RESUMEN
Epigenetic mechanisms are believed to play key roles in the establishment of cell-specific transcription programs. Accordingly, the modified bases 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been observed in DNA of genomic regulatory regions such as enhancers, and oxidation of 5mC into 5hmC by Ten-eleven translocation (TET) proteins correlates with enhancer activation. However, the functional relationship between cytosine modifications and the chromatin architecture of enhancers remains elusive. To gain insights into their function, 5mC and 5hmC levels were perturbed by inhibiting DNA methyltransferases and TETs during differentiation of mouse embryonal carcinoma cells into neural progenitors, and chromatin characteristics of enhancers bound by the pioneer transcription factors FOXA1, MEIS1, and PBX1 were interrogated. In a large fraction of the tested enhancers, inhibition of DNA methylation was associated with a significant increase in monomethylation of H3K4, a characteristic mark of enhancer priming. In addition, at some specific enhancers, 5mC oxidation by TETs facilitated chromatin opening, a process that may stabilize MEIS1 binding to these genomic regions.
Asunto(s)
5-Metilcitosina/metabolismo , Cromatina/metabolismo , Células Madre de Carcinoma Embrionario/metabolismo , Elementos de Facilitación Genéticos , Epigénesis Genética , 5-Metilcitosina/análogos & derivados , Animales , Diferenciación Celular , Cromatina/ultraestructura , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Células Madre de Carcinoma Embrionario/citología , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Histonas/genética , Histonas/metabolismo , Ratones , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Factor de Transcripción 1 de la Leucemia de Células Pre-B/genética , Factor de Transcripción 1 de la Leucemia de Células Pre-B/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The antitumor action of immune checkpoint blockade (ICB) is primarily mediated by CD8+ T cells. How sensitivity to ICB varies across CD8+ T cell subsets and clonotypes and the relationship of these with clinical outcome is unclear. To explore this, we used single-cell V(D)J and RNA-sequencing to track gene expression changes elicited by ICB across individual peripheral CD8+ T cell clones, identify baseline markers of CD8+ T cell clonal sensitivity, and chart how CD8+ T cell transcriptional changes vary according to phenotypic subset and clonal size. We identified seven subsets of CD8+ T cells with divergent reactivity to ICB and found that the cytotoxic effector subset showed the greatest number of differentially expressed genes while remaining stable in clonal size after ICB. At the level of CD8+ T cell clonotypes, we found a relationship between transcriptional changes and clone size, with large clones showing a greater number of differentially regulated genes enriched for pathways including T cell receptor (TCR) signaling. Cytotoxic CD8+ effector clones were more likely to persist following ICB and were more likely to correspond with public tumor-infiltrating lymphocyte clonotypes. Last, we demonstrated that individuals whose CD8+ T cell pretreatment showed low cytotoxicity and had fewer expanded clones typically had worse outcomes after ICB treatment. This work further advances understanding of the molecular determinants of ICB response, assisting in the search for peripheral prognostic biomarkers and highlighting the importance of the baseline CD8+ immune landscape in determining ICB response in metastatic melanoma.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Inhibidores de Puntos de Control Inmunológico/farmacología , Ipilimumab/farmacología , Nivolumab/farmacología , Linfocitos T CD8-positivos/inmunología , Humanos , Supervivencia sin ProgresiónRESUMEN
Immune checkpoint blockade (ICB) of PD-1 and CTLA-4 to treat metastatic melanoma (MM) has variable therapeutic benefit. To explore this in peripheral samples, we characterized CD8+ T cell gene expression across a cohort of patients with MM receiving anti-PD-1 alone (sICB) or in combination with anti-CTLA-4 (cICB). Whereas CD8+ transcriptional responses to sICB and cICB involve a shared gene set, the magnitude of cICB response is over fourfold greater, with preferential induction of mitosis- and interferon-related genes. Early samples from patients with durable clinical benefit demonstrated overexpression of T cell receptor-encoding genes. By mapping T cell receptor clonality, we find that responding patients have more large clones (those occupying >0.5% of repertoire) post-treatment than non-responding patients or controls, and this correlates with effector memory T cell percentage. Single-cell RNA-sequencing of eight post-treatment samples demonstrates that large clones overexpress genes implicated in cytotoxicity and characteristic of effector memory T cells, including CCL4, GNLY and NKG7. The 6-month clinical response to ICB in patients with MM is associated with the large CD8+ T cell clone count 21 d after treatment and agnostic to clonal specificity, suggesting that post-ICB peripheral CD8+ clonality can provide information regarding long-term treatment response and, potentially, facilitate treatment stratification.
