RESUMEN
T cell specification and commitment require Notch signaling. Although the requirement for Notch signaling during intrathymic T cell development is known, it is still unclear whether the onset of T cell priming can occur in a prethymic niche and whether RBPJ-dependent Notch signaling has a role during this event. Here, we established an Rbpj-inducible system that allowed temporal and tissue-specific control of the responsiveness to Notch in all hematopoietic cells. Using this system, we found that Notch signaling was required before the early T cell progenitor stage in the thymus. Lymphoid-primed multipotent progenitors in the bone marrow underwent Notch signaling with Rbpj induction, which inhibited development towards the myeloid lineage in thymus-seeding progenitors. Thus, our results indicated that the onset of T cell differentiation occurred in a prethymic setting, and that Notch played an important role during this event.
Asunto(s)
Diferenciación Celular/inmunología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Células Precursoras de Linfocitos T/fisiología , Receptores Notch/metabolismo , Subgrupos de Linfocitos T/inmunología , Animales , Linaje de la Célula/inmunología , Separación Celular , Femenino , Citometría de Flujo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Masculino , Ratones , Ratones Transgénicos , Cultivo Primario de Células , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/metabolismo , Timo/citología , Timo/inmunologíaRESUMEN
A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.
Asunto(s)
Neoplasias Encefálicas , Encéfalo , Malformaciones Vasculares del Sistema Nervioso Central , Células Endoteliales , Feto , RNA-Seq , Análisis de Expresión Génica de una Sola Célula , Femenino , Humanos , Masculino , Encéfalo/irrigación sanguínea , Encéfalo/patología , Encéfalo/embriología , Encéfalo/metabolismo , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/patología , Comunicación Celular , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/citología , Feto/irrigación sanguínea , Feto/citología , Feto/embriología , Malformaciones Vasculares del Sistema Nervioso Central/patología , Antígenos HLA-D/metabolismo , Adulto , SaludRESUMEN
The zinc-finger transcription factor GATA-3 plays a crucial role during early T cell development and also dictates later T cell differentiation outcomes. However, its role and collaboration with the Notch signaling pathway in the induction of T lineage specification and commitment have not been fully elucidated. We show that GATA-3 deficiency in mouse hematopoietic progenitors results in an early block in T cell development despite the presence of Notch signals, with a failure to upregulate Bcl11b expression, leading to a diversion along a myeloid, but not a B cell, lineage fate. GATA-3 deficiency in the presence of Notch signaling results in the apoptosis of early T lineage cells, as seen with inhibition of CDK4/6 (cyclin-dependent kinases 4 and 6) function, and dysregulated cyclin-dependent kinase inhibitor 2b (Cdkn2b) expression. We also show that GATA-3 induces Bcl11b, and together with Bcl11b represses Cdkn2b expression; however, loss of Cdkn2b failed to rescue the developmental block of GATA-3-deficient T cell progenitor. Our findings provide a signaling and transcriptional network by which the T lineage program in response to Notch signals is realized.
Asunto(s)
Factor de Transcripción GATA3/metabolismo , Transducción de Señal , Linfocitos T , Animales , Diferenciación Celular , Linaje de la Célula , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Redes Reguladoras de Genes , Ratones , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Linfocitos T/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The cortical and medullary thymic epithelial cell (cTEC and mTEC) lineages are essential for inducing T cell lineage commitment, T cell positive selection and the establishment of self-tolerance, but the mechanisms controlling their fetal specification and differentiation are poorly understood. Here, we show that notch signaling is required to specify and expand the mTEC lineage. Notch1 is expressed by and active in TEC progenitors. Deletion of Notch1 in TECs resulted in depletion of mTEC progenitors and dramatic reductions in mTECs during fetal stages, consistent with defects in mTEC specification and progenitor expansion. Conversely, forced notch signaling in all TECs resulted in widespread expression of mTEC progenitor markers and profound defects in TEC differentiation. In addition, lineage-tracing analysis indicated that all mTECs have a history of receiving a notch signal, consistent with notch signaling occurring in mTEC progenitors. These data provide strong evidence for a requirement for notch signaling in specification of the mTEC lineage.
Asunto(s)
Desarrollo Fetal/genética , Receptor Notch1/metabolismo , Timo/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Células Epiteliales/citología , Células Epiteliales/metabolismo , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Organogénesis , Receptor Notch1/deficiencia , Receptor Notch1/genética , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Timo/citología , Timo/crecimiento & desarrolloRESUMEN
T cell development is predicated on the successful rearrangement of the TCR gene loci, which encode for Ag-specific receptors. Recombination-activating gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T cell development. In this study, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCR ß-chain in mediating ß-selection during human T cell development. In stark contrast to mice, human RAG2-KO T lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRß rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR ß-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRß- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide important insights as to the distinct requirement for the TCR ß-chain during human T cell development.
