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1.
PLoS Pathog ; 16(9): e1008870, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991634

RESUMO

The two T-box transcription factors T-bet and Eomesodermin (Eomes) are important regulators of cytotoxic lymphocytes (CTLs), such as activated CD8 T cells, which are essential in the fight against intracellular pathogens and tumors. Both transcription factors share a great degree of homology based on sequence analysis and as a result exert partial functional redundancy during viral infection. However, the actual degree of redundancy between T-bet and Eomes remains a matter of debate and is further confounded by their distinct spatiotemporal expression pattern in activated CD8 T cells. To directly investigate the functional overlap of these transcription factors, we generated a new mouse model in which Eomes expression is under the transcriptional control of the endogenous Tbx21 (encoding for T-bet) locus. Applying this model, we demonstrate that the induction of Eomes in lieu of T-bet cannot rescue T-bet deficiency in CD8 T cells during acute lymphocytic choriomeningitis virus (LCMV) infection. We found that the expression of Eomes instead of T-bet was not sufficient for early cell expansion or effector cell differentiation. Finally, we show that imposed expression of Eomes after acute viral infection promotes some features of exhaustion but must act in concert with other factors during chronic viral infection to establish all hallmarks of exhaustion. In summary, our results clearly underline the importance of T-bet in guiding canonical CTL development during acute viral infections.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/fisiologia , Proteínas com Domínio T/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica/fisiologia , Interferon gama/metabolismo , Camundongos Transgênicos
2.
PLoS Genet ; 16(9): e1009011, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986715

RESUMO

Neuronal precursor cells undergo self-renewing and non-self-renewing asymmetric divisions to generate a large number of neurons of distinct identities. In Drosophila, primary precursor neuroblasts undergo a varying number of self-renewing asymmetric divisions, with one known exception, the MP2 lineage, which undergoes just one terminal asymmetric division similar to the secondary precursor cells. The mechanism and the genes that regulate the transition from self-renewing to non-self-renewing asymmetric division or the number of times a precursor divides is unknown. Here, we show that the T-box transcription factor, Midline (Mid), couples these events. We find that in mid loss of function mutants, MP2 undergoes additional self-renewing asymmetric divisions, the identity of progeny neurons generated dependent upon Numb localization in the parent MP2. MP2 expresses Mid transiently and an over-expression of mid in MP2 can block its division. The mechanism which directs the self-renewing asymmetric division of MP2 in mid involves an upregulation of Cyclin E. Our results indicate that Mid inhibits cyclin E gene expression by binding to a variant Mid-binding site in the cyclin E promoter and represses its expression without entirely abolishing it. Consistent with this, over-expression of cyclin E in MP2 causes its multiple self-renewing asymmetric division. These results reveal a Mid-regulated pathway that restricts the self-renewing asymmetric division potential of cells via inhibiting cyclin E and facilitating their exit from cell cycle.


Assuntos
Divisão Celular/genética , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Ciclo Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Hormônios Juvenis/genética , Hormônios Juvenis/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteínas com Domínio T/genética , Fatores de Transcrição/genética
3.
Nat Commun ; 11(1): 4159, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855415

RESUMO

The periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. At the initiation of tracheal development, endoderm begins to express Nkx2.1, and then mesoderm expresses the Tbx4 gene. Loss of ß-catenin in fetal mouse mesoderm causes loss of Tbx4+ tracheal mesoderm and tracheal cartilage agenesis. The mesenchymal Tbx4 expression relies on endodermal Wnt activation and Wnt ligand secretion but is independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generates tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation is required along with WNT to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair.


Assuntos
Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Traqueia/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Endoderma/citologia , Endoderma/embriologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fator Nuclear 1 de Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo , Traqueia/citologia , Traqueia/embriologia , beta Catenina/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(28): 16409-16417, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601185

RESUMO

The polar trophoblast overlays the epiblast in eutherian mammals and, depending on the species, has one of two different fates. It either remains a single-layered, thinning epithelium called "Rauber's layer," which soon disintegrates, or, alternatively, it keeps proliferating, contributing heavily to the population of differentiating, invasive trophoblast cells and, at least in mice, to the induction of gastrulation. While loss of the persistent polar trophoblast in mice leads to reduced induction of gastrulation, we show here that prevention of the loss of the polar trophoblast in cattle results in ectopic domains of the gastrulation marker, BRACHYURY This phenotype, and increased epiblast proliferation, arose when Rauber's layer was maintained for a day longer by countering apoptosis through BCL2 overexpression. This suggests that the disappearance of Rauber's layer is a necessity, presumably to avoid excessive signaling interactions between this layer and the subjacent epiblast. We note that, in all species in which the polar trophoblast persists, including humans and mice, ectopic polar trophoblast signaling is prevented via epiblast cavitation which leads to the (pro)amniotic cavity, whose function is to distance the central epiblast from such signaling interactions.


