RESUMEN
αß lineage T cells, most of which are CD4+ or CD8+ and recognize MHC I- or MHC II-presented antigens, are essential for immune responses and develop from CD4+CD8+ thymocytes. The absence of in vitro models and the heterogeneity of αß thymocytes have hampered analyses of their intrathymic differentiation. Here, combining single-cell RNA and ATAC (chromatin accessibility) sequencing, we identified mouse and human αß thymocyte developmental trajectories. We demonstrated asymmetric emergence of CD4+ and CD8+ lineages, matched differentiation programs of agonist-signaled cells to their MHC specificity, and identified correspondences between mouse and human transcriptomic and epigenomic patterns. Through computational analysis of single-cell data and binding sites for the CD4+-lineage transcription factor Thpok, we inferred transcriptional networks associated with CD4+- or CD8+-lineage differentiation, and with expression of Thpok or of the CD8+-lineage factor Runx3. Our findings provide insight into the mechanisms of CD4+ and CD8+ T cell differentiation and a foundation for mechanistic investigations of αß T cell development.
Asunto(s)
Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Subgrupos de Linfocitos T/inmunología , Timocitos/inmunología , Animales , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Linaje de la Célula/genética , Epigenoma , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/inmunología , Antígenos de Histocompatibilidad/metabolismo , Humanos , Ratones , Subgrupos de Linfocitos T/metabolismo , Timocitos/metabolismo , Timo/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T-cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for the stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies.
Asunto(s)
Receptores de Glucocorticoides , Humanos , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Glucocorticoides/metabolismo , Glucocorticoides/farmacología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Células Jurkat , Proteolisis , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Apoptosis , Sistemas CRISPR-Cas , Línea Celular TumoralRESUMEN
Immature T cells undergo a process of positive selection in the thymus when their new T cell receptor (TCR) engages and signals in response to self-peptides. As the T cell matures, a slew of negative regulatory molecules, including the inhibitory surface glycoprotein CD5, are up-regulated in proportion to the strength of the self-peptide signal. Together these regulators dampen TCR-proximal signaling and help avoid any subsequent peripheral activation of T cells by self-peptides. Paradoxically, antigen-specific T cells initially expressing more CD5 (CD5hi) have been found to better persist as effector/memory cells after a peripheral challenge. The molecular mechanisms underlying such a duality in CD5 function is not clear. We found that CD5 alters the basal activity of the NF-κB signaling in resting peripheral T cells. When CD5 was conditionally ablated, T cells were unable to maintain higher expression of the cytoplasmic NF-κB inhibitor IκBα. Consistent with this, resting CD5hi T cells expressed more of the NF-κB p65 protein than CD5lo cells, without significant increases in transcript levels, in the absence of TCR signals. This posttranslationally stabilized cellular NF-κB depot potentially confers a survival advantage to CD5hi T cells over CD5lo ones. Taken together, these data suggest a two-step model whereby the strength of self-peptide-induced TCR signal lead to the up-regulation of CD5, which subsequently maintains a proportional reserve of NF-κB in peripheral T cells poised for responding to agonistic antigen-driven T cell activation.
Asunto(s)
Antígenos CD5/metabolismo , Regulación del Desarrollo de la Expresión Génica/inmunología , Inhibidor NF-kappaB alfa/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Presentación de Antígeno/inmunología , Antígenos CD5/genética , Línea Celular Tumoral , Separación Celular , Supervivencia Celular/inmunología , Femenino , Citometría de Flujo , Lipopolisacáridos/inmunología , Activación de Linfocitos , Ratones , Ratones Noqueados , Modelos Animales , Cultivo Primario de Células , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplante , Timo/citología , Timo/crecimiento & desarrollo , Timo/inmunología , Factor de Transcripción ReIA/metabolismo , Regulación hacia ArribaRESUMEN
Melanocyte stem cells (McSCs) are the undifferentiated melanocytic cells of the mammalian hair follicle (HF) responsible for recurrent generation of a large number of differentiated melanocytes during each HF cycle. HF McSCs reside in both the CD34+ bulge/lower permanent portion (LPP) and the CD34- secondary hair germ (SHG) regions of the HF during telogen. Using Dct-H2BGFP mice, we separate bulge/LPP and SHG McSCs using FACS with GFP and anti-CD34 to show that these two subsets of McSCs are functionally distinct. Genome-wide expression profiling results support the distinct nature of these populations, with CD34- McSCs exhibiting higher expression of melanocyte differentiation genes and with CD34+ McSCs demonstrating a profile more consistent with a neural crest stem cell. In culture and in vivo, CD34- McSCs regenerate pigmentation more efficiently whereas CD34+ McSCs selectively exhibit the ability to myelinate neurons. CD34+ McSCs, and their counterparts in human skin, may be useful for myelinating neurons in vivo, leading to new therapeutic opportunities for demyelinating diseases and traumatic nerve injury.