Asunto(s)
Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Diferenciación Celular/genética , Células Madre Embrionarias Humanas/citología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Técnicas de Inactivación de Genes , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Hematopoyesis/genética , Humanos , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Transducción GenéticaRESUMEN
During T cell development, progenitor thymocytes undergo a large proliferative burst immediately following successful TCRß rearrangement, and defects in genes that regulate this proliferation have a profound effect on thymus cellularity and output. Although the signaling pathways that initiate cell cycling and nutrient uptake after TCRß selection are understood, less is known about the transcriptional programs that regulate the metabolic machinery to promote biomass accumulation during this process. In this article, we report that mice with whole body deficiency in the nuclear receptor peroxisome proliferator-activated receptor-δ (PPARδmut) exhibit a reduction in spleen and thymus cellularity, with a decrease in thymocyte cell number starting at the double-negative 4 stage of thymocyte development. Although in vivo DNA synthesis was normal in PPARδmut thymocytes, studies in the OP9-delta-like 4 in vitro system of differentiation revealed that PPARδmut double-negative 3 cells underwent fewer cell divisions. Naive CD4+ T cells from PPARδmut mice also exhibited reduced proliferation upon TCR and CD28 stimulation in vitro. Growth defects in PPAR-δ-deficient thymocytes and peripheral CD4+ T cells correlated with decreases in extracellular acidification rate, mitochondrial reserve, and expression of a host of genes involved in glycolysis, oxidative phosphorylation, and lipogenesis. By contrast, mice with T cell-restricted deficiency of Ppard starting at the double-positive stage of thymocyte development, although exhibiting defective CD4+ T cell growth, possessed a normal T cell compartment, pointing to developmental defects as a cause of peripheral T cell lymphopenia in PPARδmut mice. These findings implicate PPAR-δ as a regulator of the metabolic program during thymocyte and T cell growth.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/inmunología , Receptores Citoplasmáticos y Nucleares/metabolismo , Timocitos/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular/fisiología , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Timocitos/inmunologíaRESUMEN
γδ T-cells boast an impressive functional repertoire that can paint them as either champions or villains depending on the environmental and immunological cues. Understanding the function of the various effector γδ subsets necessitates tracing the developmental program of these subsets, including the point of lineage bifurcation from αß T-cells. Here, we review the importance of signals from the T-cell receptor (TCR) in determining αß versus γδ lineage fate, and further discuss how the molecular components of this pathway may influence the developmental programming of γδ T-cells functional subsets. Additionally, we discuss the role of temporal windows in restricting the development of IL-17 producing γδ T-cell subtypes, and explore whether fetal and adult hematopoietic progenitors maintain the same potential for giving rise to this important subset.
Asunto(s)
Células Madre Hematopoyéticas/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Diferenciación Celular , Linaje de la Célula/inmunología , Células Madre Hematopoyéticas/citología , Humanos , Interleucina-17/inmunología , Activación de Linfocitos/inmunología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/citologíaRESUMEN
How the genetic landscape governs a tumor's response to immunotherapy remains poorly understood. To assess the immune-modulatory capabilities of 573 genes associated with altered cytotoxicity in human cancers, here we perform CRISPR/Cas9 screens directly in mouse lung cancer models. We recover the known immune evasion factors Stat1 and Serpinb9 and identify the cancer testis antigen Adam2 as an immune modulator, whose expression is induced by KrasG12D and further elevated by immunotherapy. Using loss- and gain-of-function experiments, we show that ADAM2 functions as an oncogene by restraining interferon and TNF cytokine signaling causing reduced presentation of tumor-associated antigens. ADAM2 also restricts expression of the immune checkpoint inhibitors PDL1, LAG3, TIGIT and TIM3 in the tumor microenvironment, which might explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy in ADAM2 overexpressing tumors. Together, direct in vivo CRISPR/Cas9 screens can uncover genetic alterations that control responses to immunotherapies.
Asunto(s)
Antineoplásicos , Fertilinas , Neoplasias Pulmonares , Serpinas , Animales , Humanos , Masculino , Ratones , Antígenos de Neoplasias , Fertilinas/genética , Inmunoterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Proteínas de la Membrana/genética , Serpinas/genética , Linfocitos T Citotóxicos , Microambiente TumoralRESUMEN
γδ T cells form an integral arm of the immune system and are critical during protective and destructive immunity. However, how γδ T cells are functionally programmed in vivo remains unclear. Here, we employ RBPJ-inducible and KN6-transgenic mice to assess the roles of ontogenic timing, T cell receptor (TCR) signal strength, and Notch signaling. We find skewing of Vγ1+ cells toward the PLZF+Lin28b+ lineage at the fetal stage. Generation of interleukin-17 (IL-17)-producing γδ T cells is favored during, although not exclusive to, the fetal stage. Surprisingly, Notch signaling is dispensable for peripheral γδ T cell IL-17 production. Strong TCR signals, together with Notch, promote IL-4 differentiation. Conversely, less strong TCR signals promote Notch-independent IL-17 differentiation. Single-cell transcriptomic analysis reveals differential programming instilled by TCR signal strength and Notch for specific subsets. Thus, our results precisely define the roles of ontogenic timing, TCR signal strength, and Notch signaling in γδ T cell functional programming in vivo.