Assuntos
Trofoblastos/citologia , Animais , Apoptose , Bovinos , Diferenciação Celular , Proliferação de Células , Feminino , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Gastrulação , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiopatologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Trofoblastos/metabolismo
5.
Nat Commun ; 11(1): 3366, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632165

RESUMO

CD4+ T lymphocytes consist of naïve, antigen-specific memory, and memory-phenotype (MP) cell compartments at homeostasis. We recently showed that MP cells exert innate-like effector function during host defense, but whether MP CD4+ T cells are functionally heterogeneous and, if so, what signals specify the differentiation of MP cell subpopulations under homeostatic conditions is still unclear. Here we characterize MP lymphocytes as consisting of T-bethigh, T-betlow, and T-bet- subsets, with innate, Th1-like effector activity exclusively associated with T-bethigh cells. We further show that the latter population depends on IL-12 produced by CD8α+ type 1 dendritic cells (DC1) for its differentiation. Finally, our data demonstrate that this tonic IL-12 production requires TLR-MyD88 signaling independent of foreign agonists, and is further enhanced by CD40-CD40L interactions between DC1 and CD4+ T lymphocytes. We propose that optimal differentiation of T-bethigh MP lymphocytes at homeostasis is driven by self-recognition signals at both the DC and Tcell levels.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Homeostase/imunologia , Memória Imunológica/imunologia , Proteínas com Domínio T/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Comunicação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-12/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais/imunologia , Proteínas com Domínio T/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(31): 18617-18626, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675240

RESUMO

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3 +/-) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3 +/- mutants. Notably, Tbx3 +/- atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.


Assuntos
Nó Atrioventricular , Proteínas com Domínio T , Transcriptoma/genética , Animais , Arritmias Cardíacas , Nó Atrioventricular/metabolismo , Nó Atrioventricular/fisiologia , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
7.
Am J Respir Crit Care Med ; 202(7): 1013-1023, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32501729

RESUMO

Rationale: A subpopulation of B cells (age-associated B cells [ABCs]) is increased in mice and humans with infections or autoimmune diseases. Because depletion of these cells might be valuable in patients with certain lung diseases, the goal was to find out if ABC-like cells were at elevated levels in such patients.Objectives: To measure ABC-like cell percentages in patients with lung granulomatous diseases.Methods: Peripheral blood and BAL cells from patients with sarcoidosis, beryllium sensitivity, or hypersensitivity pneumonitis and healthy subjects were analyzed for the percentage of B cells that were ABC-like, defined by expression of CD11c, low levels of CD21, FcRL 1-5 (Fc receptor-like protein 1-5) expression, and, in some cases, T-bet.Measurements and Main Results: ABC-like cells in blood were at low percentages in healthy subjects and higher percentages in patients with sarcoidosis as well as at high percentages among BAL cells of patients with sarcoidosis, beryllium disease, and hypersensitivity pneumonitis. Treatment of patients with sarcoidosis led to reduced percentages of ABC-like cells in blood.Conclusions: Increased levels of ABC-like cells in patients with sarcoidosis may be useful in diagnosis. The increase in percentage of ABC-like cells in patients with lung granulomatous diseases and decrease in treated patients suggests that depletion of these cells may be valuable.


