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1.
Cancers (Basel) ; 15(19)2023 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-37835401

RESUMEN

BACKGROUND: Embryonic antigens (EA) regulate pluripotency, self-renewal, and differentiation in embryonic stem (ES) cells during their development. In adult somatic cells, EA expression is normally inhibited; however, EAs can be re-expressed by cancer cells and are involved in the deregulation of different signaling pathways (SPs). In the context of AML, data concerning the expression of EAs are scarce and contradictory. METHODS: We used mass cytometry to explore the expression of EAs and three SPs in myeloid cells from AML patients and normal bone marrow (NBM). Imaging flow cytometry was used for morphological assessment of cells in association with their OCT3/4 expression status (positive vs. negative). RESULTS: An overall reduction in or absence of EA expression was observed in immature myeloid cells from AML patients compared to their normal counterparts. Stage-specific embryonic antigen-3 (SSEA-3) was consistently expressed at low levels in immature myeloid cells, whereas SSEA-1 was overexpressed in hematopoietic stem cells (HSCs) and myeloblasts from AML with monocytic differentiation (AML M4/M5). Therefore, these markers are valuable for distinguishing between normal and abnormal myeloid cells. These preliminary results show that the exploration of myeloid cell intracellular SPs in the setting of AML is very informative. Deregulation of three important leukemogenic SPs was also observed in myeloid cells from AML. CONCLUSIONS: Exploring EAs and SPs in myeloid cells from AML patients by mass cytometry may help identify characteristic phenotypes and facilitate AML follow-up.

2.
Br J Pharmacol ; 178(21): 4291-4315, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34302297

RESUMEN

Tumour diagnosis relies on the visual examination of histological slides by pathologists through a microscope eyepiece. Digital pathology, the digitalization of histological slides at high magnification with slides scanners, has raised the opportunity to extract quantitative information due to image analysis. In the last decade, medical image analysis has made exceptional progress due to the development of artificial intelligence (AI) algorithms. AI has been successfully used in the field of medical imaging and more recently in digital pathology. The feasibility and usefulness of AI assisted pathology tasks have been demonstrated in the very last years and we can expect those developments to be applied to routine histopathology in the future. In this review, we will describe and illustrate this technique and present the most recent applications in the field of tumour histopathology. LINKED ARTICLES: This article is part of a themed issue on Molecular imaging - visual themed issue. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.21/issuetoc.


Asunto(s)
Inteligencia Artificial , Neoplasias , Algoritmos , Humanos , Procesamiento de Imagen Asistido por Computador , Neoplasias/diagnóstico
3.
Acta Physiol (Oxf) ; 232(3): e13661, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33840159

RESUMEN

AIM: Type A intercalated cells of the renal collecting duct participate in the maintenance of the acid/base balance through their capacity to adapt proton secretion to homeostatic requirements. We previously showed that increased proton secretion stems in part from the enlargement of the population of proton secreting cells in the outer medullary collecting duct through division of fully differentiated cells, and that this response is triggered by growth/differentiation factor 15. This study aimed at deciphering the mechanism of acid load-induced secretion of Gdf15 and its mechanism of action. METHODS: We developed an original method to evaluate the proliferation of intercalated cells and applied it to genetically modified or pharmacologically treated mice under basal and acid-loaded conditions. RESULTS: Gdf15 is secreted by principal cells of the collecting duct in response to the stimulation of vasopressin receptors. Vasopressin-induced production of cAMP triggers activation of AMP-stimulated kinases and of Na,K-ATPase, and induction of p53 and Gdf15. Gdf15 action on intercalated cells is mediated by ErbB2 receptors, the activation of which triggers the expression of cyclin d1, of p53 and anti-proliferative genes, and of Egr1. CONCLUSION: Acidosis-induced proliferation of intercalated cells results from a cross talk with principal cells which secrete Gdf15 in response to their stimulation by vasopressin. Thus, vasopressin is a major determinant of the collecting duct cellular homeostasis as it promotes proliferation of intercalated cells under acidosis conditions and of principal cells under normal acid-base status.