Asunto(s)
Antígenos CD34/metabolismo , Melanocitos/inmunología , Melanocitos/fisiología , Células Madre/inmunología , Células Madre/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Color del Cabello/fisiología , Folículo Piloso/citología , Folículo Piloso/fisiología , Melanocitos/clasificación , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Desnudos , Ratones Transgénicos , Proteína Básica de Mielina/deficiencia , Proteína Básica de Mielina/genética , Cresta Neural/citología , Cresta Neural/inmunología , Cresta Neural/fisiología , Pigmentación/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regeneración/fisiología , Células Madre/clasificaciónRESUMEN
Intratumor molecular heterogeneity of hepatocellular carcinoma is partly attributed to the presence of hepatic cancer stem cells (CSCs). Different CSC populations defined by various cell surface markers may contain different oncogenic drivers, posing a challenge in defining molecularly targeted therapeutics. We combined transcriptomic and functional analyses of hepatocellular carcinoma cells at the single-cell level to assess the degree of CSC heterogeneity. We provide evidence that hepatic CSCs at the single-cell level are phenotypically, functionally, and transcriptomically heterogeneous. We found that different CSC subpopulations contain distinct molecular signatures. Interestingly, distinct genes within different CSC subpopulations are independently associated with hepatocellular carcinoma prognosis, suggesting that a diverse hepatic CSC transcriptome affects intratumor heterogeneity and tumor progression. CONCLUSION: Our work provides unique perspectives into the biodiversity of CSC subpopulations, whose molecular heterogeneity further highlights their role in tumor heterogeneity, prognosis, and hepatic CSC therapy. (Hepatology 2018;68:127-140).
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Heterogeneidad Genética , Neoplasias Hepáticas/metabolismo , Células Madre Neoplásicas/metabolismo , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Estudios de Factibilidad , Perfilación de la Expresión Génica , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Células Madre Neoplásicas/citología , Fenotipo , Pronóstico , Análisis de la Célula IndividualRESUMEN
Human diffuse large B-cell lymphomas (DLBCLs) often aberrantly express oncogenes that generally contain complex secondary structures in their 5' untranslated region (UTR). Oncogenes with complex 5'UTRs require enhanced eIF4A RNA helicase activity for translation. PDCD4 inhibits eIF4A, and PDCD4 knockout mice have a high penetrance for B-cell lymphomas. Here, we show that on B-cell receptor (BCR)-mediated p70s6K activation, PDCD4 is degraded, and eIF4A activity is greatly enhanced. We identified a subset of genes involved in BCR signaling, including CARD11, BCL10, and MALT1, that have complex 5'UTRs and encode proteins with short half-lives. Expression of these known oncogenic proteins is enhanced on BCR activation and is attenuated by the eIF4A inhibitor Silvestrol. Antigen-experienced immunoglobulin (Ig)G(+) splenic B cells, from which most DLBCLs are derived, have higher levels of eIF4A cap-binding activity and protein translation than IgM(+) B cells. Our results suggest that eIF4A-mediated enhancement of oncogene translation may be a critical component for lymphoma progression, and specific targeting of eIF4A may be an attractive therapeutic approach in the management of human B-cell lymphomas.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Guanilato Ciclasa/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Regiones no Traducidas 5'/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 10 de la LLC-Linfoma de Células B , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Western Blotting , Proteínas Adaptadoras de Señalización CARD/genética , Caspasas/genética , Caspasas/metabolismo , Línea Celular Tumoral , Células Cultivadas , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/genética , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4A Eucariótico de Iniciación/genética , Guanilato Ciclasa/genética , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Persona de Mediana Edad , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Triterpenos/farmacologíaRESUMEN
αß T cells, which express the α-ß TCR heterodimer, express CD4 or CD8 coreceptors on cells that are MHC class I or MHC class II dependent. In contrast, γδ T cells do not express CD4 or CD8 and develop independently of MHC interaction. The factors that determine αß and γδ lineage choice are not fully understood, and the determinants of MHC restriction of TCR specificity have been controversial. In this study we have identified a naturally occurring population of T cells expressing Vγ-Cß receptor chains on the cell surface, the products of genomic trans-rearrangement between the Vγ2 gene and a variety of Dß or Jß genes, in place of an intact TCRß-chain and in association with TCRα. Identification of this population allowed an analysis of the role of TCR variable regions in determining T cell lineage choice and MHC restriction. We found that Vγ2(+)Cß(+) cells are positive for either CD4 or CD8 and are selected in an MHC class II- or MHC class I-dependent manner, respectively, thus following the differentiation pathway of αß and not γδ cells and demonstrating that Vß V region sequences are not required for selection of an MHC-restricted repertoire.