Assuntos
Alveolite Alérgica Extrínseca/sangue , Subpopulações de Linfócitos B/metabolismo , Beriliose/sangue , Líquido da Lavagem Broncoalveolar/citologia , Sarcoidose Pulmonar/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Alveolite Alérgica Extrínseca/imunologia , Subpopulações de Linfócitos B/imunologia , Beriliose/imunologia , Antígeno CD11c/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Receptores de Superfície Celular/metabolismo , Receptores de Complemento 3d/metabolismo , Receptores Fc/metabolismo , Receptores Imunológicos/metabolismo , Sarcoidose Pulmonar/imunologia , Proteínas com Domínio T/metabolismo , Adulto Jovem
8.
PLoS One ; 15(5): e0226539, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413046

RESUMO

A murine model to study the effect of cold-induced stress (CIS) on Chlamydia muridarum genital infection and immune response has been developed in our laboratory. Previous results in the lab show that CIS increases the intensity of chlamydia genital infection, but little is known about the effects and mechanisms of CIS on the differentiation and activities of CD4+ T cell subpopulations and bone marrow-derived dendritic cells (BMDCs). The factors that regulate the production of T helper 1 (Th1) or T helper 2 (Th2) cytokines are not well defined. In this study, we examined whether CIS modulates the expressions of beta-adrenergic receptor (ß-AR), transcription factors, hallmark cytokines of Th1 and Th2, and differentiation of BMDCs during C. muridarum genital infection in the murine model. Our results show that the mRNA level of the beta2-adrenergic receptor (ß2-AR) compared to ß1-AR and ß3-AR was high in the mixed populations of CD4+ T cells and BMDCs. Furthermore, we observed decreased expression of T-bet, low level of Interferon-gamma (IFN-γ) production, increased expression of GATA-3, and Interleukin-4 (IL-4) production in CD4+ T cells of stressed mice. Exposure of BMDCs to Fenoterol, ß2-AR agonist, or ICI118,551, ß2-AR antagonist, revealed significant ß2-AR stimulation or inhibition, respectively, in stressed mice. Moreover, co-culturing of mature BMDCs and naïve CD4+ T cells increased the production of IL-4, IL-10, L-17, and IL-23 cytokines, suggesting that stimulation of ß2-AR leads to the increased production of Th2 cytokines. Overall, our results show for the first time that CIS promotes the switching from a Th1 to Th2 cytokine environment. This was evidenced in the murine stress model by the overexpression of GATA-3 concurrent with elevated IL-4 production, reduced T-bet expression, and IFN-γ secretion.


Assuntos
Infecções por Chlamydia/imunologia , Resposta ao Choque Frio , Células Th1/imunologia , Células Th2/imunologia , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Células Cultivadas , Chlamydia muridarum , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Fenoterol/farmacologia , Interferon gama/genética , Interferon gama/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Propanolaminas/farmacologia , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Nat Immunol ; 21(7): 766-776, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32424367

RESUMO

Tissue-resident memory T (TRM) cells, functionally distinct from circulating memory T cells, have a critical role in protective immunity in tissues, are more efficacious when elicited after vaccination and yield more effective antitumor immunity, yet the signals that direct development of TRM cells are incompletely understood. Here we show that type 1 regulatory T (Treg) cells, which express the transcription factor T-bet, promote the generation of CD8+ TRM cells. The absence of T-bet-expressing type 1 Treg cells reduces the presence of TRM cells in multiple tissues and increases pathogen burden upon infectious challenge. Using infection models, we show that type 1 Treg cells are specifically recruited to local inflammatory sites via the chemokine receptor CXCR3. Close proximity with effector CD8+ T cells and Treg cell expression of integrin-ß8 endows the bioavailability of transforming growth factor-ß in the microenvironment, thereby promoting the generation of CD8+ TRM cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Memória Imunológica , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/transplante , Coccidiose/imunologia , Coccidiose/parasitologia , Modelos Animais de Doenças , Eimeria/imunologia , Feminino , Humanos , Cadeias beta de Integrinas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Receptores CXCR3/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/transplante , Fator de Crescimento Transformador beta/metabolismo
10.
Immunity ; 52(5): 726-728, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32433944

RESUMO

Memory B cells (MBCs) expressing the transcription factor T-bet have been described in normal and dysregulated immune responses. In this issue of Immunity, Johnson et al. report that T-bet+ MBCs, formed in response to a primary influenza infection, contribute to protective antibody titers and persist mainly in the spleen with restricted trafficking between tissues.