Asunto(s)
Acidosis , Túbulos Renales Colectores , Animales , Proliferación Celular , Ratones , Nefronas , ATPasa Intercambiadora de Sodio-Potasio
4.
Curr Alzheimer Res ; 15(7): 643-654, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29357794

RESUMEN

BACKGROUND: Alzheimer's disease (AD) and age-related macular degeneration (AMD) present similarities, particularly with respect to oxidative stress, including production of 4-Hydroxy-2- nonenal (HNE). AMD has been named the AD in the eye. The Müller cells (MC) function as a principal glia of the retina and maintain water/potassium, glutamate homeostasis and redox status. Any MC dysfunction results in retinal neurodegeneration. OBJECTIVES: We investigated the effects of HNE in human MC. RESULTS: HNE induced an increase of the reactive oxygen species associated with mitochondrial dysfunction and apoptosis. HNE induced endoplasmic reticulum (ER) stress (upregulation of GRP78/Bip, and the proapoptotic factor, CHOP). HNE also impaired expression of genes controlling potassium homeostasis (KCNJ10), glutamate detoxification (GS), and the visual cycle (RLBP1). MC adaptive response to HNE included upregulation of amyloid-ß protein precursor (AßPP). To determine the role of AßPP, we overexpressed AßPP in MC. Overexpression of AßPP induced strong antioxidant and anti-ER stress (PERK downregulation and GADD34 upregulation) responses accompanied by activation of the prosurvival branch of the unfolded protein response. It was also associated with upregulation of major genes involved in MC-controlled retinal homeostasis (KCNJ10, GS, and RLBP1) and protection against HNE-induced apoptosis. Therefore, AßPP is an ER and oxidative stress responsive molecule, and is able to stimulate the transcription of major genes involved in MC functions impaired by HNE. CONCLUSION: Our study suggests that targeting oxidative and ER stress might be a potential therapeutic strategy against glia impairment in AMD and AD, in light of the common features between the two pathologies.


Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Supervivencia Celular/fisiología , Neuroglía/metabolismo , Estrés Oxidativo/fisiología , Transcriptoma , Respuesta de Proteína Desplegada/fisiología , Precursor de Proteína beta-Amiloide/genética , Muerte Celular/fisiología , Línea Celular , Membrana Celular/metabolismo , Chaperón BiP del Retículo Endoplásmico , Humanos , Mitocondrias/metabolismo , Neuroprotección/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética/fisiología
5.
Mol Vis ; 21: 1051-9, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26392743

RESUMEN

PURPOSE: Thinning of the RPE and the underlying vascular layer, the choroid, is observed with age in many human eye disorders. The reasons for this thinning are ill-defined. Here, we highlight the possible role of T lymphocyte recruitment in choroidoretinal thinning in aged and light-challenged mice. METHODS: In age and light challenge models, we measured chemokine concentrations using enzyme-linked immunosorbent assay and used flow cytometry to characterize lymphocyte populations. We quantified thinning in eye immunosections and RPE65 expression using quantitative PCR. RESULTS: Age and light challenge led to increased levels of the lymphotactic protein CXCL10 alone (aging) or in conjunction with CXCL9 (light challenge). Increased numbers of CD3+ T lymphocytes, most of them CD8+ cytotoxic T lymphocytes, were also observed in the choroid and retina of old mice and following light challenge. Influx of T lymphocytes was associated with RPE and choroidal thinning and diminished expression of RPE65 mRNA, an essential enzyme of the visual cycle. CONCLUSIONS: The observations from this study suggest that cytotoxic CD8(+) T lymphocytes might participate in choroidal and RPE degeneration and that modulation of T lymphocyte recruitment might be a novel strategy to reduce choroidoretinal dysfunctions observed with age and following photo-oxidative stress.