Asunto(s)
Linaje de la Célula/genética , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T , Complejo Mayor de Histocompatibilidad/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Proteínas de la Ataxia Telangiectasia Mutada/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Expresión Génica , Genes MHC Clase I , Genes MHC Clase II , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Activación TranscripcionalRESUMEN
During the CD4(-)CD8(-) (DN) stage of T-cell development, RAG-dependent DNA breaks and V(D)J recombination occur at three T-cell receptor (TCR) loci: TCRß, TCRγ and TCRδ. During this stage, abnormal trans-rearrangements also take place between TCR loci, occurring at increased frequency in absence of the DNA damage response mediator ataxia telangiectasia mutated (ATM). Here, we use this model of physiologic trans-rearrangement to study factors that predispose to rearrangement and the role of ATM in preventing chromosomal translocations. The frequency of DN thymocytes with DNA damage foci at multiple TCR loci simultaneously is increased 2- to 3-fold in the absence of ATM. However, trans-rearrangement is increased 10 000- to 100 000-fold, indicating that ATM function extends beyond timely resolution of DNA breaks. RAG-mediated synaptic complex formation occurs between recombination signal sequences with unequal 12 and 23 base spacer sequences (12/23 rule). TCR trans-rearrangements violate this rule, as we observed similar frequencies of 12/23 and aberrant 12/12 or 23/23 recombination products. This suggests that trans-rearrangements are not the result of trans-synaptic complex formation, but they are instead because of unstable cis synaptic complexes that form simultaneously at distinct TCR loci. Thus, ATM suppresses trans-rearrangement primarily through stabilization of DNA breaks at TCR loci.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas de Unión al ADN/fisiología , Reordenamiento Génico de Linfocito T , Proteínas Serina-Treonina Quinasas/fisiología , Timocitos/inmunología , Proteínas Supresoras de Tumor/fisiología , Recombinación V(D)J , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , ADN/metabolismo , Roturas del ADN , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Linfocitos T/inmunología , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
Bax, a pro-apoptotic protein from the Bcl-2 family, is central to apoptosis regulation. To suppress spontaneous apoptosis, Bax must be under stringent control that may include regulation of Bax conformation and expression levels. We report that IBRDC2, an IBR-type RING-finger E3 ubiquitin ligase, regulates the levels of Bax and protects cells from unprompted Bax activation and cell death. Downregulation of IBRDC2 induces increased cellular levels and accumulation of the active form of Bax. The ubiquitination-dependent regulation of Bax stability is suppressed by IBRDC2 downregulation and stimulated by IBRDC2 overexpression in both healthy and apoptotic cells. Although mostly cytosolic in healthy cells, upon induction of apoptosis, IBRDC2 accumulates in mitochondrial domains enriched with Bax. Mitochondrial accumulation of IBRDC2 occurs in parallel with Bax activation and also depends on the expression levels of Bcl-xL. Furthermore, IBRDC2 physically interacts with activated Bax. By applying Bax mutants in HCT116 Bax(-/-) cells, combined with the use of active Bax-specific antibodies, we have established that both mitochondrial localization and apoptotic activation of Bax are required for IBRDC2 translocation to the mitochondria.
