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1.
J Exp Clin Cancer Res ; 38(1): 446, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676012

RESUMO

BACKGROUND: NOTCH1 gene mutations in mantle cell lymphoma (MCL) have been described in about 5-10% of cases and are associated with significantly shorter survival rates. The present study aimed to investigate the biological impact of this mutation in MCL and its potential as a therapeutic target. METHODS: Activation of Notch1 signaling upon ligand-stimulation and inhibitory effects of the monoclonal anti-Notch1 antibody OMP-52M51 in NOTCH1-mutated and -unmutated MCL cells were assessed by Western Blot and gene expression profiling. Effects of OMP-52M51 treatment on tumor cell migration and tumor angiogenesis were evaluated with chemotaxis and HUVEC tube formation assays. The expression of Delta-like ligand 4 (DLL4) in MCL lymph nodes was analyzed by immunofluorescence staining and confocal microscopy. A MCL mouse model was used to assess the activity of OMP-52M51 in vivo. RESULTS: Notch1 expression can be effectively stimulated in NOTCH1-mutated Mino cells by DLL4, whereas in the NOTCH1-unmutated cell line JeKo-1, less effect was observed upon any ligand-stimulation. DLL4 was expressed by histiocytes in both, NOTCH1-mutated and -unmutated MCL lymph nodes. Treatment of NOTCH1-mutated MCL cells with the monoclonal anti-Notch1 antibody OMP-52M51 effectively prevented DLL4-dependent activation of Notch1 and suppressed the induction of numerous direct Notch target genes involved in lymphoid biology, lymphomagenesis and disease progression. Importantly, in lymph nodes from primary MCL cases with NOTCH1/2 mutations, we detected an upregulation of the same gene sets as observed in DLL4-stimulated Mino cells. Furthermore, DLL4 stimulation of NOTCH1-mutated Mino cells enhanced tumor cell migration and angiogenesis, which could be abolished by treatment with OMP-52M51. Importantly, the effects observed were specific for NOTCH1-mutated cells as they did not occur in the NOTCH1-wt cell line JeKo-1. Finally, we confirmed the potential activity of OMP-52M51 to inhibit DLL4-induced Notch1-Signaling in vivo in a xenograft mouse model of MCL. CONCLUSION: DLL4 effectively stimulates Notch1 signaling in NOTCH1-mutated MCL and is expressed by the microenvironment in MCL lymph nodes. Our results indicate that specific inhibition of the Notch1-ligand-receptor interaction might provide a therapeutic alternative for a subset of MCL patients.

2.
Oncogene ; 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31616059

RESUMO

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.

3.
Blood ; 134(24): 2171-2182, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31530562

RESUMO

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the dysregulation of signaling pathways that control intrathymic T-cell development. Relapse rates are still significant, and prognosis is particularly bleak for relapsed patients. Therefore, development of novel therapies specifically targeting pathways controlling leukemia-initiating cell (LIC) activity is mandatory for fighting refractory T-ALL. The interleukin-7 receptor (IL-7R) is a crucial T-cell developmental pathway that is commonly expressed in T-ALL and has been implicated in leukemia progression; however, the significance of IL-7R/IL-7 signaling in T-ALL pathogenesis and its contribution to disease relapse remain unknown. To directly explore whether IL-7R targeting may be therapeutically efficient against T-ALL relapse, we focused on a known Notch1-induced T-ALL model, because a majority of T-ALL patients harbor activating mutations in NOTCH1, which is a transcriptional regulator of IL-7R expression. Using loss-of-function approaches, we show that Il7r-deficient, but not wild-type, mouse hematopoietic progenitors transduced with constitutively active Notch1 failed to generate leukemia upon transplantation into immunodeficient mice, thus providing formal evidence that IL-7R function is essential for Notch1-induced T-cell leukemogenesis. Moreover, we demonstrate that IL-7R expression is an early functional biomarker of T-ALL cells with LIC potential and report that impaired IL-7R signaling hampers engraftment and progression of patient-derived T-ALL xenografts. Notably, we show that IL-7R-dependent LIC activity and leukemia progression can be extended to human B-cell acute lymphoblastic leukemia (B-ALL). These results have important therapeutic implications, highlighting the relevance that targeting normal IL-7R signaling may have in future therapeutic interventions, particularly for preventing T-ALL (and B-ALL) relapse.