Asunto(s)
Envejecimiento/genética , Coroides/efectos de la radiación , ARN Mensajero/genética , Epitelio Pigmentado de la Retina/efectos de la radiación , Linfocitos T Citotóxicos/efectos de la radiación , cis-trans-Isomerasas/genética , Envejecimiento/inmunología , Envejecimiento/patología , Animales , Movimiento Celular/efectos de la radiación , Quimiocina CXCL10/genética , Quimiocina CXCL10/inmunología , Quimiocina CXCL9/genética , Quimiocina CXCL9/inmunología , Coroides/inmunología , Coroides/patología , Regulación de la Expresión Génica , Humanos , Luz/efectos adversos , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Procesos Fotoquímicos , ARN Mensajero/inmunología , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/patología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , cis-trans-Isomerasas/inmunología
6.
J Immunol ; 194(8): 3917-23, 2015 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-25786686

RESUMEN

Three subpopulations of circulating monocytes have been described: CD14(2+)CD16(-) (classical monocytes [CM]), CD14(2+)CD16(+) (intermediate monocytes [IM]), and CD14(+)CD16(2+) (nonclassical monocytes [NCM]). We previously showed that obesity is associated with an increased proportion of IM and NCM. Our objective is to decipher the migratory and inflammatory functions of each monocyte subset in obesity-related low-grade inflammation. Twenty-six healthy, normal-weight and nondiabetic volunteers (C) and 40 obese nondiabetic (Ob) individuals were included in this study. We explored the gene expression profile of 18 inflammatory genes in each subset of C and Ob subjects and measured protein expression of the upregulated genes. We then tested their functional response to TLR signaling in both groups. We showed an increased expression of CX3CR1 in all monocyte subpopulations and of CCR2 and CCR5 in CM and IM in the Ob group. We found negative correlation between CCR2 and CX3CR1 expressions and high-density lipoprotein-cholesterol, whereas CCR5 expression was positively linked to obesity-related metabolic traits. Production of inflammatory proteins upon bacterial LPS and viral ssRNA stimulation was higher in CM and NCM of the Ob group compared with the C group. Our work highlights an enhanced inflammatory phenotype of monocytes with a higher response to TLR4 and TLR8 stimulations in obesity. Moreover, it suggests an increased migration capacity of CM and IM subpopulations.


Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Monocitos/inmunología , Adulto , Receptor 1 de Quimiocinas CX3C , Femenino , Proteínas Ligadas a GPI/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mediadores de Inflamación/inmunología , Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Masculino , Monocitos/patología , Obesidad , Receptores CCR2/inmunología , Receptores CCR5/inmunología , Receptores de Quimiocina/inmunología , Receptores de IgG/inmunología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/inmunología
7.
Angiogenesis ; 15(4): 609-22, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22869002

RESUMEN

Inflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1ß expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1ß expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV.


Asunto(s)
Neovascularización Coroidal/prevención & control , Endotelio Vascular/fisiopatología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Macrófagos Peritoneales/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Secuencia de Bases , Western Blotting , Proteínas de Unión al Calcio , Cartilla de ADN , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Oncoimmunology ; 1(4): 467-530, 2012 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-22754765

RESUMEN

The relatively low frequencies of tumor Ag-specific T-cells in PBMC and metastases from cancer patients have long precluded the analysis of their direct ex vivo cytolytic capacity. Using a new composite technique that works well with low cell numbers, we aimed at determining the functional competence of melanoma-specific CD8(+) T-cells. A multiparameter flow cytometry based technique was applied to assess the cytolytic function, degranulation and IFNγ production by tumor Ag-specific CD8(+) T-cells from PBMC and tumor-infiltrated lymph nodes (TILN) of melanoma patients. We found strong cytotoxicity by T-cells not only when they were isolated from PBMC but also from TILN. Cytotoxicity was observed against peptide-pulsed target cells and melanoma cells presenting the naturally processed endogenous antigen. However, unlike their PBMC-derived counterparts, T-cells from TILN produced only minimal amounts of IFNγ, while exhibiting similar levels of degranulation, revealing a critical functional dichotomy in metastatic lesions. Our finding of partial functional impairment fits well with the current knowledge that T-cells from cancer metastases are so-called exhausted, a state of T-cell hyporesponsiveness also found in chronic viral infections. The identification of responsible mechanisms in the tumor microenvironment is important for improving cancer therapies.