Asunto(s)
Apoptosis , Ubiquitina-Proteína Ligasas/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Secuencia de Aminoácidos , Citocromos c/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Transporte de Proteínas , Ubiquitina-Proteína Ligasas/análisis , UbiquitinaciónRESUMEN
Variable, diversity and joining (V(D)J) recombination and class-switch recombination use overlapping but distinct non-homologous end joining pathways to repair DNA double-strand-break intermediates. 53BP1 is a DNA-damage-response protein that is rapidly recruited to sites of chromosomal double-strand breaks, where it seems to function in a subset of ataxia telangiectasia mutated (ATM) kinase-, H2A histone family member X (H2AX, also known as H2AFX)- and mediator of DNA damage checkpoint 1 (MDC1)-dependent events. A 53BP1-dependent end-joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination. Here we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1-deficient lymphocytes that is distinct from that found in classical non-homologous-end-joining-, H2ax-, Mdc1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of unrepaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes that have antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long-range joining of DNA breaks.
Asunto(s)
ADN/metabolismo , Reordenamiento Génico de Linfocito T/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Recombinación Genética , Animales , Apoptosis , Proteínas Cromosómicas no Histona , ADN/genética , Roturas del ADN , Proteínas de Unión al ADN , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T/genética , Inestabilidad Genómica , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Linfopenia/genética , Linfopenia/patología , Ratones , Modelos Genéticos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Homología de Secuencia , Linfocitos T/citología , Linfocitos T/metabolismo , Timo/citología , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
IFN-regulatory factor 5 (IRF-5), a member of the IRF family, is a transcription factor that has a key role in the induction of the antiviral and inflammatory response. When compared with C57BL/6 mice, Irf5(-/-) mice show higher susceptibility to viral infection and decreased serum levels of type I IFN and the inflammatory cytokines IL-6 and TNF-alpha. Here, we demonstrate that IRF-5 is involved in B-cell maturation and the stimulation of Blimp-1 expression. The Irf5(-/-) mice develop an age-related splenomegaly, associated with a dramatic accumulation of CD19(+)B220(-) B cells and a disruption of normal splenic architecture. Splenic B cells from Irf5(-/-) mice also exhibited a decreased level of plasma cells. The CD19(+) Irf5(-/-) B cells show a defect in Toll-like receptor (TLR) 7- and TLR9-induced IL-6 production, and the aged Irf5(-/-) mice have decreased serum levels of natural antibodies; however, the antigen-specific IgG1 primary response was already dependent in IRF-5 in young mice, although the IgM response was not. Analysis of the profile of transcription factors associated with plasma cell differentiation shows down-regulation of Blimp-1 expression, a master regulator of plasma cell differentiation, which can be reconstituted with ectopic IRF-5. IRF-5 stimulates transcription of the Prdm1 gene encoding Blimp-1 and binds to the IRF site in the Prdm1 promoter. Collectively, these results reveal that the age-related splenomegaly in Irf5(-/-) mice is associated with an accumulation of CD19(+)B220(-) B cells with impaired functions and show the role of IRF-5 in the direct regulation of the plasma cell commitment factor Blimp-1 and in B-cell terminal differentiation.
Asunto(s)
Linfocitos B/metabolismo , Diferenciación Celular , Factores Reguladores del Interferón/fisiología , Factores de Edad , Animales , Antígenos CD19/metabolismo , Linfocitos B/patología , Sitios de Unión/genética , Línea Celular , Femenino , Citometría de Flujo , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/metabolismo , Bazo/patología , Esplenomegalia/genética , Esplenomegalia/patología , Factores de Transcripción/genéticaRESUMEN
Recent advances have revolutionized the oldest high-throughput single-cell analytical tool, flow cytometry. Fluorescent analyzers and sorters with up to seven lasers and the potential to detect up to 50 parameters are changing the way flow cytometry is used, but old school practices which are inadequate for new technologies remain alive. This chapter summarizes recent advances, explains the most salient new features and offers a step-by-step guide to develop and successfully execute high-dimensional fluorescent flow cytometry experiments.
Asunto(s)
Rayos Láser , Luz , Citometría de Flujo/métodosRESUMEN
How CD4+ lineage gene expression is initiated in differentiating thymocytes remains poorly understood. Here, we show that the paralog transcription factors Zfp281 and Zfp148 control both this process and cytokine expression by T helper cell type 2 (TH2) effector cells. Genetic, single-cell, and spatial transcriptomic analyses showed that these factors promote the intrathymic CD4+ T cell differentiation of class II major histocompatibility complex (MHC II)-restricted thymocytes, including expression of the CD4+ lineage-committing factor Thpok. In peripheral T cells, Zfp281 and Zfp148 promoted chromatin opening at and expression of TH2 cytokine genes but not of the TH2 lineage-determining transcription factor Gata3. We found that Zfp281 interacts with Gata3 and is recruited to Gata3 genomic binding sites at loci encoding Thpok and TH2 cytokines. Thus, Zfp148 and Zfp281 collaborate with Gata3 to promote CD4+ T cell development and TH2 cell responses.
