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1.
Oncotarget ; 8(40): 67380-67393, 2017 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-28978040

RESUMEN

The cytosolic 5'-nucleotidase cN-II is a highly conserved enzyme implicated in nucleotide metabolism. Based on recent observations suggesting additional roles not directly associated to its enzymatic activity, we studied human cancer cell models with basal or decreased cN-II expression. We developed cancer cells with stable inhibition of cN-II expression by transfection of shRNA-coding plasmids, and studied their biology. We show that human breast cancer cells MDA-MB-231 with decreased cN-II expression better adapt to the disappearance of glucose in growth medium under normoxic conditions than cells with a baseline expression level. This is associated with enhanced in vivo growth and a lower content of ROS in cells cultivated in absence of glucose due to more efficient mechanisms of elimination of ROS. Conversely, cells with low cN-II expression are more sensitive to glucose deprivation in hypoxic conditions. Overall, our results show that cN-II regulates the cellular response to glucose deprivation through a mechanism related to ROS metabolism and defence.

2.
Nucleosides Nucleotides Nucleic Acids ; 35(10-12): 604-612, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27906612

RESUMEN

The 5'-nucleotidase cN-II has been shown to be associated with the sensitivity to nucleoside analogues, the survival of cytarabine treated leukemia patients and to cell proliferation. Due to the lack of relevant cell models for solid tumors, we developed four cell lines with low cN-II expression and characterized them concerning their in vitro sensitivity to cancer drugs and their intracellular nucleotide pools. All four cell models had an important decrease of cN-II expression but did not show modified sensitivity, cell proliferation or nucleotide pools. Our cell models will be important for the study of the role of cN-II in human cancer cells.


Asunto(s)
5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Antineoplásicos/farmacología , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Desoxirribonucleótidos/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Concentración 50 Inhibidora , Interferencia de ARN , ARN Interferente Pequeño/genética , Ribonucleótidos/metabolismo , Transfección
3.
J Exp Med ; 213(5): 841-57, 2016 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-27069116

RESUMEN

Peripheral T cell lymphomas (PTCLs) are a heterogeneous entity of neoplasms with poor prognosis, lack of effective therapies, and a largely unknown pathophysiology. Identifying the mechanism of lymphomagenesis and cell-of-origin from which PTCLs arise is crucial for the development of efficient treatment strategies. In addition to the well-described thymic lymphomas, we found that p53-deficient mice also developed mature PTCLs that did not originate from conventional T cells but from CD1d-restricted NKT cells. PTCLs showed phenotypic features of activated NKT cells, such as PD-1 up-regulation and loss of NK1.1 expression. Injections of heat-killed Streptococcus pneumonia, known to express glycolipid antigens activating NKT cells, increased the incidence of these PTCLs, whereas Escherichia coli injection did not. Gene expression profile analyses indicated a significant down-regulation of genes in the TCR signaling pathway in PTCL, a common feature of chronically activated T cells. Targeting TCR signaling pathway in lymphoma cells, either with cyclosporine A or anti-CD1d blocking antibody, prolonged mice survival. Importantly, we identified human CD1d-restricted lymphoma cells within Vδ1 TCR-expressing PTCL. These results define a new subtype of PTCL and pave the way for the development of blocking anti-CD1d antibody for therapeutic purposes in humans.


Asunto(s)
Antígenos CD1d/inmunología , Linfoma de Células T Periférico/inmunología , Transducción de Señal/inmunología , Animales , Antígenos CD1d/genética , Antígenos Ly/genética , Antígenos Ly/inmunología , Femenino , Humanos , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/patología , Masculino , Ratones , Ratones Noqueados , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/genética , Streptococcus pneumoniae/inmunología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunología
4.
PLoS One ; 6(12): e28648, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22174854

RESUMEN

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.


Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/inmunología , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Células HeLa , Humanos , Ratones , Mitógenos/farmacología , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/metabolismo , Neoplasias/inmunología , Fenotipo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología
6.
J Am Chem Soc ; 133(14): 5198-201, 2011 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-21425779

RESUMEN

Structural variants of α-galactosylceramide (αGC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct αGC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-γ secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with αGC, we screened a library of ∼200 synthetic αGC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Galactosilceramidas/farmacología , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/efectos de los fármacos , Animales , Línea Celular , Citometría de Flujo , Galactosilceramidas/química , Humanos , Ratones , Espectrometría de Fluorescencia , Factores de Tiempo
7.
PLoS One ; 5(12): e14374, 2010 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-21179412

RESUMEN

CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.