9.
Cancer Immunol Immunother ; 61(6): 817-26, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22080404

RESUMEN

In mice, vaccination with high peptide doses generates higher frequencies of specific CD8+ T cells, but with lower avidity compared to vaccination with lower peptide doses. To investigate the impact of peptide dose on CD8+ T cell responses in humans, melanoma patients were vaccinated with 0.1 or 0.5 mg Melan-A/MART-1 peptide, mixed with CpG 7909 and Incomplete Freund's adjuvant. Neither the kinetics nor the amplitude of the Melan-A-specific CD8+ T cell responses differed between the two vaccination groups. Also, CD8+ T cell differentiation and cytokine production ex vivo were similar in the two groups. Interestingly, after low peptide dose vaccination, Melan-A-specific CD8+ T cells showed enhanced degranulation upon peptide stimulation, as assessed by CD107a upregulation and perforin release ex vivo. In accordance, CD8+ T cell clones derived from low peptide dose-vaccinated patients showed significantly increased degranulation and stronger cytotoxicity. In parallel, Melan-A-specific CD8+ T cells and clones from low peptide dose-vaccinated patients expressed lower CD8 levels, despite similar or even stronger binding to tetramers. Furthermore, CD8+ T cell clones from low peptide dose-vaccinated patients bound CD8 binding-deficient tetramers more efficiently, suggesting that they may express higher affinity TCRs. We conclude that low peptide dose vaccination generated CD8+ T cell responses with stronger cytotoxicity and lower CD8 dependence.


Asunto(s)
Vacunas contra el Cáncer/inmunología , Antígeno MART-1/inmunología , Melanoma/inmunología , Melanoma/terapia , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunación , Vacunas contra el Cáncer/administración & dosificación , Quimioterapia Adyuvante , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Femenino , Antígeno HLA-A2/inmunología , Humanos , Masculino , Melanoma/patología , Estadificación de Neoplasias , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , Péptidos/administración & dosificación
10.
J Clin Invest ; 121(6): 2350-60, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21555851

RESUMEN

In chronic viral infections, CD8⁺ T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8⁺ T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8⁺ T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy.


Asunto(s)
Linfocitos T CD8-positivos/patología , Islas de CpG/inmunología , Perfilación de la Expresión Génica , Antígeno MART-1/inmunología , Melanoma/inmunología , Melanoma/secundario , Subgrupos de Linfocitos T/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/terapia , Datos de Secuencia Molecular , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Especificidad del Receptor de Antígeno de Linfocitos T , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Vacunación , Latencia del Virus/inmunología
11.
J Transl Med ; 9: 44, 2011 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-21507256

RESUMEN

BACKGROUND: The differentiation of CD8+ T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown. METHODS: In order to explore the potential implication of microRNAs in CD8+ T cell differentiation in humans, microRNA expression profiles were analysed using microarrays and quantitative PCR in several human CD8+ T cell subsets defining the major steps of the T cell differentiation pathway. RESULTS: We found expression of a limited set of microRNAs, including the miR-17~92 cluster. Moreover, we reveal the existence of differentiation-associated regulation of specific microRNAs. When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease. CONCLUSIONS: This study establishes for the first time in a large panel of individuals the existence of differentiation associated regulation of microRNA expression in human CD8+ T lymphocytes in vivo, which is likely to impact on specific cellular functions.


Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Regulación de la Expresión Génica , MicroARNs/genética , Humanos , Subgrupos de Linfocitos T
12.
J Immunother ; 33(8): 848-58, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20842051

RESUMEN

Induction of cytotoxic CD8 T-cell responses is enhanced by the exclusive presentation of antigen through dendritic cells, and by innate stimuli, such as toll-like receptor ligands. On the basis of these 2 principles, we designed a vaccine against melanoma. Specifically, we linked the melanoma-specific Melan-A/Mart-1 peptide to virus-like nanoparticles loaded with A-type CpG, a ligand for toll-like receptor 9. Melan-A/Mart-1 peptide was cross-presented, as shown in vitro with human dendritic cells and in HLA-A2 transgenic mice. A phase I/II study in stage II-IV melanoma patients showed that the vaccine was well tolerated, and that 14/22 patients generated ex vivo detectable T-cell responses, with in part multifunctional T cells capable to degranulate and produce IFN-γ, TNF-α, and IL-2. No significant influence of the route of immunization (subcutaneous versus intradermal) nor dosing regimen (weekly versus daily clusters) could be observed. It is interesting to note that, relatively large fractions of responding specific T cells exhibited a central memory phenotype, more than what is achieved by other nonlive vaccines. We conclude that vaccination with CpG loaded virus-like nanoparticles is associated with a human CD8 T-cell response with properties of a potential long-term immune protection from the disease.


Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer , Melanoma/terapia , Neoplasias Cutáneas/terapia , Subgrupos de Linfocitos T/metabolismo , Vacunas de Partículas Similares a Virus , Adulto , Anciano , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Diferenciación Celular , Células Cultivadas , Femenino , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Memoria Inmunológica , Activación de Linfocitos , Antígeno MART-1/química , Antígeno MART-1/inmunología , Masculino , Melanoma/inmunología , Melanoma/patología , Melanoma/fisiopatología , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Nanopartículas/química , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/fisiopatología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Resultado del Tratamiento
13.
Methods Mol Biol ; 626: 231-49, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20099132

RESUMEN

Large numbers and functionally competent T cells are required to protect from diseases for which antibody-based vaccines have consistently failed (1), which is the case for many chronic viral infections and solid tumors. Therefore, therapeutic vaccines aim at the induction of strong antigen-specific T-cell responses. Novel adjuvants have considerably improved the capacity of synthetic vaccines to activate T cells, but more research is necessary to identify optimal compositions of potent vaccine formulations. Consequently, there is a great need to develop accurate methods for the efficient identification of antigen-specific T cells and the assessment of their functional characteristics directly ex vivo. In this regard, hundreds of clinical vaccination trials have been implemented during the last 15 years, and monitoring techniques become more and more standardized.


Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Evaluación Preclínica de Medicamentos/métodos , Activación de Linfocitos/efectos de los fármacos , Linfocitos T CD8-positivos/citología , Proliferación Celular , Células Cultivadas , Humanos
14.
J Immunol ; 183(1): 319-31, 2009 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-19542443

RESUMEN

Protection from reactivation of persistent herpes virus infection is mediated by Ag-specific CD8 T cell responses, which are highly regulated by still poorly understood mechanisms. In this study, we analyzed differentiation and clonotypic dynamics of EBV- and CMV-specific T cells from healthy adults. Although these T lymphocytes included all subsets, from early-differentiated (EM/CD28(pos)) to late-differentiated (EMRA/CD28(neg)) stages, they varied in the sizes/proportions of these subsets. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28(neg) subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, because some clonotypes were selected with differentiation while others were not. Late-differentiated CMV-specific clonotypes were mostly characterized by TCR with lower dependency on CD8 coreceptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of 4 years. Remarkably, clonotypic distribution was stable not only in late-differentiated but also in less-differentiated T cell subsets. Thus, T cell clonotypes segregate with differentiation, but the clonal composition once established is kept constant for at least several years. These findings reveal novel features of the highly sophisticated control of steady state protective T cell activity in healthy adults.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Diferenciación Celular/inmunología , Senescencia Celular/inmunología , Citomegalovirus/inmunología , Epítopos de Linfocito T/inmunología , Herpesvirus Humano 4/inmunología , Adulto , Linfocitos T CD8-positivos/clasificación , Vacunas contra el Cáncer/síntesis química , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Diferenciación Celular/genética , Células Cultivadas , Senescencia Celular/genética , Células Clonales , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/prevención & control , Infecciones por Citomegalovirus/virología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/prevención & control , Infecciones por Virus de Epstein-Barr/virología , Regulación Viral de la Expresión Génica/inmunología , Herpesvirus Humano 4/patogenicidad , Vacunas contra Herpesvirus/síntesis química , Vacunas contra Herpesvirus/genética , Vacunas contra Herpesvirus/inmunología , Humanos , Persona de Mediana Edad , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Factores de Tiempo , Transactivadores/química , Transactivadores/genética , Transactivadores/inmunología , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología
15.
J Immunol ; 182(11): 6718-26, 2009 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-19454666