Asunto(s)
Linfocitos T CD4-Positivos , Factores de Transcripción , Animales , Ratones , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/genética , Citocinas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Modulating telomere dynamics may be a useful strategy for targeting prostate cancer cells, because they generally have short telomeres. Because a plateau has been reached in the development of taxane-based treatments for prostate cancer, this study was undertaken to evaluate the relative efficacy of targeting telomeres and microtubules in taxane-sensitive, taxane-resistant, androgen-sensitive, and androgen-insensitive prostate cancer cells. Paclitaxel- and docetaxel-resistant DU145 cells were developed and their underlying adaptive responses were evaluated. Telomere dynamics and the effects of targeting telomeres with sodium meta-arsenite (KML001) (an agent undergoing early clinical trials), including combinations with paclitaxel and docetaxel, were evaluated in parental and drug-resistant cells. The studies were extended to androgen-sensitive LNCaP cells and androgen-insensitive LNCaP/C81 cells. Both P-glycoprotein (Pgp)-dependent and non-Pgp-dependent mechanisms of resistance were recruited within the same population of DU145 cells with selection for drug resistance. Wild-type DU145 cells have a small side population (SP) (0.4-1.2%). The SP fraction increased with increasing drug resistance, which was correlated with enhanced expression of Pgp but not breast cancer resistance protein. Telomere dynamics remained unchanged in taxane-resistant cells, which retained sensitivity to KML001. Furthermore, KML001 targeted SP and non-SP fractions, inducing DNA damage signaling in both fractions. KML001 induced telomere erosion, decreased telomerase gene expression, and was highly synergistic with the taxanes in wild-type and drug-resistant DU145 cells. This synergism extended to androgen-sensitive and androgen-insensitive LNCaP cells under basal and androgen-deprived conditions. These studies demonstrate that KML001 plus docetaxel and KML001 plus paclitaxel represent highly synergistic drug combinations that should be explored further in the different disease states of prostate cancer.
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Sistemas de Liberación de Medicamentos/métodos , Resistencia a Antineoplásicos/fisiología , Microtúbulos/metabolismo , Neoplasias de la Próstata/metabolismo , Telómero/metabolismo , Línea Celular Tumoral , Docetaxel , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Masculino , Microtúbulos/efectos de los fármacos , Paclitaxel/administración & dosificación , Paclitaxel/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Taxoides/administración & dosificación , Taxoides/metabolismo , Telómero/efectos de los fármacos , Resultado del TratamientoRESUMEN
CD47 regulates the trafficking of specific coding and noncoding RNAs into extracellular vesicles (EVs), and the RNA contents of CD47+ EVs differ from that of CD63+ EVs released by the same cells. Single particle interferometric reflectance imaging sensing combined with immunofluorescent imaging was used to analyse the colocalization of tetraspanins, integrins, and CD47 on EVs produced by wild type and CD47-deficient Jurkat T lymphoblast and PC3 prostate carcinoma cell lines. On Jurkat cell-derived EVs, ß1 and α4 integrin subunits colocalized predominantly with CD47 and CD81 but not with CD63 and CD9, conserving the known lateral interactions between these proteins in the plasma membrane. Although PC3 cell-derived EVs lacked detectable α4 integrin, specific association of CD81 with ß1 and CD47 was preserved. Loss of CD47 expression in Jurkat cells significantly reduced ß1 and α4 levels on EVs produced by these cells while elevating CD9+ , CD63+ , and CD81+ EVs. In contrast, loss of CD47 in PC3 cells decreased the abundance of CD63+ and CD81+ EVs. These data establish that CD47+ EVs are mostly distinct from EVs bearing the tetraspanins CD63 and CD9, but CD47 also indirectly regulates the abundance of EVs bearing these non-interacting tetraspanins via mechanisms that remain to be determined.
