis and diet-induced insulin resistance (IR) was recently established, the impact of STAT4 on atherogenesis in conditions of IR is not known. In this study, we generated Stat4-/-Ldlr-/- mice that were fed a diabetogenic diet with added cholesterol (DDC). DDC-fed Stat4-/-Ldlr-/- mice demonstrated improved glucose tolerance, insulin sensitivity, and a 36% reduction in atherosclerosis compared with Ldlr-/- controls. Interestingly, we detected a reduction in T follicular helper (Tfh) cells and plasma B cells but a sharp elevation in CD8+ regulatory T cells (Tregs) in spleens and aortas of Stat4-/-Ldlr-/- mice compared with Ldlr-/- mice. Similarly, STAT4 deficiency supported CD8+ Treg differentiation in vitro. STAT4-deficient CD8+ Tregs suppressed Tfh cell and germinal center B cell development upon immunization with keyhole limpet hemocyanin, indicating an important role for STAT4 in CD8+ Treg functions in vivo. Furthermore, adoptive transfer of Stat4-/-Ldlr-/- CD8+ Tregs versus Ldlr-/- CD8+ Tregs resulted in a significant reduction in plaque burden and suppression of Tfh cell and germinal center B cells in DDC-fed Ldlr-/- recipients. STAT4 expression in macrophages (MΦs) also affected the Tfh/CD8+ Treg axis, because conditioned media from Stat4-/-Ldlr-/- MΦs supported CD8+ Treg differentiation, but not Tfh cell differentiation, in a TGF-ß-dependent manner. These findings suggest a novel mechanism by which STAT4 supports atherosclerosis in IR Ldlr-/- mice via STAT4-dependent MΦs, as well as cell-intrinsic suppression of CD8+ Treg generation and functions and maintenance of Tfh cell generation and the accompanying humoral immune response.


Assuntos
Aterosclerose/imunologia , Receptores de LDL/metabolismo , Fator de Transcrição STAT4/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD8/metabolismo , Células Cultivadas , Colesterol/metabolismo , Dieta Aterogênica , Centro Germinativo/imunologia , Humanos , Resistência à Insulina , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/genética , Fator de Transcrição STAT4/genética
20.
J Immunol ; 199(8): 2815-2822, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28855309

RESUMO

In this study, we determined the role of IL-21R signaling in Mycobacterium tuberculosis infection, using IL-21R knockout (KO) mice. A total of 50% of M. tuberculosis H37Rv-infected IL-21R KO mice died in 6 mo compared with no deaths in infected wild type (WT) mice. M. tuberculosis-infected IL-21R KO mice had enhanced bacterial burden and reduced infiltration of Ag-specific T cells in lungs compared with M. tuberculosis-infected WT mice. Ag-specific T cells from the lungs of M. tuberculosis-infected IL-21R KO mice had increased expression of T cell inhibitory receptors, reduced expression of chemokine receptors, proliferated less, and produced less IFN- γ, compared with Ag-specific T cells from the lungs of M. tuberculosis-infected WT mice. T cells from M. tuberculosis-infected IL-21R KO mice were unable to induce optimal macrophage responses to M. tuberculosis. This may be due to a decrease in the Ag-specific T cell population. We also found that IL-21R signaling is associated with reduced expression of a transcriptional factor Eomesodermin and enhanced functional capacity of Ag-specific T cells of M. tuberculosis-infected mice. The sum of our findings suggests that IL-21R signaling is essential for the optimal control of M. tuberculosis infection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Pulmão/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Receptores de Interleucina-21/metabolismo , Tuberculose/imunologia , Animais , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Interferon gama/metabolismo , Pulmão/microbiologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-21/genética , Transdução de Sinais , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
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