4.
Development ; 145(16)2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30042180

RESUMO

T-cell development is a complex dynamic process that relies on ordered stromal signals delivered to thymus-seeding progenitors that migrate throughout different thymus microenvironments (TMEs). Particularly, Notch signaling provided by thymic epithelial cells (TECs) is crucial for T-cell fate specification and generation of mature T cells. Four canonical Notch ligands (Dll1, Dll4, Jag1 and Jag2) are expressed in the thymus, but their spatial distribution in functional TMEs is largely unknown, especially in humans, and their impact on Notch1 activation during T-lymphopoiesis remains undefined. Based on immunohistochemistry and quantitative confocal microscopy of fetal, postnatal and adult human and mouse thymus samples, we show that spatial regulation of Notch ligand expression defines discrete Notch signaling niches and dynamic species-specific TMEs. We further show that Notch ligand expression, particularly DLL4, is tightly regulated in cortical TECs during human thymus ontogeny and involution. Also, we provide the first evidence that NOTCH1 activation is induced in vivo in CD34+ progenitors and developing thymocytes at particular cortical niches of the human fetal and postnatal thymus. Collectively, our results show that human thymopoiesis involves complex spatiotemporal regulation of Notch ligand expression, which ensures the coordinated delivery of niche-specific NOTCH1 signals required for dynamic T-cell development.


Assuntos
Receptor Notch1/metabolismo , Timo/crescimento & desenvolvimento , Timo/metabolismo , Adolescente , Adulto , Envelhecimento/metabolismo , Animais , Antígenos CD34/metabolismo , Criança , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lactente , Recém-Nascido , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Organogênese , Proteínas Serrate-Jagged/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Timo/citologia , Timo/embriologia
5.
J Clin Invest ; 128(7): 2802-2818, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29781813

RESUMO

NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T-ALL), but crucial NOTCH1 downstream signals and target genes contributing to T-ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia-initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T-ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T-ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T-ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T-ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T-ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T-ALL pathogenesis. The observed therapeutic benefit of anti-CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T-ALL relapse.


Assuntos
Receptores de Hialuronatos/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiologia , Receptor Notch1/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Transplante de Células-Tronco Hematopoéticas , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mutação , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/genética , Transdução de Sinais
6.
Cancer Cell ; 32(5): 608-623.e9, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-29136506

RESUMO

Rho guanine exchange factors (GEFs), the enzymes that stimulate Rho GTPases, are deemed as potential therapeutic targets owing to their protumorigenic functions. However, the understanding of the spectrum of their pathobiological roles in tumors is still very limited. We report here that the GEF Vav1 unexpectedly possesses tumor-suppressor functions in immature T cells. This function entails the noncatalytic nucleation of complexes between the ubiquitin ligase Cbl-b and the intracellular domain of Notch1 (ICN1) that favors ICN1 ubiquitinylation and degradation. Ablation of Vav1 promotes ICN1 signaling and the development of T cell acute lymphoblastic leukemia (T-ALL). The downregulation of Vav1 is essential for the pathogenesis of human T-ALL of the TLX+ clinical subtype, further underscoring the suppressor role of this pathway.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas c-vav/genética , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Western Blotting , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Células Jurkat , Camundongos Knockout , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais/genética , Linfócitos T/patologia , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo
7.
J Exp Med ; 214(11): 3361-3379, 2017 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-28947612

