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1.
Immunity ; 53(2): 335-352.e8, 2020 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-32610077

RESUMEN

Dendritic cells (DCs) are antigen-presenting cells controlling T cell activation. In humans, the diversity, ontogeny, and functional capabilities of DC subsets are not fully understood. Here, we identified circulating CD88-CD1c+CD163+ DCs (called DC3s) as immediate precursors of inflammatory CD88-CD14+CD1c+CD163+FcεRI+ DCs. DC3s develop via a specific pathway activated by GM-CSF, independent of cDC-restricted (CDP) and monocyte-restricted (cMoP) progenitors. Like classical DCs but unlike monocytes, DC3s drove activation of naive T cells. In vitro, DC3s displayed a distinctive ability to prime CD8+ T cells expressing a tissue homing signature and the epithelial homing alpha-E integrin (CD103) through transforming growth factor ß (TGF-ß) signaling. In vivo, DC3s infiltrated luminal breast cancer primary tumors, and DC3 infiltration correlated positively with CD8+CD103+CD69+ tissue-resident memory T cells. Together, these findings define DC3s as a lineage of inflammatory DCs endowed with a strong potential to regulate tumor immunity.


Asunto(s)
Antígenos CD1/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Neoplasias de la Mama/inmunología , Linfocitos T CD8-positivos/citología , Células Dendríticas/inmunología , Glicoproteínas/metabolismo , Cadenas alfa de Integrinas/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Factor de Crecimiento Transformador beta1/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo
2.
Immunity ; 45(6): 1205-1218, 2016 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-28002729

RESUMEN

Inflammation triggers the differentiation of Ly6Chi monocytes into microbicidal macrophages or monocyte-derived dendritic cells (moDCs). Yet, it is unclear whether environmental inflammatory cues control the polarization of monocytes toward each of these fates or whether specialized monocyte progenitor subsets exist before inflammation. Here, we have shown that naive monocytes are phenotypically heterogeneous and contain an NR4A1- and Flt3L-independent, CCR2-dependent, Flt3+CD11c-MHCII+PU.1hi subset. This subset acted as a precursor for FcγRIII+PD-L2+CD209a+, GM-CSF-dependent moDCs but was distal from the DC lineage, as shown by fate-mapping experiments using Zbtb46. By contrast, Flt3-CD11c-MHCII-PU.1lo monocytes differentiated into FcγRIII+PD-L2-CD209a-iNOS+ macrophages upon microbial stimulation. Importantly, Sfpi1 haploinsufficiency genetically distinguished the precursor activities of monocytes toward moDCs or microbicidal macrophages. Indeed, Sfpi1+/- mice had reduced Flt3+CD11c-MHCII+ monocytes and GM-CSF-dependent FcγRIII+PD-L2+CD209a+ moDCs but generated iNOS+ macrophages more efficiently. Therefore, intercellular disparities of PU.1 expression within naive monocytes segregate progenitor activity for inflammatory iNOS+ macrophages or moDCs.


Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Traslado Adoptivo , Animales , Antígenos Ly/inmunología , Separación Celular , Células Dendríticas/citología , Citometría de Flujo , Macrófagos/citología , Ratones , Monocitos/citología , Óxido Nítrico Sintasa de Tipo II/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa
3.
Methods Mol Biol ; 2618: 121-132, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36905513

RESUMEN

Dendritic cells (DCs) are professional antigen-presenting cells controlling the activation of T cells and thus regulating adaptive immune response against pathogens or tumors. Modeling human DC differentiation and function is crucial for our understanding of immune response and the development of new therapies. Considering DC rarity in human blood, in vitro systems allowing their faithful generation are needed. This chapter will describe a DC differentiation method based on the co-culture of CD34+ cord blood progenitors together with mesenchymal stromal cells (eMSCs) engineered to deliver growth factors and chemokines.


Asunto(s)
Células Dendríticas , Sangre Fetal , Humanos , Células Cultivadas , Antígenos CD34/metabolismo , Diferenciación Celular , Moléculas de Adhesión Celular
4.
bioRxiv ; 2023 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-37662317

RESUMEN

During embryogenesis, yolk-sac and intra-embryonic-derived hematopoietic progenitors, comprising the precursors of adult hematopoietic stem cells, converge into the fetal liver. With a new staining strategy, we defined all non-hematopoietic components of the fetal liver and found that hepatoblasts are the major producers of hematopoietic growth factors. We identified mesothelial cells, a novel component of the stromal compartment, producing Kit ligand, a major hematopoietic cytokine. A high-definition imaging dataset analyzed using a deep-learning based pipeline allowed the unambiguous identification of hematopoietic and stromal populations, and enabled determining a neighboring network composition, at the single cell resolution. Throughout active hematopoiesis, progenitors preferentially associate with hepatoblasts, but not with stellate or endothelial cells. We found that, unlike yolk sac-derived progenitors, intra-embryonic progenitors respond to a chemokine gradient created by CXCL12-producing stellate cells. These results revealed that FL hematopoiesis is a spatiotemporal dynamic process, defined by an environment characterized by low cytokine concentrations.

5.
Nat Commun ; 13(1): 773, 2022 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-35140205

RESUMEN

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increase in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells is associated with the formation of tissue-specific sub-TADs over Runx1, a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletion of promoter-proximal CTCF sites at the sub-TAD boundaries has no obvious effects on E-P interactions but leads to partial loss of domain structure, mildly affects gene expression, and delays hematopoietic development. Together, our analysis of gene regulation at a large multi-promoter developmental gene reveals that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.