Assuntos
Subpopulações de Linfócitos B , Animais , Especificidade de Anticorpos , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Humanos , Memória Imunológica , Camundongos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Distribuição Tecidual
11.
Immunity ; 52(5): 842-855.e6, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32353250

RESUMO

B cell subsets expressing the transcription factor T-bet are associated with humoral immune responses and autoimmunity. Here, we examined the anatomic distribution, clonal relationships, and functional properties of T-bet+ and T-bet- memory B cells (MBCs) in the context of the influenza-specific immune response. In mice, both T-bet- and T-bet+ hemagglutinin (HA)-specific B cells arose in germinal centers, acquired memory B cell markers, and persisted indefinitely. Lineage tracing and IgH repertoire analyses revealed minimal interconversion between T-bet- and T-bet+ MBCs, and parabionts showed differential tissue residency and recirculation properties. T-bet+ MBCs could be subdivided into recirculating T-betlo MBCs and spleen-resident T-bethi MBCs. Human MBCs displayed similar features. Conditional gene deletion studies revealed that T-bet expression in B cells was required for nearly all HA stalk-specific IgG2c antibodies and for durable neutralizing titers to influenza. Thus, T-bet expression distinguishes MBC subsets that have profoundly different homing, residency, and functional properties, and mediate distinct aspects of humoral immune memory.


Assuntos
Especificidade de Anticorpos/imunologia , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Memória Imunológica/imunologia , Especificidade de Órgãos/imunologia , Proteínas com Domínio T/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Anticorpos Anti-HIV/imunologia , Humanos , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
12.
PLoS Biol ; 18(4): e3000696, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275659

RESUMO

It is well known that various developmental signals play diverse roles in hematopoietic stem and progenitor cell (HSPC) production; however, how these signaling pathways are orchestrated remains incompletely understood. Here, we report that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos. Rab5c deficiency leads to defects in HSPC production. Mechanistically, Rab5c regulates hemogenic endothelium (HE) specification by endocytic trafficking of Notch ligands and receptor. We further show that the interaction between Rab5c and Appl1 in the endosome is required for the survival of HE in the ventral wall of the dorsal aorta through AKT signaling. Interestingly, Rab5c overactivation can also lead to defects in HSPC production, which is attributed to excessive endolysosomal trafficking inducing Notch signaling defect. Taken together, our findings establish a previously unrecognized role of Rab5c-mediated endocytic trafficking in HSPC development and provide new insights into how spatiotemporal signals are orchestrated to accurately execute cell fate transition.


Assuntos
Células-Tronco Hematopoéticas/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Notch/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Endocitose , Endotélio/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Receptores Notch/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/genética
13.
Nat Immunol ; 21(5): 567-577, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284593

RESUMO

Unprimed mice harbor a substantial population of 'memory-phenotype' CD8+ T cells (CD8-MP cells) that exhibit hallmarks of activation and innate-like functional properties. Due to the lack of faithful markers to distinguish CD8-MP cells from bona fide CD8+ memory T cells, the developmental origins and antigen specificities of CD8-MP cells remain incompletely defined. Using deep T cell antigen receptor (TCR) sequencing, we found that the TCRs expressed by CD8-MP cells are highly recurrent and distinct from the TCRs expressed by naive-phenotype CD8+ T cells. CD8-MP clones exhibited reactivity to widely expressed self-ligands. T cell precursors expressing CD8-MP TCRs showed upregulation of the transcription factor Eomes during maturation in the thymus, prior to induction of the full memory phenotype, which is suggestive of a unique program triggered by recognition of self-ligands. Moreover, CD8-MP cells infiltrate oncogene-driven prostate tumors and express high densities of PD-1, which suggests potential roles in antitumor immunity and the response to immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias da Próstata/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas com Domínio T/metabolismo , Timo/fisiologia , Animais , Autoantígenos/imunologia , Diferenciação Celular , Seleção Clonal Mediada por Antígeno , Células Clonais , Memória Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/metabolismo , Proteínas com Domínio T/genética , Regulação para Cima
14.
J Pathol ; 251(1): 87-99, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32154590