Asunto(s)
Antiinflamatorios/farmacología , Galactosilceramidas/química , Células T Asesinas Naturales/efectos de los fármacos , Ligando de CD40/metabolismo , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo/métodos , Galactosilceramidas/agonistas , Glucolípidos/química , Células HeLa , Humanos , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Leucocitos Mononucleares/citología , Modelos Químicos , Células Th2/metabolismo
8.
Bioorg Med Chem Lett ; 20(12): 3475-8, 2010 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-20529677

RESUMEN

Alpha-glucosyl ceramides 4 and 5 have been synthesised and evaluated for their ability to stimulate the activation and expansion of human iNKT cells. The key challenge in the synthesis of both target molecules was the stereoselective synthesis of the alpha-glycosidic linkage. Of the methods examined, glycosylation using per-TMS-protected glucosyl iodide 16 was completely alpha-selective and provided gram quantities of amine 11, from which alpha-glucosyl ceramides 4 and 5 were obtained by N-acylation. alpha-GlcCer 4, containing a C24 saturated acyl chain, stimulated a marked proliferation and expansion of human circulating iNKT cells in short-term cultures. alpha-GlcCer 5, which contains a C20 11,14-cis-diene acyl chain (C20:2), induced extremely similar levels of iNKT cell activation and expansion.


Asunto(s)
Glucosilceramidas/síntesis química , Glucosilceramidas/farmacología , Células T Asesinas Naturales/efectos de los fármacos , Acilación , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Glicosilación , Humanos , Activación de Linfocitos/efectos de los fármacos , Células T Asesinas Naturales/citología , Relación Estructura-Actividad
9.
Bioorg Med Chem Lett ; 20(11): 3223-6, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-20462758

RESUMEN

Several L-fucoglycolipids are associated with diseases such as cancer, cystic fibrosis and rheumatoid arthritis. Activation of iNKT cells is known to lead to the production of cytokines that can help alleviate or exacerbate these conditions. alpha-Galactosyl ceramide (alpha-GalCer) is a known agonist of iNKT cells and it is believed that its fucosyl counterpart might have similar immunogenic properties. We herein report the synthesis of alpha-L-fucosyl ceramide derivatives and describe their biological evaluation. The key challenge in the synthesis of the target molecules involved the stereoselective synthesis of the alpha-glycosidic linkage. Of the methods examined, the per-TMS-protected glycosyl iodide donor was completely alpha-selective, and could be scaled up to provide gram quantities of the azide precursor 11, from which a range of N-acylated alpha-L-fucosyl ceramides were readily obtained and evaluated for ex vivo expansion of human iNKT cells.


Asunto(s)
Ceramidas/síntesis química , Ceramidas/farmacología , Galactosilceramidas/farmacología , Acilación , Humanos , Técnicas In Vitro , Monocitos/efectos de los fármacos
10.
Bioorg Med Chem Lett ; 20(3): 814-8, 2010 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-20061147

RESUMEN

KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can stimulate NKT cells to release a broad range of bioactive cytokines. In an effort to understand the structure-activity relationships, we have carried out syntheses of 26 new KRN7000 analogues incorporating aromatic residues in either or both side chains. Structural variations of the phytosphingosine moiety also include varying stereochemistry at C3 and C4, and 4-deoxy and 3,4-dideoxy versions. Their biological activities are described.


Asunto(s)
Galactosilceramidas/síntesis química , Galactosilceramidas/farmacología , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Hidrocarburos Aromáticos/síntesis química , Hidrocarburos Aromáticos/farmacología , Interleucina-13/biosíntesis , Estereoisomerismo
11.
J Immunol ; 183(3): 1644-56, 2009 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-19620317

