RESUMO
Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers1. The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.52-7 contains a distal enhancer that is functional in CD4+ regulatory T (Treg) cells and required for Treg-mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3+ Treg cells, which are unable to control colitis in a cell-transfer model of the disease. In human Treg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy.
Assuntos
Cromossomos Humanos Par 11/genética , Colite/genética , Colite/imunologia , Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença/genética , Linfócitos T Reguladores/imunologia , Acetilação , Alelos , Animais , Cromossomos de Mamíferos/genética , Feminino , Fatores de Transcrição Forkhead/metabolismo , Histonas/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Sintenia/genéticaRESUMO
CD4+ regulatory T (Treg) cells, dependent upon the transcription factor Foxp3, contribute to tumour immunosuppression but are also required for immune homeostasis. There is interest in developing therapies that selectively target the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti-inflammatory function. High levels of expression of chemokine (C-C motif) receptor 8 (CCR8) discriminate Treg cells within tumours from those found in systemic lymphoid tissues. It has recently been proposed that disruption of CCR8 function using blocking anti-CCR8 antibodies results in reduced accumulation of Treg cells within tumours and disruption of their immunosuppressive function. Here, using Ccr8-/- mice, we show that CCR8 function is not required for Treg cell accumulation or immunosuppression in the context of syngeneic MC38 colorectal adenocarcinoma and B16 melanoma tumours. We observed high levels of CCR8 expression on tumour-infiltrating Treg cells which were abolished in Ccr8-/- mice. High levels of CCR8 marked cells with high levels of suppressive function. However, whereas systemic ablation of Treg cells resulted in strikingly diminished tumour burden, growth of subcutaneously implanted tumours was unaffected by systemic CCR8 loss. Consistently, we observed minimal impact of systemic CCR8 ablation on the frequency, phenotype and function of tumour-infiltrating Treg cells and conventional T (Tconv) function. These findings suggest that CCR8 is not required for Treg cell accumulation and immunosuppressive function within tumours and that depletion of CCR8+ Treg cells rather than blockade of CCR8 function is a more promising avenue for selective immunotherapy.
Assuntos
Adenocarcinoma/imunologia , Neoplasias Colorretais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Receptores CCR8/metabolismo , Neoplasias Cutâneas/imunologia , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição Forkhead/metabolismo , Humanos , Tolerância Imunológica , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR8/genéticaRESUMO
Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.
Assuntos
Azacitidina/farmacologia , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Células Cultivadas , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/fisiologiaRESUMO
Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8-kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.
Assuntos
Embrião de Mamíferos/metabolismo , Endoglina/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Hematopoese/fisiologia , Sondas Moleculares/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Endoglina/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Sondas Moleculares/genética , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismoRESUMO
Adoptive cell transfer therapy using CD8+ T lymphocytes showed promising results eradicating metastatic malignancies. However, several regulatory mechanisms limit its efficacy. We studied the role of the expression of the transcription factor FOXP3 on CD8+ T cell function and anti-tumor immunity. Here we show that suboptimal T cell receptor stimulation of CD8+ T cells upregulates FOXP3 in vitro. Similarly, CD8 T cells transferred into tumor-bearing mice upregulate FOXP3 in vivo. Cell-intrinsic loss of FOXP3 by CD8+ T cells resulted in improved functionality after TCR stimulation and better antitumor responses in vivo. Inhibition of the FOXP3/NFAT interaction likewise improved CD8+ T cell functionality. Transcriptomic analysis of cells after TCR stimulation revealed an enrichment of genes implicated in the response to IFN-γ, IFN-α, inflammatory response, IL-6/JAK/STAT, G2M checkpoint and IL-2/STAT signaling in FOXP3-deficient CD8+ T cells with respect to FOXP3-wt CD8+ T cells. Our results suggest that transient expression of FOXP3 by CD8+ T cells in the tumor microenvironment restrains their anti-tumor activity, with clear implications for improving T cell responses during immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição Forkhead/metabolismo , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Humanos , CamundongosRESUMO
Regulatory T (Treg) cell populations are composed of functionally quiescent resting Treg (rTreg) cells which differentiate into activated Treg (aTreg) cells upon antigen stimulation. How rTreg cells remain quiescent despite chronic exposure to cognate self- and foreign antigens is unclear. The transcription factor BACH2 is critical for early Treg lineage specification, but its function following lineage commitment is unresolved. Here, we show that BACH2 is repurposed following Treg lineage commitment and promotes the quiescence and long-term maintenance of rTreg cells. Bach2 is highly expressed in rTreg cells but is down-regulated in aTreg cells and during inflammation. In rTreg cells, BACH2 binds to enhancers of genes involved in aTreg differentiation and represses their TCR-driven induction by competing with AP-1 factors for DNA binding. This function promotes rTreg cell quiescence and long-term maintenance and is required for immune homeostasis and durable immunosuppression in cancer. Thus, BACH2 supports a "division of labor" between quiescent rTreg cells and their activated progeny in Treg maintenance and function, respectively.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Ciclo Celular , Homeostase , Terapia de Imunossupressão , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Linhagem da Célula , Citocinas/metabolismo , Regulação para Baixo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/patologia , Integrases/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias/genética , Fenótipo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Fator de Transcrição AP-1/metabolismoRESUMO
During embryonic development, hematopoietic cells develop by a process of endothelial-to hematopoietic transition of a specialized population of endothelial cells. These hemogenic endothelium (HE) cells in turn develop from a primitive population of FLK1(+) mesodermal cells. Endoglin (ENG) is an accessory TGF-ß receptor that is enriched on the surface of endothelial and hematopoietic stem cells and is also required for the normal development of hemogenic precursors. However, the functional role of ENG during the transition of FLK1(+) mesoderm to hematopoietic cells is ill defined. To address this we used a murine embryonic stem cell model that has been shown to mirror the temporal emergence of these cells in the embryo. We noted that FLK1(+) mesodermal cells expressing ENG generated fewer blast colony-forming cells but had increased hemogenic potential when compared with ENG non-expressing cells. TIE2(+)/CD117(+) HE cells expressing ENG also showed increased hemogenic potential compared with non-expressing cells. To evaluate whether high ENG expression accelerates hematopoiesis, we generated an inducible ENG expressing ES cell line and forced expression in FLK1(+) mesodermal or TIE2(+)/CD117(+) HE cells. High ENG expression at both stages accelerated the emergence of CD45(+) definitive hematopoietic cells. High ENG expression was associated with increased pSMAD2/eNOS expression and NO synthesis in hemogenic precursors. Inhibition of eNOS blunted the ENG induced increase in definitive hematopoiesis. Taken together, these data show that ENG potentiates the emergence of definitive hematopoietic cells by modulating TGF-ß/pSMAD2 signalling and increasing eNOS/NO synthesis.