Asunto(s)
Carcinoma , Vesículas Extracelulares , Neoplasias de la Próstata , Antígeno CD47/metabolismo , Carcinoma/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Integrina alfa4/metabolismo , Integrinas/metabolismo , Masculino , Próstata , Neoplasias de la Próstata/metabolismo , ARN/metabolismo , Tetraspaninas/metabolismoRESUMEN
Neurons harbor high levels of single-strand DNA breaks (SSBs) that are targeted to neuronal enhancers, but the source of this endogenous damage remains unclear. Using two systems of postmitotic lineage specification-induced pluripotent stem cell-derived neurons and transdifferentiated macrophages-we show that thymidine DNA glycosylase (TDG)-driven excision of methylcytosines oxidized with ten-eleven translocation enzymes (TET) is a source of SSBs. Although macrophage differentiation favors short-patch base excision repair to fill in single-nucleotide gaps, neurons also frequently use the long-patch subpathway. Disrupting this gap-filling process using anti-neoplastic cytosine analogs triggers a DNA damage response and neuronal cell death, which is dependent on TDG. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage, a process that normally safeguards cell identity but can also provoke neurotoxicity after anticancer treatments.
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Roturas del ADN de Cadena Simple , Desmetilación del ADN , Reparación del ADN , Elementos de Facilitación Genéticos , Células Madre Pluripotentes Inducidas , Neuronas , Timina ADN Glicosilasa , Diferenciación Celular , Neuronas/enzimología , 5-Metilcitosina/metabolismo , Humanos , Transdiferenciación CelularRESUMEN
Despite breakthroughs in cancer immunotherapy, most tumor-reactive T cells cannot persist in solid tumors due to an immunosuppressive environment. We developed Tres (tumor-resilient T cell), a computational model utilizing single-cell transcriptomic data to identify signatures of T cells that are resilient to immunosuppressive signals, such as transforming growth factor-ß1, tumor necrosis factor-related apoptosis-inducing ligand and prostaglandin E2. Tres reliably predicts clinical responses to immunotherapy in melanoma, lung cancer, triple-negative breast cancer and B cell malignancies using bulk T cell transcriptomic data from pre-treatment tumors from patients who received immune-checkpoint inhibitors (n = 38), infusion products for chimeric antigen receptor T cell therapies (n = 34) and pre-manufacture samples for chimeric antigen receptor T cell or tumor-infiltrating lymphocyte therapies (n = 84). Further, Tres identified FIBP, whose functions are largely unknown, as the top negative marker of tumor-resilient T cells across many solid tumor types. FIBP knockouts in murine and human donor CD8+ T cells significantly enhanced T cell-mediated cancer killing in in vitro co-cultures. Further, Fibp knockout in murine T cells potentiated the in vivo efficacy of adoptive cell transfer in the B16 tumor model. Fibp knockout T cells exhibit reduced cholesterol metabolism, which inhibits effector T cell function. These results demonstrate the utility of Tres in identifying biomarkers of T cell effectiveness and potential therapeutic targets for immunotherapies in solid tumors.
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Melanoma , Receptores Quiméricos de Antígenos , Animales , Linfocitos T CD8-positivos , Proteínas Portadoras , Humanos , Inmunoterapia/métodos , Inmunoterapia Adoptiva/métodos , Proteínas de la Membrana , RatonesRESUMEN
T cells and endothelial cells engage in bidirectional communication that regulates angiogenesis and T cell transmigration. Extracellular vesicles (EVs) mediate intercellular communication by the transfer of bioactive molecules including RNAs. EVs produced by a given cell type are heterogeneous in their RNA content, but it is unclear how specific EV surface markers relate to their functional effects on target cells. Our previous work established that Jurkat T cell EVs bearing CD63, MHC-I, or CD47 surface markers contain distinct noncoding RNA populations. The present study reveals that CD63+ and MHC-I+ EVs from CD47-deficient Jurkat T cells are enriched in small non-coding RNAs relative to EVs from wild-type Jurkat T cells. CD47-deficient Jurkat T cells secrete more CD63+ and MHC-I+ EVs, but MHC-I+ EVs are selectively taken up more by human umbilical vein endothelial cells. Transcriptomics analysis of endothelial cells treated with CD63+ or MHC-I+ EVs showed surface marker- and CD47-dependent changes in gene expression in the target cells. Gene set enrichment analysis identified CD47-dependent, and surface marker-dependent effects of T cell EVs on VEGF and inflammatory signaling, cell cycle, and lipid and cholesterol metabolism. Thus, subsets of T cell EVs differentially regulate endothelial cell metabolism and inflammatory and angiogenic responses.
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53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for "classic" nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.