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1.
Methods Enzymol ; 631: 239-255, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31948550

RESUMO

Lymphocytes proliferate in response to several stimuli. In many situations, a rapid lymphocyte expansion, or the identification of a slow dividing cell subpopulation may be of great interest. Thus, it is necessary to perform reliable assays to study and compare lymphocyte subsets proliferation. For this purpose, carboxifluorescein diacetate succinimidyl ester (CFSE) dilution assay has been stablished as a very useful tool that provides cumulative information about cell proliferation. Unlike other techniques that measure a static parameter of a specific time-point, CFSE staining allows to distinguish between subsequent cell divisions. Here, we show a simple protocol to study human T and NK cell proliferation with CFSE dilution assay by flow cytometry.

2.
Front Immunol ; 10: 2278, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31616440

RESUMO

Natural Killer (NK) cells are characterized by their potential to kill tumor cells by different means without previous sensitization and have, therefore, become a valuable tool in cancer immunotherapy. However, their efficacy against solid tumors is still poor and further studies are required to improve it. One of the major restrictions for NK cell activity is the immunosuppressive tumor microenvironment (TME). There, tumor and other immune cells create the appropriate conditions for tumor proliferation while, among others, preventing NK cell activation. Furthermore, NK cell metabolism is impaired in the TME, presumably due to nutrient and oxygen deprivation, and the higher concentration of tumor-derived metabolic end products, such as lactate. This metabolic restriction of NK cells limits their effector functions, and it could represent a potential target to focus on to improve the efficacy of NK cell-based therapies against solid tumors. In this review, we discuss the potential effect of TME into NK cell metabolism and its influence in NK cell effector functions.

5.
Eur J Immunol ; 49(3): 364-374, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30485414

RESUMO

The CD300 molecules constitute an evolutionarily significant family of receptors that are expressed on myeloid and lymphoid cells, but also on other cell types, such as tuft cells. Many of the CD300 receptors have been shown to recognize lipids, e.g. phosphatidylserine and phosphatidylethanolamine. Over the past couple of years, accumulating evidence has shown that this family of receptors is involved in the pathogenesis of many diseases. Specifically, CD300 molecules participate in the mechanisms that viruses employ to develop immune evasion strategies and to infect host cells. The participation of CD300 molecules in viral infection includes both lipid dependent and independent mechanisms, as for example in infections with dengue virus (DENV) and murine norovirus (MNV), respectively. CD300 receptors are also involved in viral escape mechanisms, for instance inhibiting NK cell-mediated cytotoxicity against infected cells. Moreover, it is becoming increasingly recognized that the expression of CD300 receptors is altered during viral diseases. Here, we review the involvement of human and murine CD300 molecules in viral binding and entry and in cellular responses to viruses, which highlights the potential of CD300 molecules in the search of new biomarkers for various stages of infection and therapeutic targets for the treatment of viral infections.

6.
J Allergy Clin Immunol ; 143(2): 700-711.e5, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29906528

RESUMO

BACKGROUND: Basophils express high-affinity IgE receptors (FcεRI), which play an essential role in allergic diseases. It is important to characterize new cell-surface receptors that modulate IgE-mediated basophil activation threshold to design promising immunomodulatory therapies. OBJECTIVES: We sought to analyze the expression of CD300 receptors on human basophils and their implication in IgE-mediated basophil activation processes. METHODS: Blood samples from healthy subjects and patients with cow's milk allergy were collected through the Basque Biobank under an institutional review board-approved protocol. PBMCs were obtained by means of density centrifugation, basophils were purified with a specific isolation kit, and phenotypic and functional studies were performed by using flow cytometry. RESULTS: We demonstrate that basophils express the activating receptor CD300c, which is specifically upregulated in response to IL-3. CD300c works as a costimulatory molecule during IgE-mediated basophil activation, as shown by a significant increase in degranulation and cytokine production when basophils are activated in the presence of CD300c cross-linking compared with activation through the IgE/FcεRI axis alone. Coligation of FcεRI and CD300c increased intracellular calcium mobilization and phosphorylation of signaling intermediates evoked only by FcεRI ligation. We show that the natural ligands of CD300c, phosphatidylserine and phosphatidylethanolamine, modulate IgE-mediated basophil activation. Furthermore, we have observed that CD300c expression in children with cow's milk allergy is increased compared with that in healthy control subjects and that the intensity of expression correlates with the severity of the hypersensitivity symptoms. CONCLUSION: CD300c could be considered a biomarker and therapeutic target in patients with IgE-mediated allergic diseases because it seems to be involved in the modulation of IgE-mediated basophil activation.