RESUMEN

The attenuated strain of Mycobacterium bovis known as bacille Calmette-Guérin (BCG) has been widely used as a vaccine for prevention of disease by Mycobacterium tuberculosis, but with relatively little evidence of success. Recent studies suggest that the failure of BCG may be due to its retention of immune evasion mechanisms that delay or prevent the priming of robust protective cell-mediated immunity. In this study, we describe an approach to enhance the immunogenicity of BCG by incorporating glycolipid activators of CD1d-restricted NKT cells, a conserved T cell subset with the potential to augment many types of immune responses. A method was developed for stably incorporating two forms of the NKT cell activator alpha-galactosylceramide into live BCG organisms, and the impact of this on stimulation of T cell responses and protective antimycobacterial immunity was evaluated. We found that live BCG containing relatively small amounts of incorporated alpha-galactosylceramide retained the ability to robustly activate NKT cells. Compared with immunization with unmodified BCG, the glycolipid-modified BCG stimulated increased maturation of dendritic cells and markedly augmented the priming of Ag-specific CD8(+) T cells responses. These effects were correlated with improved protective effects of vaccination in mice challenged with virulent M. tuberculosis. These results support the view that mycobacteria possess mechanisms to avoid stimulation of CD8(+) T cell responses and that such responses contribute significantly to protective immunity against these pathogens. Our findings raise the possibility of a simple modification of BCG that could yield a more effective vaccine for control of tuberculosis.


Asunto(s)
Galactosilceramidas/inmunología , Mycobacterium bovis/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Adyuvantes Inmunológicos , Animales , Vacuna BCG/inmunología , Galactosilceramidas/farmacología , Inmunidad , Inmunización , Activación de Linfocitos/inmunología , Ratones , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/inmunología , Subgrupos de Linfocitos T
12.
Bioorg Med Chem Lett ; 19(13): 3386-8, 2009 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-19481452

RESUMEN

An alpha-galactosyl ceramide (alpha-GalCer) 2 was synthesized and evaluated for its ability to stimulate iNKT-cell proliferation and elicit T-helper cytokines, IL-4 and IFNgamma. Compound 2 combines the acyl chain of the potent, Th2 biasing alpha-GalCer 1 with a sphingoid base of the same length as that found in OCH, which also exhibits Th2 skewing, Such complementation may enhance cytokine bias, which is thought to be important for therapeutic applications of iNKT cell stimulation. Two related alpha-GalCers, 3 and 4, with saturated acyl chains were prepared for comparison.


Asunto(s)
Adyuvantes Inmunológicos/síntesis química , Alcanos/síntesis química , Galactosilceramidas/química , Monosacáridos/síntesis química , Células Th2/inmunología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Alcanos/química , Alcanos/farmacología , Animales , Antígenos CD1d/inmunología , Antígenos CD1d/metabolismo , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos C57BL , Monosacáridos/química , Monosacáridos/farmacología , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Células Th2/efectos de los fármacos
13.
Immunity ; 30(6): 888-98, 2009 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-19538930

RESUMEN

CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.


Asunto(s)
Células Presentadoras de Antígenos/inmunología , Antígenos CD1d/inmunología , Galactosilceramidas/inmunología , Activación de Linfocitos/inmunología , Células T Asesinas Naturales/inmunología , Células Th2/inmunología , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/metabolismo , Antígenos CD1d/metabolismo , Citocinas/biosíntesis , Citocinas/inmunología , Femenino , Galactosilceramidas/farmacología , Humanos , Cinética , Activación de Linfocitos/efectos de los fármacos , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células T Asesinas Naturales/efectos de los fármacos , Células Th2/efectos de los fármacos
14.
J Immunol ; 182(8): 5140-51, 2009 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-19342695

RESUMEN

Invariant NKT cells (iNKT cells) recognize glycolipid Ags via an invariant TCR alpha-chain and play a central role in various immune responses. Although human CD4(+) and CD4(-) iNKT cell subsets both produce Th1 cytokines, the CD4(+) subset displays an enhanced ability to secrete Th2 cytokines and shows regulatory activity. We performed an ex vivo analysis of blood, liver, and tumor iNKT cells from patients with hepatocellular carcinoma and metastases from uveal melanoma or colon carcinoma. Frequencies of Valpha24/Vbeta11 iNKT cells were increased in tumors, especially in patients with hepatocellular carcinoma. The proportions of CD4(+), double negative, and CD8alpha(+) iNKT cell subsets in the blood of patients were similar to those of healthy donors. However, we consistently found that the proportion of CD4(+) iNKT cells increased gradually from blood to liver to tumor. Furthermore, CD4(+) iNKT cell clones generated from healthy donors were functionally distinct from their CD4(-) counterparts, exhibiting higher Th2 cytokine production and lower cytolytic activity. Thus, in the tumor microenvironment the iNKT cell repertoire is modified by the enrichment of CD4(+) iNKT cells, a subset able to generate Th2 cytokines that can inhibit the expansion of tumor Ag-specific CD8(+) T cells. Because CD4(+) iNKT cells appear inefficient in tumor defense and may even favor tumor growth and recurrence, novel iNKT-targeted therapies should restore CD4(-) iNKT cells at the tumor site and specifically induce Th1 cytokine production from all iNKT cell subsets.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Neoplasias Hepáticas/inmunología , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD1d/inmunología , Complejo CD3/inmunología , Clonación Molecular , Femenino , Células HeLa , Salud , Humanos , Masculino , Persona de Mediana Edad
15.
Bioorg Med Chem Lett ; 18(14): 3906-9, 2008 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-18586489