RESUMO

The oncogene brachyury (TBXT) is a T-box transcription factor that is overexpressed in multiple solid tumors and is associated with tumor aggressiveness and poor patient prognosis. Gliomas comprise the most common and aggressive group of brain tumors, and at the present time the functional and clinical impact of brachyury expression has not been investigated previously in these neoplasms. Brachyury expression (mRNA and protein) was assessed in normal brain (n = 67), glioma tissues (n = 716) and cell lines (n = 42), and further in silico studies were undertaken using genomic databases totaling 3115 samples. Our glioma samples were analyzed for copy number (n = 372), promoter methylation status (n = 170), and mutation status (n = 1569 tissues and n = 52 cell lines) of the brachyury gene. The prognostic impact of brachyury expression was studied in 1524 glioma patient tumors. The functional impact of brachyury on glioma proliferation, viability, and cell death was evaluated both in vitro and in vivo. Brachyury was expressed in the normal brain, and significantly downregulated in glioma tissues. Loss of brachyury was associated with tumor aggressiveness and poor survival in glioma patients. Downregulation of brachyury was not associated with gene deletion, promoter methylation, or inactivating point mutations. Brachyury re-expression in glioma cells was found to decrease glioma tumorigenesis by induction of autophagy. These data strongly suggest that brachyury behaves as a tumor suppressor gene in gliomas by modulating autophagy. It is important to note that brachyury constitutes an independent positive biomarker of patient prognosis. Our findings indicate that the role of brachyury in tumorigenesis may be tissue-dependent and demands additional investigation to guide rational interventions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Neoplasias Encefálicas/patologia , Proteínas Fetais/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Proteínas com Domínio T/metabolismo , Animais , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proteínas Fetais/genética , Genes Supressores de Tumor/fisiologia , Glioma/patologia , Humanos , Camundongos , Prognóstico , Regiões Promotoras Genéticas , Proteínas com Domínio T/genética , Fatores de Transcrição/metabolismo
15.
Development ; 147(3)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32014863

RESUMO

Cardiopharyngeal mesoderm (CPM) gives rise to muscles of the head and heart. Using genetic lineage analysis in mice, we show that CPM develops into a broad range of pharyngeal structures and cell types encompassing musculoskeletal and connective tissues. We demonstrate that CPM contributes to medial pharyngeal skeletal and connective tissues associated with both branchiomeric and somite-derived neck muscles. CPM and neural crest cells (NCC) make complementary mediolateral contributions to pharyngeal structures, in a distribution established in the early embryo. We further show that biallelic expression of the CPM regulatory gene Tbx1, haploinsufficient in 22q11.2 deletion syndrome patients, is required for the correct patterning of muscles with CPM-derived connective tissue. Our results suggest that CPM plays a patterning role during muscle development, similar to that of NCC during craniofacial myogenesis. The broad lineage contributions of CPM to pharyngeal structures provide new insights into congenital disorders and evolution of the mammalian pharynx.


Assuntos
Tecido Conjuntivo/embriologia , Desenvolvimento Muscular/genética , Faringe/embriologia , Somitos/fisiologia , Animais , Padronização Corporal/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Crista Neural/metabolismo , Faringe/citologia , Somitos/citologia , Proteínas com Domínio T/metabolismo
16.
Malar J ; 19(1): 62, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32033605

RESUMO

BACKGROUND: Malaria is a worldwide problem that affects millions of people yearly. In rural areas where anti-malarial drugs are not easily accessible, many people use herbal treatments, such as Moringa oleifera, to treat a variety of diseases and ailments including malaria. While Moringa is reported to possess potent and curative anti-malarial properties, previous studies have mostly been restricted to assessment of parasitaemia. In this study, the effect of Moringa on malaria immunity in a murine model was investigated. METHODS: Using a high dose (60 mg/mouse) for a short time (7 days) or low dose Moringa (30 mg/mouse) for a longer time (3 weeks), cytokine production, and Tbet expression by effector CD4+ T cells (Teff) were determined. Mice were also treated with Moringa after infection (curatively) or before infection (prophylactically) to determine the effect of the plant extract on parasitaemia and immunity. Given that Moringa also possess many nutritional benefits, the contribution of Moringa on malnourished malaria infected mice was determined. Malnutrition was induced by limiting access to food to only 4 h a day for 4 weeks, while control mice had unlimited access to mouse laboratory chow. All data was collected by flow cytometry and analysed using one-Way ANOVA or two tailed Student's t test. RESULTS: Moringa-treated mice had increased numbers of effector CD4+ T cells accompanied by an increase in Tbet expression compared to control untreated mice. Mice that were treated with Moringa curatively also exhibited increased effector CD4+ T cell numbers, IFN-gamma and TNF secretion. Interestingly, the mice that were treated prophylactically had significantly higher Tbet expression. In the absence of adaptive immunity, high parasitaemia was observed in the RAG1 knockout mice. The food limited mice (malnourished) had reduced numbers of CD4+ T cells, TNF proportions, and significantly greater Tbet expression compared to the control group. Supplementation with Moringa in the limited group slightly restored CD4+ T cell activation, IL-2, and IL-10 production. CONCLUSIONS: Taken together, these data suggest that Moringa treatment leads to increased CD4+ T cell activation, Th1 differentiation and production of pro-inflammatory cytokines after malaria infection. Thus, Moringa may be immunologically useful in the treatment of malaria and malnutrition. Further investigations are required to identify the active components in Moringa.