RESUMO

A key unsolved question regarding the developmental origin of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) resident in the steady-state thymus is whether early thymic progenitors (ETPs) could escape T cell fate constraints imposed normally by a Notch-inductive microenvironment and undergo DC development. By modeling DC generation in bulk and clonal cultures, we show here that Jagged1 (JAG1)-mediated Notch signaling allows human ETPs to undertake a myeloid transcriptional program, resulting in GATA2-dependent generation of CD34+ CD123+ progenitors with restricted pDC, cDC, and monocyte potential, whereas Delta-like1 signaling down-regulates GATA2 and impairs myeloid development. Progressive commitment to the DC lineage also occurs intrathymically, as myeloid-primed CD123+ monocyte/DC and common DC progenitors, equivalent to those previously identified in the bone marrow, are resident in the normal human thymus. The identification of a discrete JAG1+ thymic medullary niche enriched for DC-lineage cells expressing Notch receptors further validates the human thymus as a DC-poietic organ, which provides selective microenvironments permissive for DC development.


Assuntos
Células Dendríticas/metabolismo , Proteína Jagged-1/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Nicho de Células-Tronco , Timo/metabolismo , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Proteína Jagged-1/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Monócitos/citologia , Monócitos/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Receptores Notch/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Timo/citologia
8.
Mol Cell Biol ; 37(9)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28167605

RESUMO

Thymus-derived regulatory T (tTreg) cells are key to preventing autoimmune diseases, but the mechanisms involved in their development remain unsolved. Here, we show that the C-type lectin receptor CD69 controls tTreg cell development and peripheral Treg cell homeostasis through the regulation of BIC/microRNA 155 (miR-155) and its target, suppressor of cytokine signaling 1 (SOCS-1). Using Foxp3-mRFP/cd69+/- or Foxp3-mRFP/cd69-/- reporter mice and short hairpin RNA (shRNA)-mediated silencing and miR-155 transfection approaches, we found that CD69 deficiency impaired the signal transducer and activator of transcription 5 (STAT5) pathway in Foxp3+ cells. This results in BIC/miR-155 inhibition, increased SOCS-1 expression, and severely impaired tTreg cell development in embryos, adults, and Rag2-/- γc-/- hematopoietic chimeras reconstituted with cd69-/- stem cells. Accordingly, mirn155-/- mice have an impaired development of CD69+ tTreg cells and overexpression of the miR-155-induced CD69 pathway, suggesting that both molecules might be concomitantly activated in a positive-feedback loop. Moreover, in vitro-inducible CD25+ Treg (iTreg) cell development is inhibited in Il2rγ-/-/cd69-/- mice. Our data highlight the contribution of CD69 as a nonredundant key regulator of BIC/miR-155-dependent Treg cell development and homeostasis.


Assuntos
Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Lectinas Tipo C/genética , MicroRNAs/genética , Fator de Transcrição STAT5/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/biossíntese , Linfócitos T Reguladores/citologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Quimera/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/biossíntese , Técnicas de Cultura de Órgãos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Linfócitos T Reguladores/imunologia , Timo/citologia
9.
10.
Sci Rep ; 6: 20223, 2016 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-26838552

RESUMO

Evidence links aryl hydrocarbon receptor (AHR) activation to rheumatoid arthritis (RA) pathogenesis, although results are inconsistent. AHR agonists inhibit pro-inflammatory cytokine expression in macrophages, pivotal cells in RA aetiopathogenesis, which hints at specific circuits that regulate the AHR pathway in RA macrophages. We compared microRNA (miR) expression in CD14(+) cells from patients with active RA or with osteoarthritis (OA). Seven miR were downregulated and one (miR-223) upregulated in RA compared to OA cells. miR-223 upregulation correlated with reduced Notch3 and Notch effector expression in RA patients. Overexpression of the Notch-induced repressor HEY-1 and co-culture of healthy donor monocytes with Notch ligand-expressing cells showed direct Notch-mediated downregulation of miR-223. Bioinformatics predicted the AHR regulator ARNT (AHR nuclear translocator) as a miR-223 target. Pre-miR-223 overexpression silenced ARNT 3'UTR-driven reporter expression, reduced ARNT (but not AHR) protein levels and prevented AHR/ARNT-mediated inhibition of pro-inflammatory cytokine expression. miR-223 counteracted AHR/ARNT-induced Notch3 upregulation in monocytes. Levels of ARNT and of CYP1B1, an AHR/ARNT signalling effector, were reduced in RA compared to OA synovial tissue, which correlated with miR-223 levels. Our results associate Notch signalling to miR-223 downregulation in RA macrophages, and identify miR-223 as a negative regulator of the AHR/ARNT pathway through ARNT targeting.