Asunto(s)
Linfocitos T CD8-positivos/citología , Antígeno CTLA-4/inmunología , Inmunoterapia/métodos , Melanoma/inmunología , Melanoma/terapia , Adulto , Anticuerpos/uso terapéutico , Antígenos de Diferenciación de Linfocitos T/genética , Proliferación Celular , Quimiocina CCL4/genética , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Humanos , Sistema Inmunológico , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Metástasis de la Neoplasia , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Análisis de la Célula Individual , Adulto JovenRESUMEN
IL-7 is a key factor in T cell immunity and common variants at IL7R, encoding its receptor, are associated with autoimmune disease susceptibility. IL7R mRNA is induced in stimulated monocytes, yet a function for IL7R in monocyte biology remains unexplored. Here we characterize genetic regulation of IL7R at the protein level in healthy individuals, and find that monocyte surface and soluble IL7R (sIL7R) are markedly induced by lipopolysaccharide. In monocytes, both surface IL7R and sIL7R expression strongly associate with allelic carriage of rs6897932, a disease-associated IL7R polymorphism. Monocytes produce more sIL7R than CD4 + T cells, and the amount is additionally correlated with the expression of DDX39A, encoding a splicing factor. Synovial fluid-derived monocytes from patients with spondyloarthritis are enriched for IL7R+ cells with a unique transcriptional profile that overlaps with IL-7-induced gene sets. Our data thus suggest a previously unappreciated function for monocytes in IL-7 biology and IL7R-associated diseases.
Asunto(s)
Autoinmunidad/genética , Subunidad alfa del Receptor de Interleucina-7/genética , Interleucina-7/inmunología , Monocitos/inmunología , Espondilitis Anquilosante/genética , Alelos , ARN Helicasas DEAD-box/inmunología , ARN Helicasas DEAD-box/metabolismo , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Voluntarios Sanos , Humanos , Interleucina-7/metabolismo , Subunidad alfa del Receptor de Interleucina-7/inmunología , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Lipopolisacáridos/inmunología , Monocitos/metabolismo , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Espondilitis Anquilosante/inmunología , Espondilitis Anquilosante/patología , Líquido Sinovial/citología , Líquido Sinovial/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Zinc-finger and homeodomain transcription factors have been shown in vitro to bind to recognition motifs containing a methylated CpG. However, accessing these motifs in vivo might be seriously impeded by the inclusion of DNA in nucleosomes and by the condensed structure adopted by chromatin formed on methylated DNA. Here, we discuss how oxidation of 5-methylcytosine into 5-hydroxymethylcytosine could provide the initial destabilizing clue for such transcription factors to get access to nucleosomal DNA and read epigenetic information.
Asunto(s)
Cromatina/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , ADN/química , ADN/metabolismo , Cromatina/química , Cromatina/genética , Citosina/química , ADN/genética , Humanos , Oxidación-ReducciónRESUMEN
Conventional techniques for single-base resolution mapping of epigenetic modifications of DNA such as 5-hydroxymethylcytosine (5hmC) rely on the sequencing of bisulfite-modified DNA. Here we present an alternative approach called SCL-exo which combines selective chemical labeling (SCL) of 5hmC in genomic DNA with exonuclease (exo) digestion of the bead-trapped modified DNA molecules. Associated with a straightforward bioinformatic analysis, this new procedure provides an unbiased and fast method for mapping this epigenetic mark at high resolution. Implemented on mouse genomic DNA from in vitro-differentiated neural precursor cells, SCL-exo sheds light on an intrinsic lack of conservation of hydroxymethylated CpGs across vertebrates.
Asunto(s)
Citosina/análogos & derivados , ADN/metabolismo , Epigenómica/métodos , Exonucleasas/metabolismo , 5-Metilcitosina/análogos & derivados , Animales , Células Cultivadas , Islas de CpG , Citosina/metabolismo , ADN/química , Metilación de ADN , Células Madre Embrionarias/química , Células Madre Embrionarias/citología , Epigénesis Genética , Ratones , Análisis de Secuencia de ADN/métodos , Coloración y EtiquetadoRESUMEN
Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-?1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxy)methylation, and cell fate determination.