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1.
STAR Protoc ; 3(2): 101444, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35677615

RESUMO

Here, we present a protocol for flow cytometry analysis of endothelial cells (ECs) and CD8+ T cells in murine tumor models, at baseline and after cancer immunotherapy with anti-PD-1/anti-CTLA-4 antibodies. We provide gating strategies for identification of specific cell subsets including ECs from tumor-associated high endothelial venules (TA-HEVs), stem-like, and terminally exhausted CD8+ T cells. This protocol represents a valuable tool for the analysis of rare subsets of tumor ECs and CD8+ T cells with critical roles in antitumor immunity. For complete details on the use and execution of this protocol, please refer to Asrir et al. (2022).


Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Animais , Linfócitos T CD8-Positivos , Células Endoteliais , Citometria de Fluxo , Imunoterapia/métodos , Camundongos , Neoplasias/terapia
2.
Cancer Cell ; 40(3): 318-334.e9, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35120598

RESUMO

Recruitment of lymphocytes into tumors is critical for anti-tumor immunity and efficacious immunotherapy. We show in murine models that tumor-associated high endothelial venules (TA-HEVs) are major sites of lymphocyte entry into tumors at baseline and upon treatment with anti-PD-1/anti-CTLA-4 immune checkpoint blockade (ICB). TA-HEV endothelial cells (TA-HECs) derive from post-capillary venules, co-express MECA-79+ HEV sialomucins and E/P-selectins, and are associated with homing and infiltration into tumors of various T cell subsets. Intravital microscopy further shows that TA-HEVs are the main sites of lymphocyte arrest and extravasation into ICB-treated tumors. Increasing TA-HEC frequency and maturation increases the proportion of tumor-infiltrating stem-like CD8+ T cells, and ameliorates ICB efficacy. Analysis of tumor biopsies from 93 patients with metastatic melanoma reveals that TA-HEVs are predictive of better response and survival upon treatment with anti-PD-1/anti-CTLA-4 combination. These studies provide critical insights into the mechanisms governing lymphocyte trafficking in cancer immunity and immunotherapy.


Assuntos
Melanoma , Receptor de Morte Celular Programada 1 , Animais , Linfócitos T CD8-Positivos , Antígeno CTLA-4 , Células Endoteliais , Humanos , Fatores Imunológicos , Imunoterapia , Linfócitos do Interstício Tumoral , Melanoma/patologia , Camundongos , Subpopulações de Linfócitos T , Vênulas/patologia
3.
J Nanobiotechnology ; 16(1): 53, 2018 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-29921300

RESUMO

BACKGROUND: Titanium dioxide (TiO2) particles are commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, the risk of gut barrier disruption is an increasing concern because of the presence of a nano-sized fraction. Food-grade E171 may interact with mucus, a gut barrier protagonist still poorly explored in food nanotoxicology. To test this hypothesis, a comprehensive approach was performed to evaluate in vitro and in vivo interactions between TiO2 and intestinal mucus, by comparing food-grade E171 with NM-105 (Aeroxyde P25) OECD reference nanomaterial. RESULTS: We tested E171-trapping properties of mucus in vitro using HT29-MTX intestinal epithelial cells. Time-lapse confocal laser scanning microscopy was performed without labeling to avoid modification of the particle surface. Near-UV irradiation of E171 TiO2 particles at 364 nm resulted in fluorescence emission in the visible range, with a maximum at 510 nm. The penetration of E171 TiO2 into the mucoid area of HT29-MTX cells was visualized in situ. One hour after exposure, TiO2 particles accumulated inside "patchy" regions 20 µm above the substratum. The structure of mucus produced by HT29-MTX cells was characterized by MUC5AC immunofluorescence staining. The mucus layer was thin and organized into regular "islands" located approximately 20 µm above the substratum. The region-specific trapping of food-grade TiO2 particles was attributed to this mucus patchy structure. We compared TiO2-mediated effects in vivo in rats after acute or sub-chronic oral daily administration of food-grade E171 and NM-105 at relevant exposure levels for humans. Cecal short-chain fatty acid profiles and gut mucin O-glycosylation patterns remained unchanged, irrespective of treatment. CONCLUSIONS: Food-grade TiO2 is trapped by intestinal mucus in vitro but does not affect mucin O-glycosylation and short-chain fatty acid synthesis in vivo, suggesting the absence of a mucus barrier impairment under "healthy gut" conditions.