Assuntos
Artrite Reumatoide/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Citocinas/metabolismo , Macrófagos/metabolismo , MicroRNAs/genética , Receptores Notch/genética , Idoso , Artrite Reumatoide/patologia , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Técnicas de Cocultura , Citocinas/genética , Feminino , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/genética , Osteoartrite/patologia , Transdução de Sinais
12.
Nat Cell Biol ; 17(3): 241-50, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25706235

RESUMO

Epithelial organs develop through tightly coordinated events of cell proliferation and differentiation in which endocytosis plays a major role. Despite recent advances, how endocytosis regulates the development of vertebrate organs is still unknown. Here we describe a mechanism that facilitates the apical availability of endosomal SNARE receptors for epithelial morphogenesis through the developmental upregulation of plasmolipin (pllp) in a highly endocytic segment of the zebrafish posterior midgut. The protein PLLP (Pllp in fish) recruits the clathrin adaptor EpsinR to sort the SNARE machinery of the endolysosomal pathway into the subapical compartment, which is a switch for polarized endocytosis. Furthermore, PLLP expression induces apical Crumbs internalization and the activation of the Notch signalling pathway, both crucial steps in the acquisition of cell polarity and differentiation of epithelial cells. We thus postulate that differential apical endosomal SNARE sorting is a mechanism that regulates epithelial patterning.


Assuntos
Endossomos/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Lisossomos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Polaridade Celular , Proliferação de Células , Embrião não Mamífero , Endocitose , Endossomos/ultraestrutura , Células Epiteliais/ultraestrutura , Epitélio/ultraestrutura , Túbulos Renais/metabolismo , Túbulos Renais/ultraestrutura , Lisossomos/ultraestrutura , Camundongos , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transdução de Sinais , Peixe-Zebra
13.
Nucleic Acids Res ; 43(2): 760-74, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25539926

RESUMO

Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis.


Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Linfócitos T/imunologia , Transcrição Genética , Diferenciação Celular/genética , Expressão Gênica , Humanos , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timo/citologia , Timo/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
J Immunol ; 193(10): 5181-9, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25326025

RESUMO

Intravenous Igs (IVIg) therapy is widely used as an immunomodulatory strategy in inflammatory pathologies and is suggested to promote cancer regression. Because progression of tumors depends on their ability to redirect the polarization state of tumor-associated macrophages (from M1/immunogenic/proinflammatory to M2/anti-inflammatory), we have evaluated whether IVIg limits tumor progression and dissemination through modulation of macrophage polarization. In vitro, IVIg inhibited proinflammatory cytokine production from M1 macrophages and induced a M2-to-M1 polarization switch on human and murine M2 macrophages. In vivo, IVIg modified the polarization of tumor-associated myeloid cells in a Fcεr1γ chain-dependent manner, modulated cytokine blood levels in tumor-bearing animals, and impaired tumor progression via FcγRIII (CD16), FcγRIV, and FcRγ engagement, the latter two effects being macrophage mediated. Therefore, IVIg immunomodulatory activity is dependent on the polarization state of the responding macrophages, and its ability to trigger a M2-to-M1 macrophage polarization switch might be therapeutically useful in cancer, in which proinflammatory or immunogenic functions should be promoted.


Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Imunoglobulinas Intravenosas/farmacologia , Fatores Imunológicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Macrófagos/classificação , Macrófagos/imunologia , Macrófagos/patologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Transplante de Neoplasias , Receptores de IgE/genética , Receptores de IgE/imunologia , Receptores de IgG/genética , Receptores de IgG/imunologia , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos
15.
J Autoimmun ; 55: 51-62, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24934597

RESUMO

Although FoxP3(+) regulatory T cells are key players in the maintenance of immune tolerance and autoimmunity, the lack of specific markers constitute an obstacle to their use for immunotherapy protocols. In this study, we have investigated the role of the C-type lectin receptor CD69 in the suppressor function of Tregs and maintenance of immune tolerance towards harmless inhaled antigens. We identified a novel FoxP3(+)CD69(+) Treg subset capable to maintain immune tolerance and protect to developing inflammation. Although CD69(+) and CD69(-)FoxP3(+) Tregs exist in homeostasis, only CD69-expressing Tregs express high levels of CTLA-4, ICOS, CD38 and GITR suppression-associated markers, secrete high amounts of TGFß and have potent suppressor activity. This activity is regulated by STAT5 and ERK signaling pathways and is impaired by antibody-mediated down-regulation of CD69 expression. Moreover, immunotherapy with FoxP3(+)CD69(+) Tregs restores the homeostasis in Cd69(-/-) mice, that fail to induce tolerance, and is also highly proficient in the prevention of inflammation. The identification of the FoxP3(+)CD69(+) Treg subset paves the way toward the development of new therapeutic strategies to control immune homeostasis and autoimmunity.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Regulação da Expressão Gênica/imunologia , Tolerância Imunológica/fisiologia , Lectinas Tipo C/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD/genética , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Regulação da Expressão Gênica/genética , Lectinas Tipo C/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Linfócitos T Reguladores/citologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia
16.
Mol Ther Methods Clin Dev ; 1: 14021, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-26015965

RESUMO

Chimeric antigen receptor technology offers a highly effective means for increasing the anti-tumor effects of autologous adoptive T-cell immunotherapy, and could be made widely available if adapted to the use of allogeneic T-cells. Although gene-editing technology can be used to remove the alloreactive potential of third party T-cells through destruction of either the α or ß T-cell receptor (TCR) subunit genes, this approach results in the associated loss of surface expression of the CD3 complex. This is nonetheless problematic as it results in the lack of an important trophic signal normally mediated by the CD3 complex at the cell surface, potentially compromising T-cell survival in vivo, and eliminating the potential to expand TCR-knockout cells using stimulatory anti-CD3 antibodies. Here, we show that pre-TCRα, a TCRα surrogate that pairs with TCRß chains to signal proper TCRß folding during T-cell development, can be expressed in TCRα knockout mature T-cells to support CD3 expression at the cell surface. Cells expressing pre-TCR/CD3 complexes can be activated and expanded using standard CD3/CD28 T-cell activation protocols. Thus, heterologous expression of pre-TCRα represents a promising technology for use in the manufacturing of TCR-deficient T-cells for adoptive immunotherapy applications.