7.
Front Immunol ; 9: 1709, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30083165

RESUMO

The ability of the CD300a inhibitory receptor to modulate immune cell functions and its involvement in the pathogenesis of many diseases has aroused a great interest in this molecule. Within human CD4+ T lymphocytes from healthy donors, the inhibitory receptor CD300a is differentially expressed among different T helper subsets. However, there are no data about the expression and regulation of CD300a receptor on CD4+ T cells from human immunodeficiency virus (HIV)-1-infected patients. The objective of this study was to investigate the expression of CD300a on CD4+ T cells from HIV-infected patients on suppressive combined antiretroviral therapy (cART) and cART naïve patients. Our results have demonstrated that the expression levels of this inhibitory receptor were higher on CD4+ T cells from HIV-1 infected subjects compared with healthy donors, and that cART did not reverse the altered expression of CD300a receptor in these patients. We have observed an increase of CD300a expression on both PD1+CD4+ and CD38+CD4+ T cells from HIV-1 infected people. Interestingly, a triple positive (CD300a+PD1+CD38+) subset was expanded in naïve HIV-1 infected patients, while it was very rare in healthy donors and patients on cART. Finally, we found a negative correlation of CD300a expression on CD4+ T lymphocytes and some markers associated with HIV-1 disease progression. Thus, our results show that HIV-1 infection has an impact in the regulation of CD300a inhibitory receptor expression levels, and further studies will shed light into the role of this cell surface receptor in the pathogenesis of HIV infection.

8.
Front Immunol ; 9: 737, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29713323

RESUMO

A brief in vitro stimulation of natural killer (NK) cells with interleukin (IL)-12, IL-15, and IL-18 endow them a memory-like behavior, characterized by higher effector responses when they are restimulated after a resting period of time. These preactivated NK cells, also known as cytokine-induced memory-like (CIML) NK cells, have several properties that make them a promising tool in cancer immunotherapy. In the present study, we have described the effect that different combinations of IL-12, IL-15, and IL-18 have on the generation of human CIML NK cells. Our data points to a major contribution of IL-15 to CIML NK cell-mediated cytotoxicity against target cells. However, the synergistic effect of the three cytokines grant them the best polyfunctional profile, that is, cells that simultaneously degranulate (CD107a) and produce multiple cytokines and chemokines such as interferon γ, tumor necrosis factor α, and C-C motif chemokine ligand 3. We have also analyzed the involvement of each cytokine and their combinations in the expression of homing receptors CXCR4 and CD62L, as well as the expression of CD25 and IL-2-induced proliferation. Furthermore, we have tested the effects of the Jak1/2 inhibitor ruxolitinib in the generation of CIML NK cells. We found that ruxolitinib-treated CIML NK cells expressed lower levels of CD25 than non-treated CIML NK cells, but exhibited similar proliferation in response to IL-2. In addition, we have also found that ruxolitinib-treated NK cells displayed reduced effector functions after the preactivation, which can be recovered after a 4 days expansion phase in the presence of low doses of IL-2. Altogether, our results describe the impact that each cytokine and the Jak1/2 pathway have in the phenotype, IL-2-induced proliferation, and effector functions of human CIML NK cells.


Assuntos
Interleucina-12/fisiologia , Interleucina-15/fisiologia , Interleucina-18/fisiologia , Células Matadoras Naturais/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Adulto , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Fenótipo
9.
Front Immunol ; 8: 836, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28785262