RESUMEN

KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can stimulate NKT cells to release Th1 and Th2 cytokines. In an effort to understand the structure-activity relationships, we have carried out the synthesis of a complete set of the eight KRN7000 stereoisomers, and their biological activities have been examined.


Asunto(s)
Antígenos CD1/química , Galactosilceramidas/química , Galactosilceramidas/síntesis química , Células Asesinas Naturales/inmunología , Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/química , Antígenos CD1/metabolismo , Antígenos CD1d , Química Farmacéutica/métodos , Diseño de Fármacos , Humanos , Células Asesinas Naturales/metabolismo , Ligandos , Modelos Químicos , Unión Proteica , Estereoisomerismo
16.
Cancer Immun ; 8: 11, 2008 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-18581998

RESUMEN

Despite the high prevalence of colon cancer in the world and the great interest in targeted anti-cancer therapy, only few tumor-specific gene products have been identified that could serve as targets for the immunological treatment of colorectal cancers. The aim of our study was therefore to identify frequently expressed colon cancer-specific antigens. We performed a large-scale analysis of genes expressed in normal colon and colon cancer tissues isolated from colorectal cancer patients using massively parallel signal sequencing (MPSS). Candidates were additionally subjected to experimental evaluation by semi-quantitative RT-PCR on a cohort of colorectal cancer patients. From a pool of more than 6000 genes identified unambiguously in the analysis, we found 2124 genes that were selectively expressed in colon cancer tissue and 147 genes that were differentially expressed to a significant degree between normal and cancer cells. Differential expression of many genes was confirmed by RT-PCR on a cohort of patients. Despite the fact that deregulated genes were involved in many different cellular pathways, we found that genes expressed in the extracellular space were significantly over-represented in colorectal cancer. Strikingly, we identified a transcript from a chromosome X-linked member of the human endogenous retrovirus (HERV) H family that was frequently and selectively expressed in colon cancer but not in normal tissues. Our data suggest that this sequence should be considered as a target of immunological interventions against colorectal cancer.


Asunto(s)
Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Antígenos de Neoplasias/análisis , Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Regulación hacia Abajo , Retrovirus Endógenos/genética , Humanos
17.
J Comb Chem ; 9(6): 1084-93, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17896821

RESUMEN

Two 60+-membered libraries of alpha-galactosylceramides have been prepared by reactions between activated ester resins and two core, fully deprotected galactosylated sphingoid bases. The libraries were evaluated for their ability to stimulate CD1d-restricted NKT cells, using in vitro stimulation of a murine NKT cell hybridoma line and for their ability to induce the expansion of NKT cells from peripheral blood mononuclear cells (PBMC) of a normal human subject. Our results showed that many compounds constructed on a C18-phytosphingosine base had significant stimulatory activity in both assays. Because no product purification was required, this approach is particularly attractive as a method for rapid synthesis of large libraries of potential immunomodulatory glycosylceramides.


Asunto(s)
Técnicas Químicas Combinatorias , Galactosilceramidas/inmunología , Galactosilceramidas/farmacología , Células Asesinas Naturales/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Animales , Galactosa/química , Galactosilceramidas/síntesis química , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/fisiología , Ratones , Modelos Químicos , Resinas Sintéticas/química , Esfingolípidos/química , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/inmunología , Esfingosina/metabolismo
18.
Cancer Immunol Immunother ; 56(11): 1795-805, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17447064