17.
Curr Top Microbiol Immunol ; 360: 47-73, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22695916

RESUMO

Notch receptors are master regulators of many aspects of development and tissue renewal in metazoans. Notch1 activation is essential for T-cell specification of bone marrow-derived multipotent progenitors that seed the thymus, and for proliferation and further progression of early thymocytes along the T-cell lineage. Deregulated activation of Notch1 significantly contributes to the generation of T-cell acute lymphoblastic leukaemia (T-ALL). In addition to Notch1 signals, survival and proliferation signals provided by the IL-7 receptor (IL-7R) are also required during thymopoiesis. Our understanding of the molecular mechanisms controlling stage-specific survival and proliferation signals provided by Notch1 and IL-7R has recently been improved by the discovery that the IL-7R is a transcriptional target of Notch1. Thus, Notch1 controls T-cell development, in part by regulating the stage- and lineage-specific expression of IL-7R. The finding that induction of IL-7R expression downstream of Notch1 also occurs in T-ALL highlights the important contribution that deregulated IL-7R expression and function may have in this pathology. Confirming this notion, oncogenic IL7R gain-of-function mutations have recently been identified in childhood T-ALL. Here we discuss the fundamental role of Notch1 and IL-7R signalling pathways in physiological and pathological T-cell development in mice and men, highlighting their close molecular underpinnings.


Assuntos
Células-Tronco Multipotentes/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/metabolismo , Receptores de Interleucina-7/metabolismo , Linfócitos T/metabolismo , Animais , Medula Óssea/metabolismo , Diferenciação Celular , Linhagem da Célula , Regulação da Expressão Gênica , Humanos , Camundongos , Células-Tronco Multipotentes/citologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/genética , Receptores de Interleucina-7/genética , Transdução de Sinais , Linfócitos T/citologia
18.
Inmunología (1987) ; 31(2): 49-57, abr.-jun. 2012. ilus
Artigo em Inglês | IBECS | ID: ibc-108927

RESUMO

The immune system is probably one of the most complex cellular organizations in the body. Its complexity is not superfluous, but rather it is required to fulfill the complicated purpose of the immune system, namely: the recognition of the diverse repertoire of microorganisms and pathogens; the detection of neoplastic lesions originating from a range of tissues; and, while executing these tasks, the maintenance of peripheral tolerance by suppressing detrimental responses against healthy tissues. Since they were discovered by R. Steinman et al. nearly 40 years ago, dendritic cells (DCs) have emerged to be critical players in conducting the immune response to fulfill these roles. Here, we provide a general view on some aspects of DC immunology, highlighting the crucial role that R. Steinman’s research in the DC field has played during all those years. This review will also give an outline on DC research in the particular aspects that represent the focus of research groups in Spain(recently organized as the DC.esp working group within SEI). Firstly, some of the subtypes of DC will be described, particularly thymic DC and their role on tolerance; then the DC role intolerance will be examined, followed by their implications in viral infections. Finally, antigentargeting DCs will be reviewed taking into account the crucial contributions made by R. Steinman et al. This chapter will end by reviewing some DCs based therapies in viral infections (AU)


El sistema inmune es probablemente una de las estructuras más complejas del cuerpo. Esta complejidad no es superflua, sino que es necesaria para realizar todas las complicadas tareas a las que se enfrenta, tales como el reconocimiento de un amplísimo espectro demicroorganismos y patógenos, la detección de lesiones oncogénicas en un amplio rango de tejidos y, mientras ejecuta estas tareas, el mantenimiento de la tolerancia periférica mediante la supresión de respuestas perjudiciales dirigidas a tejidos sanos. Desde que fueron descubiertas por R. Steinman y colaboradores hace casi cuarenta años, las células dendríticas (CD) se han posicionado como elementos clave que dirigen el sistema inmune para acometer las tareas propias del mismo. En esta revisión, mostraremos una visión general sobre algunos aspectos de la inmunología de las CD así como subrayaremos el papel primordial que la investigación de R. Steinman ejerció en el campo de las CD durante todos estos años. Además, esta revisión da unas pinceladas sobre la investigación en CD que abordan la mayoría de los grupos de investigación en este campo en España (recientemente organizados en un grupo de trabajo llamado DC.esp, dentro de la SEI). En primer lugar, se describen la mayoría de subtipos de CD, y en concreto las CD tímicas y su papel en tolerancia, para después hablar de la tolerancia y las CD, seguido de los descubrimientos sobre las implicaciones de las CD en infecciones virales. Finalmente, se revisará el direccionamiento de antígenos a CD, teniendo en cuenta las importantes contribuciones de R. Steinman y colaboradores en el campo. Este capítulo finalizará con una revisión sobre las terapias basadas en CD en infecciones virales (AU)