Asunto(s)
Interferón gamma/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Notch/metabolismo , Animales , Diferenciación Celular , Humanos , Ratones , Transducción de SeñalRESUMEN
Broader clinical application of umbilical cord blood (UCB), as a source of hematopoietic stem/progenitor cells (HSPCs), is limited by low CD34+ and T-cell numbers, contributing to slow lymphohematopoietic recovery, infection, and relapse. Studies have evaluated the safety, feasibility, and expedited neutrophil recovery associated with the transplantation of CD34+ HSPCs from ex vivo expansion cultures using the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1). In a phase 1/2 study of 17 patients who received combined SR1-expanded and unexpanded UCB units, a considerable advantage for enhancing T-cell chimerism was not observed. We previously showed that progenitor T (proT) cells generated in vitro from HSPCs accelerated T-cell reconstitution and restored immunity after hematopoietic stem cell transplantation (HSCT). To expedite immune recovery, we hypothesized that SR1-expanded HSPCs together with proT cells could overcome the known T-cell immune deficiency that occurs post-HSCT. Here, we show that SR1-expanded UCB can induce >250-fold expansion of CD34+ HSPCs, which can generate large numbers of proT cells upon in vitro differentiation. When compared with nonexpanded naive proT cells, SR1 proT cells also showed effective thymus-seeding and peripheral T-cell functional capabilities in vivo despite having an altered phenotype. In a competitive transfer approach, both naive and SR1 proT cells showed comparable thymus-engrafting capacities. Single-cell RNA sequencing of peripheral CD3+ T cells from mice injected with either naive or SR1 proT cells revealed functional subsets of T cells with polyclonal T-cell receptor repertoires. Our findings support the use of SR1-expanded UCB grafts combined with proT-cell generation for decreasing T-cell immunodeficiency post-HSCT.
Asunto(s)
Antígenos CD34/metabolismo , Diferenciación Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Precursoras de Linfocitos T/citología , Células Precursoras de Linfocitos T/metabolismo , Purinas/metabolismo , Animales , Biomarcadores , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Movimiento Celular , Células Cultivadas , Evolución Clonal , Técnicas de Cocultivo , Sangre Fetal/citología , Perfilación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunofenotipificación , Ratones , Ratones TransgénicosRESUMEN
Antigen recognition by T cells bearing αß T cell receptors (TCRs) is restricted by major histocompatibility complex (MHC). However, how antigens are recognized by T cells bearing γδ TCRs remains unclear. Although γδ T cells can recognize nonclassical MHC, it is generally thought that recognition of antigens is not MHC restricted. Here, we took advantage of an in vitro system to generate antigen-specific human T cells and show that melanoma-associated antigens, MART-1 and gp100, can be recognized by γδ T cells in an MHC-restricted fashion. Cloning and transferring of MART-1-specific γδ TCRs restored the specific recognition of the initial antigen MHC/peptide reactivity and conferred antigen-specific functional responses. A crystal structure of a MART-1-specific γδ TCR, together with MHC/peptide, revealed distinctive but similar docking properties to those previously reported for αß TCRs, recognizing MART-1 on HLA-A*0201. Our work shows that antigen-specific and MHC-restricted γδ T cells can be generated in vitro and that MART-1-specific γδ T cells can also be found and cloned from the naïve repertoire. These findings reveal that classical MHC-restricted human γδ TCRs exist in the periphery and have the potential to be used in developing of new TCR-based immunotherapeutic approaches.
Asunto(s)
Antígeno MART-1/inmunología , Melanoma/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Adulto , Células Cultivadas , Cristalografía por Rayos X , Humanos , Antígeno MART-1/química , Modelos Moleculares , Receptores de Antígenos de Linfocitos T gamma-delta/químicaRESUMEN
IL-17-producing γδ T (γδT17) cells are critical components of the innate immune system. However, the gene networks that control their development are unclear. Here we show that HEB (HeLa E-box binding protein, encoded by Tcf12) is required for the generation of a newly defined subset of fetal-derived CD73- γδT17 cells. HEB is required in immature CD24+CD73- γδ T cells for the expression of Sox4, Sox13, and Rorc, and these genes are repressed by acute expression of the HEB antagonist Id3. HEB-deficiency also affects mature CD73+ γδ T cells, which are defective in RORγt expression and IL-17 production. Additionally, the fetal TCRγ chain repertoire is altered, and peripheral Vγ4 γδ T cells are mostly restricted to the IFNγ-producing phenotype in HEB-deficient mice. Therefore, our work identifies HEB-dependent pathways for the development of CD73+ and CD73- γδT17 cells, and provides mechanistic evidence for control of the γδT17 gene network by HEB.