RESUMEN

The adaptive immune system plays a critical role in protection at the time of secondary infection. It does so through the rapid and robust reactivation of memory T cells which are maintained long-term, in a phenotypically heterogeneous state, following their primary encounter with Ag. Although most HLA-A*0201/influenza matrix protein(58-66)-specific CD8 T cells from healthy donors display characteristics typical of memory T cells, through our extensive phenotypic analysis we have further shown that up to 20% of these cells express neither the IL-7 receptor CD127 nor the costimulatory molecule CD28. In contrast to the majority of CD28(pos) cells, granzyme B and perforin were frequently expressed by the CD28(neg) cells, suggesting that they are effector cells. Indeed, these cells were able to kill target cells, in an Ag-specific manner, directly ex vivo. Thus, our findings demonstrate the remarkable long-term persistence in healthy humans of not only influenza-specific memory cells, but also of effector T cells. We further observed that granzyme B expression in influenza-specific CD8 T cells paralleled levels in the total CD8 T cell population, suggestive of Ag-nonspecific bystander activation. Sequencing of TCR alpha- and beta-chains showed that the TCR repertoire specific for this epitope was dominated by one, or a few, T cell clonotype per healthy donor. Moreover, our sequencing analysis revealed, for the first time in humans, that identical clonotypes can coexist as both memory and effector T cells, thereby supporting the principle of multipotent clonotypic differentiation.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Memoria Inmunológica , Antígenos CD28/análisis , Diferenciación Celular , Células Clonales/inmunología , Granzimas/análisis , Humanos , Inmunofenotipificación , Subunidad alfa del Receptor de Interleucina-7/análisis , Perforina/análisis
16.
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
17.
Immunity ; 30(3): 337-47, 2009 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-19249231

RESUMEN

Natural killer (NK) cells show enhanced functional competence when they express inhibitory receptors specific for inherited major histocompatibility complex class I (MHC-I) molecules. Current models imply that NK cell education requires an interaction of inhibitory receptors with MHC-I expressed on other cells. However, the inhibitory Ly49A receptor can also bind MHC-I ligand on the NK cell itself (in cis). Here we describe a Ly49A variant, which can engage MHC-I expressed on other cells but not in cis. Even though this variant inhibited NK cell effector function, it failed to educate NK cells. The association with MHC-I in cis sequestered wild-type Ly49A, and this was found to relieve NK cells from a suppressive effect of unengaged Ly49A. These data explain how inhibitory MHC-I receptors can facilitate NK cell activation. They dissociate classical inhibitory from educating functions of Ly49A and suggest that cis interaction of Ly49A is necessary for NK cell education.


Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Citometría de Flujo , Variación Genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Subfamilia A de Receptores Similares a Lectina de Células NK/genética
18.
Proc Natl Acad Sci U S A ; 105(10): 3849-54, 2008 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-18319339

RESUMEN

Human cancer vaccines are often prepared with altered "analog" or "heteroclitic" antigens that have been optimized for HLA class I binding, resulting in enhanced immunogenicity. Here, we take advantage of CpG oligodeoxynucleotides as powerful vaccine adjuvants and demonstrate the induction of high T cell frequencies in melanoma patients, despite the use of natural (unmodified) tumor antigenic peptide. Compared with vaccination with analog peptide, natural peptide induced T cell frequencies that were approximately twofold lower. However, T cells showed superior tumor reactivity because of (i) increased functional avidity for natural antigen and (ii) enhancement of T cell activation and effector function. Thus, novel vaccine formulations comprising potent immune stimulators may allow to circumvent the need for modified antigens and can induce highly functional T cells with precise antigen specificity.