Assuntos
Humanos , Células Dendríticas/imunologia , Imunoterapia/métodos , Imunoterapia/tendências
19.
Nat Genet ; 43(10): 932-9, 2011 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-21892159

RESUMO

Interleukin 7 (IL-7) and its receptor, formed by IL-7Rα (encoded by IL7R) and γc, are essential for normal T-cell development and homeostasis. Here we show that IL7R is an oncogene mutated in T-cell acute lymphoblastic leukemia (T-ALL). We find that 9% of individuals with T-ALL have somatic gain-of-function IL7R exon 6 mutations. In most cases, these IL7R mutations introduce an unpaired cysteine in the extracellular juxtamembrane-transmembrane region and promote de novo formation of intermolecular disulfide bonds between mutant IL-7Rα subunits, thereby driving constitutive signaling via JAK1 and independently of IL-7, γc or JAK3. IL7R mutations induce a gene expression profile partially resembling that provoked by IL-7 and are enriched in the T-ALL subgroup comprising TLX3 rearranged and HOXA deregulated cases. Notably, IL7R mutations promote cell transformation and tumor formation. Overall, our findings indicate that IL7R mutational activation is involved in human T-cell leukemogenesis, paving the way for therapeutic targeting of IL-7R-mediated signaling in T-ALL.


Assuntos
Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptores de Interleucina-7/genética , Transdução de Sinais , Animais , Ciclo Celular , Linhagem Celular , Sobrevivência Celular , Criança , Cisteína/genética , Cisteína/metabolismo , Análise Mutacional de DNA , Éxons , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Interleucina-7/genética , Interleucina-7/metabolismo , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Camundongos , Camundongos Knockout , Mutação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Interleucina-7/metabolismo , Análise de Sequência de DNA , Linfócitos T/metabolismo , Transfecção , Células Tumorais Cultivadas
20.
Immunobiology ; 215(9-10): 812-20, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20615577

RESUMO

RUNX proteins are heterodimeric factors that play crucial roles during development and differentiation of cells of the immune system. The RUNX3 transcription factor controls lineage decisions during thymopoiesis and T-cell differentiation, and modulates myeloid cell effector functions. We now report the characterization of the human RUNX3/p33 isoform, generated by splicing out a Runt DNA-binding domain-encoding exon, and whose transcriptional activities differ from those of the prototypic RUNX3/p44 molecule. Unlike RUNX3/p44, RUNX3/p33 is induced upon maturation of monocyte-derived dendritic cells (MDDC), and is unable to transactivate the regulatory regions of the CD11a, CD11c and CD49e integrin genes. Overexpression of RUNX3/p33 in myeloid cell lines led to diminished expression of genes involved in inflammatory responses. Moreover, overexpression of RUNX3/p33 down-modulated the basal level of IL-8 production from immature monocyte-derived dendritic cells (MDDC). Besides, siRNA-mediated knock-down of RUNX3 led to diminished levels of IL-8 RNA in immature MDDC, and modulated the neutrophil-recruiting capacity of myeloid cell line supernatants. Since IL-8 promotes neutrophil chemotaxis and degranulation during inflammatory responses, and exerts mitogenic and angiogenic actions within tumor microenvironment, our results imply that myeloid RUNX3 expression regulates the recruitment of leukocytes towards inflammatory foci and might also contribute to human cancer progression.


Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Células Dendríticas/metabolismo , Interleucina-8/biossíntese , Isoformas de Proteínas/metabolismo , Animais , Células COS , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Quimiotaxia/genética , Quimiotaxia/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Regulação para Baixo , Células HEK293 , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Monócitos/patologia , Mutagênese Sítio-Dirigida , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , RNA Interferente Pequeno/genética , Células U937
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