Asunto(s)
Cromatina/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Expresión Génica , Animales , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , ADN/química , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Mesodermo/metabolismo , Ratones , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas
6.
Front Immunol ; 12: 631279, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33790904

RESUMEN

Tissue engineering opens multiple opportunities in regenerative medicine, drug testing, and modeling of the hematopoiesis in health and disease. Recapitulating the organization of physiological microenvironments supporting leukocyte development is essential to model faithfully the development of immune cells. Hematopoietic organs are shaped by spatially organized niches defined by multiple cellular contributions. A shared feature of immune niches is the presence of mesenchymal stromal cells endowed with unique roles in organizing niche development, maintenance, and function. Here, we review challenges and opportunities in harnessing stromal cells for the engineering of artificial immune niches and hematopoietic organoids recapitulating leukocyte ontogeny both in vitro and in vivo.


Asunto(s)
Células Madre Mesenquimatosas/fisiología , Nicho de Células Madre/fisiología , Células del Estroma/metabolismo , Ingeniería de Tejidos/métodos , Animales , Células de la Médula Ósea/metabolismo , Humanos , Células Madre Mesenquimatosas/inmunología , Ratones , Nicho de Células Madre/genética , Nicho de Células Madre/inmunología , Células del Estroma/inmunología
7.
Cell Rep ; 37(11): 110103, 2021 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-34910918

RESUMEN

Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence. Here we show that in Ncx1-/- mouse embryos devoid of circulation the HSC lineage develops until the phenotypic pro-HSC stage. However, these cells reside in an abnormal microenvironment, fail to activate the hematopoietic program downstream of Runx1, and are functionally impaired. Single-cell transcriptomics shows that during the endothelial-to-hematopoietic transition, Ncx1-/- cells fail to undergo a glycolysis to oxidative phosphorylation metabolic switch present in wild-type cells. Interestingly, experimental activation of glycolysis results in decreased intraembryonic hematopoiesis. Our results suggest that the onset of circulation triggers metabolic changes that allow HSC generation to proceed.


Asunto(s)
Diferenciación Celular , Linaje de la Célula , Endotelio Vascular/patología , Glucólisis , Hematopoyesis , Células Madre Hematopoyéticas/patología , Intercambiador de Sodio-Calcio/fisiología , Animales , Endotelio Vascular/metabolismo , Femenino , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación Oxidativa , Análisis de la Célula Individual , Transcriptoma
8.
Mol Immunol ; 125: 151-161, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32688117

RESUMEN

Dendritic cells (DCs) are sentinel cells of the immune system arising from hematopoietic stem cells. DCs play a key role in the regulation of both adaptive and innate lymphocyte responses. As such, experimental models enabling a thorough analysis of human DCs development and function are needed. Humanized mice models (termed collectively as HIS mice, or human immune system mice models) provide unique opportunities to model human hematopoiesis and tackle the function of human immune cell types in vivo. Here, we review experimental approaches enabling to recapitulate the ontogeny of DC subsets in HIS mice and discuss studies addressing the biology of human DC subsets implementing HIS mice models.


Asunto(s)
Células Dendríticas/inmunología , Modelos Animales , Animales , Humanos , Ratones
9.
Nat Commun ; 11(1): 2054, 2020 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-32345968

RESUMEN

Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo.


Asunto(s)
Células Dendríticas/citología , Nicho de Células Madre , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quimiocina CXCL12/farmacología , Análisis por Conglomerados , Colágeno/farmacología , Células Dendríticas/efectos de los fármacos , Combinación de Medicamentos , Humanos , Laminina/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Organoides/efectos de los fármacos , Organoides/metabolismo , Proteoglicanos/farmacología , Nicho de Células Madre/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo
10.
Oncotarget ; 8(39): 65900-65916, 2017 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-29029481

RESUMEN

The disulfiram and copper complex (DSF:Cu) has emerged as a potent radiosensitising anti-cancer agent. The ability of copper to stabilise DSF in a planar conformation and to inhibit DNA replication enzymes stimulated our investigation of the effect of DSF:Cu on cell cycle regulation. Flow cytometry and immunoblotting were used to assess the effect of DSF:Cu on cell cycle progression of the neuroblastoma cell line SK-N-BE(2c) and the glioma cell line UVW. Treatment with 0.1 and 0.3 µM DSF:Cu inhibited DNA synthesis in SK-N-BE(2c) and UVW cells, respectively. The increased potency of ionising radiation treatment induced by DSF:Cu and/or gemcitabine was determined by clonogenic assay. Treatment with 0.3 µM DSF:Cu resulted in greater radiation kill, exemplified by dose enhancement factor values of 2.64 and 2.84 in SK-N-BE(2c) and UVW cells, respectively. Although DSF:Cu failed to sensitise S phase cells to irradiation, we observed that DSF:Cu radiosensitisation was potentiated by the S phase-specific cytotoxic drug gemcitabine. The efficacy of the combination treatment consisting of DSF:Cu, gemcitabine and ionising radiation was schedule-dependent. Together, these results describe cell cycle specific radiosensitisation by DSF:Cu. The well-established toxicity profiles of DSF and gemcitabine should facilitate their evaluation as a combination treatment in patients undergoing radiotherapy.

11.
Front Immunol ; 6: 363, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26236315

RESUMEN

Dendritic cells (DCs) have the unique ability to pick up dead cells carrying antigens in tissue and migrate to the lymph nodes where they can cross-present cell-associated antigens by MHC class I to CD8(+) T cells. There is strong in vivo evidence that the mouse XCR1(+) DCs subset acts as a key player in this process. The intracellular processes underlying cross-presentation remain controversial and several pathways have been proposed. Indeed, a wide number of studies have addressed the cellular process of cross-presentation in vitro using a variety of sources of antigen and antigen-presenting cells. Here, we review the in vivo and in vitro evidence supporting the current mechanistic models and disscuss their physiological relevance to the cross-presentation of cell-associated antigens by DCs subsets.

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