Assuntos
Ácidos Graxos Voláteis/biossíntese , Aditivos Alimentares/química , Mucosa Intestinal/metabolismo , Mucinas/metabolismo , Muco/metabolismo , Nanopartículas/química , Titânio/química , Animais , Ceco/efeitos dos fármacos , Ceco/metabolismo , Aditivos Alimentares/toxicidade , Glicosilação , Células HT29 , Humanos , Absorção Intestinal , Masculino , Nanopartículas/toxicidade , Tamanho da Partícula , Ratos Wistar , Propriedades de Superfície , Distribuição Tecidual , Titânio/toxicidade
4.
Sci Rep ; 7: 40373, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-28106049

RESUMO

Food-grade titanium dioxide (TiO2) containing a nanoscale particle fraction (TiO2-NPs) is approved as a white pigment (E171 in Europe) in common foodstuffs, including confectionary. There are growing concerns that daily oral TiO2-NP intake is associated with an increased risk of chronic intestinal inflammation and carcinogenesis. In rats orally exposed for one week to E171 at human relevant levels, titanium was detected in the immune cells of Peyer's patches (PP) as observed with the TiO2-NP model NM-105. Dendritic cell frequency increased in PP regardless of the TiO2 treatment, while regulatory T cells involved in dampening inflammatory responses decreased with E171 only, an effect still observed after 100 days of treatment. In all TiO2-treated rats, stimulation of immune cells isolated from PP showed a decrease in Thelper (Th)-1 IFN-γ secretion, while splenic Th1/Th17 inflammatory responses sharply increased. E171 or NM-105 for one week did not initiate intestinal inflammation, while a 100-day E171 treatment promoted colon microinflammation and initiated preneoplastic lesions while also fostering the growth of aberrant crypt foci in a chemically induced carcinogenesis model. These data should be considered for risk assessments of the susceptibility to Th17-driven autoimmune diseases and to colorectal cancer in humans exposed to TiO2 from dietary sources.


Assuntos
Colo/imunologia , Colo/patologia , Alimentos , Homeostase , Sistema Imunitário/imunologia , Lesões Pré-Cancerosas/patologia , Titânio/química , Administração Oral , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Contagem de Células , Separação Celular , Citocinas/metabolismo , Dano ao DNA , Células Dendríticas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Inflamação/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Permeabilidade , Nódulos Linfáticos Agregados/patologia , Ratos Wistar , Frações Subcelulares/metabolismo , Linfócitos T/imunologia , Distribuição Tecidual , Titânio/administração & dosagem
5.
Eur J Immunol ; 46(3): 609-18, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26626316

RESUMO

The elimination of solid tumors largely depends on effective T-cell priming by dendritic cells (DCs). For decades, studies focusing on antitumoral immune responses have been performed with tumors transplanted subcutaneously (s.c.). These studies however do not take into account the heterogeneous tissue distribution and functionality of the different DC subsets. Given the crucial role of DCs in inducing protective immune response, we postulated that the anatomic location of tumor development may greatly impact tumor immunogenicity. We therefore implanted tumor cells either in the DC-rich dermis environment or in the s.c. tissue that mainly contains macrophages and monocytes. We showed that intradermal (i.d.), but not s.c. tumors are rapidly rejected in a T-cell-dependent manner and induce protective T-cell responses. The rejection of i.d. tumors correlates with rapid recruitment of dermal DCs presenting the tumor antigen to both CD4 and CD8 T cells in the draining lymph nodes (dLNs). The same DC subsets were mobilized upon s.c. tumor transplantation but with delayed kinetics. Altogether, our results show that the anatomical site of tumor development influences tumor immunogenicity, notably by controlling the kinetics of DC mobilization in the draining LNs.


Assuntos
Células Dendríticas/imunologia , Linfonodos/citologia , Neoplasias/imunologia , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/fisiologia , Derme/imunologia , Células de Langerhans/imunologia , Ativação Linfocitária , Camundongos , Neoplasias/fisiopatologia , Tela Subcutânea/imunologia
6.
Biol Aujourdhui ; 208(2): 167-75, 2014.
Artigo em Francês | MEDLINE | ID: mdl-25190576

RESUMO

As we are faced with the exponential use of nanomaterials in consumer products, including food, the consequences of daily exposure to nanoparticles at low doses set public health issues for humans. Among the different routes of exposure, the oral route remains the less documented, although nanomaterials are commonly used as food additives, or incorporated into packaging in contact with food or water, to provide their texturing and anti-microbial properties, or as simple colorant agents. The oral and gastrointestinal mucosa are the first regions in contact with the ingested nanoparticles. The latter cross these biological barriers, and distribute to the systemic compartment. Although differences exist between categories of nanoparticles, given differences in their physico-chemical properties, primary particle size and solubility, the example given in this review with titanium dioxide (TiO2) is intended to illustrate oral toxicity studies conducted in vivo and in vitro in order to contribute to the risk assessment in humans.


Assuntos
Mucosa Intestinal/metabolismo , Nanopartículas Metálicas , Mucosa Bucal/metabolismo , Exposição Ocupacional/efeitos adversos , Titânio , Administração Oral , Animais , Disponibilidade Biológica , Humanos , Sistema Imunitário/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/toxicidade , Mucosa Bucal/efeitos dos fármacos , Titânio/administração & dosagem , Titânio/farmacocinética , Titânio/toxicidade
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