RESUMO

A modified vaccinia Ankara-based HIV-1 vaccine clade B (MVA-B) has been tested for safety and immunogenicity in low-risk human immunodeficiency virus (HIV)-uninfected individuals and as a therapeutic vaccine in HIV-1-infected individuals on combined antiretroviral therapy (cART). As a therapeutic vaccine, MVA-B was safe and broadly immunogenic; however, patients still showed a viral rebound upon treatment interruption. Monocytes are an important part of the viral reservoir and several studies suggest that they are partly responsible for the chronic inflammation observed in cART-treated HIV-infected people. The CD300 family of receptors has an important role in several diseases, including viral infections. Monocytes express CD300a, c, e, and f molecules and lipopolysaccharide (LPS) and other stimuli regulate their expression. However, the expression and function of CD300 receptors on monocytes in HIV infection is still unknown. In this work, we investigated for the first time the expression of CD300 molecules and the cytokine production in response to LPS on monocytes from HIV-1-infected patients before and after vaccination with MVA-B. Our results showed that CD300 receptors expression on monocytes from HIV-1-infected patients correlates with markers of HIV infection progression and immune inflammation. Specifically, we observed a positive correlation between the expression of CD300e and CD300f receptors on monocytes with the number of CD4+ T cells of HIV-1-infected patients before vaccination. We also saw a positive correlation between the expression of the inhibitory receptor CD300f and the expression of CD163 on monocytes from HIV-1-infected individuals before and after vaccination. In addition, monocytes exhibited a higher cytokine production in response to LPS after vaccination, almost at the same levels of monocytes from healthy donors. Furthermore, we also described a correlation in the expression of CD300e and CD300f receptors with TNF-α production in response to LPS, only in monocytes of HIV-1-infected patients before vaccination. Altogether, our results describe the impact of HIV-1 and of the MVA-B vaccine in cytokine production and monocytes phenotype.

10.
Clin Cancer Res ; 23(3): 615-617, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27872101

RESUMO

TGFß in the tumor microenvironment diminishes natural killer (NK) cell-mediated anti-disialoganglioside (anti-GD2) mAb elimination of neuroblastoma cells. Consequently, blockade of TGFß signaling with galunisertib in combination with the anti-GD2 mAb dinutuximab plus adoptively transferred NK cells is a promising tool for the treatment of neuroblastoma. Clin Cancer Res; 23(3); 615-7. ©2016 AACRSee related article by Tran et al., p. 804.


Assuntos
Anticorpos Monoclonais , Neuroblastoma/imunologia , Humanos , Células Matadoras Naturais/imunologia , Pirazóis , Quinolinas , Microambiente Tumoral
11.
Sci Rep ; 6: 32693, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27595670

RESUMO

Neonates are more susceptible to infections than adults. This susceptibility is thought to reflect neonates' qualitative and quantitative defects in the adaptive and innate immune responses. Differential expression of cell surface receptors may result in altered thresholds of neonatal immune cell activation. We determined whether the expression and function of the lipid-binding CD300 family of receptors are different on neonatal immune cells compared to adult immune cells. A multiparametric flow cytometry analysis was performed to determine the expression of CD300 receptors on adult peripheral blood mononuclear cells and neonatal cord blood mononuclear cells. The expression of the CD300a inhibitory receptor was significantly reduced on cells from the newborn adaptive immune system, and neonatal antigen presenting cells exhibited a different CD300 receptors expression pattern. We also found differential LPS-mediated regulation of CD300 receptors expression on adult monocytes compared to cord blood monocytes, and that CD300c and CD300e-mediated activation was quantitatively different in neonatal monocytes. This is the first complete study examining the expression of CD300 receptors on human neonatal immune cells compared with adult immune cells. Significant differences in the expression and function of CD300 receptors may help to explain the peculiarities and distinctness of the neonatal immune responses.


Assuntos
Monócitos/metabolismo , Receptores Imunológicos/metabolismo , Adulto , Humanos , Imunofenotipagem , Recém-Nascido , Monócitos/imunologia , Receptores Imunológicos/imunologia
12.
J Immunol ; 194(11): 5053-60, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25980030

RESUMO

The CD300a inhibitory receptor belongs to the CD300 family of cell surface molecules that regulate a diverse array of immune cell processes. The inhibitory signal of CD300a depends on the phosphorylation of tyrosine residues embedded in ITIMs of the cytoplasmic tail. CD300a is broadly expressed on myeloid and lymphoid cells, and its expression is differentially regulated depending on the cell type. The finding that CD300a recognizes phosphatidylserine and phosphatidylethanolamine, two aminophospholipids exposed on the outer leaflet of dead and activated cells, has shed new light on its role in the modulation of immune functions and in its participation in the host response to several diseases states, such as infectious diseases, cancer, allergy, and chronic inflammatory diseases. This review summarizes the literature on CD300a expression, regulation, signaling pathways, and ligand interaction, as well as its role in fine tuning immune cell functions and its clinical relevance.


Assuntos
Antígenos CD/metabolismo , Motivo de Inibição do Imunorreceptor Baseado em Tirosina/genética , Fosfatidiletanolaminas/antagonistas & inibidores , Fosfatidilserinas/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Humanos , Ligantes , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Transdução de Sinais , Viroses/genética , Viroses/metabolismo
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