Assuntos
Malária/tratamento farmacológico , Desnutrição/imunologia , Moringa oleifera/química , Plasmodium chabaudi/efeitos dos fármacos , Proteínas com Domínio T/metabolismo , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Feminino , Citometria de Fluxo , Malária/complicações , Malária/imunologia , Desnutrição/complicações , Desnutrição/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Parasitemia/parasitologia , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Baço/citologia , Subpopulações de Linfócitos T/efeitos dos fármacos
17.
RNA ; 26(4): 481-491, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31953255

RESUMO

Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators and play important roles in cardiac development and congenital heart disease. In a previous study, we identified a novel lncRNA, Ppp1r1b, with expression highly correlated with myogenesis. However, the molecular mechanism that underlies Ppp1r1b-lncRNA function in myogenic regulation is unknown. By silencing Ppp1r1b-lncRNA, mouse C2C12 and human skeletal myoblasts failed to develop fully differentiated myotubes. Myogenic differentiation was also impaired in PPP1R1B-lncRNA deficient human-induced pluripotent stem cell-derived cardiomyocytes (hiPSCs-CMs). The expression of myogenic transcription factors, including MyoD, Myogenin, and Tbx5, as well as sarcomere proteins, was significantly suppressed in Ppp1r1b-lncRNA inhibited myoblast cells and neonatal mouse heart. Histone modification analysis revealed increased H3K27 tri-methylation at MyoD1 and Myogenin promoters in GapmeR treated C2C12 cells. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and chromatin isolation by RNA purification (ChIRP) assay revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic transcription factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Therefore, our results suggest that Ppp1r1b-lncRNA promotes myogenic differentiation through competing for PRC2 binding with chromatin of myogenic master regulators during heart and skeletal muscle development.


Assuntos
Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Miócitos Cardíacos/metabolismo , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/genética , Animais , Linhagem Celular , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Inativação Gênica , Código das Histonas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
18.
Immunity ; 52(1): 151-166.e6, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31924474

RESUMO

In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation.


Assuntos
Granzimas/imunologia , Neoplasias/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Humanos , Interferon gama/imunologia , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/citologia , Microambiente Tumoral/imunologia
19.
Int J Mol Sci ; 21(2)2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31963474

RESUMO

Early events of basal cell carcinoma (BCC) tumorigenesis are triggered by inappropriate activation of SHH signaling, via the loss of Patched1 (Ptch1) or by activating mutations of Smoothened (Smo). TBX1 is a key regulator of pharyngeal development, mainly through expression in multipotent progenitor cells of the cardiopharyngeal lineage. This transcription factor is connected to several major signaling systems, such as FGF, WNT, and SHH, and it has been linked to cell proliferation and to the regulation of cell shape and cell dynamics. Here, we show that TBX1 was expressed in all of the 51 BCC samples that we have tested, while in healthy human skin it was only expressed in the hair follicle. Signal intensity and distribution was heterogeneous among tumor samples. Experiments performed on a cellular model of mouse BCC showed that Tbx1 is downstream to GLI2, a factor in the SHH signaling, and that, in turn, it regulates the expression of Dvl2, which encodes an adaptor protein that is necessary for the transduction of WNT signaling. Consistently, Tbx1 depletion in the cellular model significantly reduced cell migration. These results suggest that TBX1 is part of a core transcription network that promotes BCC tumorigenesis.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Basocelular/patologia , Proteínas Desgrenhadas/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/patologia , Proteínas com Domínio T/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Proteínas Desgrenhadas/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Prognóstico , Estudos Retrospectivos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Proteínas com Domínio T/genética , Células Tumorais Cultivadas , Proteína Gli2 com Dedos de Zinco/genética
20.
Int J Mol Sci ; 21(2)2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31963264

RESUMO

Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1. Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.


Assuntos
Cartilagem/química , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Células Cultivadas , Colágeno/metabolismo , Proteínas Fetais/metabolismo , Camundongos , Osteogênese/genética , Osteogênese/fisiologia , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Proteínas com Domínio T/metabolismo
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