RESUMEN

Carcinoembryonic antigen (CEACAM5) is commonly overexpressed in human colon cancer. Several antigenic peptides recognized by cytolytic CD8+ T-cells have been identified and used in colon cancer phase-I vaccination clinical trials. The HLA-A*0201-binding CEA(694-702) peptide was recently isolated from acid eluted MHC-I associated peptides from a human colon tumor cell line. However, the immunogenicity of this peptide in humans remains unknown. We found that the peptide CEA(694-702) binds weakly to HLA-A*0201 molecules and is ineffective at inducing specific CD8+ T-cell responses in healthy donors. Immunogenic-altered peptide ligands with increased affinity for HLA-A*0201 were identified. Importantly, the elicited cytolytic T lymphocyte (CTL) lines and clones cross-reacted with the wild-type CEA(694-702) peptide. Tumor cells expressing CEA were recognized in a peptide and HLA-A*0201 restricted fashion, but high-CEA expression levels appear to be required for CTL recognition. Finally, CEA-specific T-cell precursors could be readily expanded by in vitro stimulation of peripheral blood mononuclear cell (PBMC) from colon cancer patients with altered CEA peptide. However, the CEA-specific CD8+ T-cell clones derived from cancer patients revealed low-functional avidity and impaired tumor-cell recognition. Together, using T-cells to demonstrate the processing and presentation of the peptide CEA694-702, we were able to corroborate its presentation by tumor cells. However, the low avidity of the specific CTLs generated from cancer patients as well as the high-antigen expression levels required for CTL recognition pose serious concerns for the use of CEA694-702 in cancer immunotherapy.


Asunto(s)
Antígeno Carcinoembrionario/inmunología , Carcinoma/terapia , Neoplasias Colorrectales/terapia , Antígeno HLA-A2/metabolismo , Leucocitos Mononucleares/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/uso terapéutico , Secuencia de Aminoácidos , Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Antígeno Carcinoembrionario/genética , Antígeno Carcinoembrionario/metabolismo , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/inmunología , Humanos , Activación de Linfocitos , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Unión Proteica
19.
J Immunol ; 178(6): 3566-74, 2007 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-17339453

RESUMEN

Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28(-)) T cells strongly expressing granzyme/perforin, and two EM28(+) subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28(-)-derived clones lysed target cells with high efficacy. In contrast, EM28(+)-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28(+) conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.


Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos de Neoplasias/inmunología , Antígenos CD28/inmunología , Linfocitos T CD8-positivos/inmunología , Interleucina-12/farmacología , Isoantígenos/inmunología , Melanoma/inmunología , Fragmentos de Péptidos/inmunología , Antígenos de Neoplasias/administración & dosificación , Antígenos CD28/biosíntesis , Linfocitos T CD8-positivos/metabolismo , Células Clonales , Femenino , Granzimas/biosíntesis , Granzimas/inmunología , Humanos , Inmunidad Celular/efectos de los fármacos , Isoantígenos/administración & dosificación , Masculino , Melanoma/metabolismo , Melanoma/terapia , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/inmunología , Fragmentos de Péptidos/administración & dosificación , Perforina , Proteínas Citotóxicas Formadoras de Poros/biosíntesis , Proteínas Citotóxicas Formadoras de Poros/inmunología , Factores de Tiempo , Vacunación
20.
Cancer Immunol Immunother ; 56(6): 839-47, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16960690

RESUMEN

Tumor-specific gene products, such as cancer/testis (CT) antigens, constitute promising targets for the development of T cell vaccines. Whereas CT antigens are frequently expressed in melanoma, their expression in colorectal cancers (CRC) remains poorly characterized. Here, we have studied the expression of the CT antigens MAGE-A3, MAGE-A4, MAGE-A10, NY-ESO-1 and SSX2 in CRC because of the presence of well-described HLA-A2-restricted epitopes in their sequences. Our analyses of 41 primary CRC and 14 metastatic liver lesions confirmed the low frequency of expression of these CT antigens. No increased expression frequencies were observed in metastatic tumors compared to primary tumors. Histological analyses of CRC samples revealed heterogeneous expression of individual CT antigens. Finally, evidence of a naturally acquired CT antigen-specific CD8(+) T cell response could be demonstrated. These results show that the expression of CT antigens in a subset of CRC patients induces readily detectable T cell responses.


Asunto(s)
Antígenos de Neoplasias/biosíntesis , Linfocitos T CD8-positivos/inmunología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Proteínas de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Neoplasias Colorrectales/genética , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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