Asunto(s)
Antígenos de Neoplasias/inmunología , Autoantígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias/inmunología , Secuencia de Aminoácidos , Antígenos de Neoplasias/química , Autoantígenos/química , Vacunas contra el Cáncer/inmunología , Células Clonales , Epítopos/inmunología , Granzimas/metabolismo , Humanos , Interferón gamma/biosíntesis , Activación de Linfocitos/inmunología , Antígeno MART-1 , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/inmunología , Péptidos/química , Péptidos/inmunología , Perforina/metabolismo , Vacunación
19.
J Immunol ; 179(11): 7406-14, 2007 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-18025184

RESUMEN

The differentiation of CD4(+) or CD8(+) T cells following priming of naive cells is central in the establishment of the immune response against pathogens or tumors. However, our understanding of this complex process and the significance of the multiple subsets of differentiation remains controversial. Gene expression profiling has opened new directions of investigation in immunobiology. Nonetheless, the need for substantial amount of biological material often limits its application range. In this study, we have developed procedures to perform microarray analysis on amplified cDNA from low numbers of cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4(+) or CD8(+) T cell differentiation. Surprisingly, our data revealed that while CD4(+) and CD8(+) T cell gene expression programs diverge at early stages of differentiation, they become increasingly similar as cells reach a late differentiation stage. This suggests that functional heterogeneity between Ag experienced CD4(+) and CD8(+) T cells is more likely to be located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Perfilación de la Expresión Génica , Subgrupos de Linfocitos T/inmunología , Timo/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Línea Celular , Linaje de la Célula/inmunología , ADN Complementario/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Timo/citología
20.
J Immunol ; 179(11): 7635-45, 2007 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-18025209

RESUMEN

HLA-A2-restricted cytolytic T cells specific for the immunodominant human tumor Ag Melan-A(MART-1) can kill most HLA-matched melanoma cells, through recognition of two naturally occurring antigenic variants, i.e., Melan-A nonamer AAGIGILTV and decamer EAAGIGILTV peptides. Several previous studies have suggested a high degree of TCR cross-reactivity to the two peptides. In this study, we describe for the first time that some T cell clones are exclusively nonamer specific, because they are not labeled by A2/decamer-tetramers and do not recognize the decamer when presented endogenously. Functional assays with peptides gave misleading results, possibly because decamers were cleaved by exopeptidases. Interestingly, nonapeptide-specific T cell clones were rarely Valpha2.1 positive (only 1 of 19 clones), in contrast to the known strong bias for Valpha2.1-positive TCRs found in decamer-specific clones (59 of 69 clones). Molecular modeling revealed that nonapeptide-specific TCRs formed unfavorable interactions with the decapeptide, whereas decapeptide-specific TCRs productively created a hydrogen bond between CDR1alpha and glutamic acid (E) of the decapeptide. Ex vivo analysis of T cells from melanoma metastases demonstrated that both nonamer and decamer-specific T cells were enriched to substantial frequencies in vivo, and representative clones showed efficient tumor cell recognition and killing. We conclude that the two peptides should be regarded as distinct epitopes when analyzing tumor immunity and developing immunotherapy against melanoma.


Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos Inmunodominantes/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/inmunología , Sitios de Unión , Línea Celular Tumoral , Clonación Molecular , Humanos , Antígeno MART-1 , Modelos Moleculares , Fragmentos de Péptidos/síntesis química , Conformación Proteica , Estructura Secundaria de Proteína , Receptores de Antígenos de Linfocitos T/inmunología
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