RESUMO
Vertebrate cell identity depends on the combined activity of scores of transcription factors (TF). While TFs have often been studied in isolation, a systematic perspective on their integration has been missing. Focusing on FoxP3+ regulatory T cells (Tregs), key guardians of immune tolerance, we combined single-cell chromatin accessibility, machine learning, and high-density genetic variation, to resolve a validated framework of diverse Treg chromatin programs, each shaped by multi-TF inputs. This framework identified previously unrecognized Treg controllers (Smarcc1) and illuminated the mechanism of action of FoxP3, which amplified a pre-existing Treg identity, diversely activating or repressing distinct programs, dependent on different regulatory partners. Treg subpopulations in the colon relied variably on FoxP3, Helios+ Tregs being completely dependent, but RORγ+ Tregs largely independent. These differences were rooted in intrinsic biases decoded by the integrated framework. Moving beyond master regulators, this work unravels how overlapping TF activities coalesce into Treg identity and diversity.
Assuntos
Fatores de Transcrição Forkhead , Linfócitos T Reguladores , Fatores de Transcrição , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Cromatina/metabolismo , Cromatina/genética , Análise de Célula Única/métodosRESUMO
Foxp3+ regulatory T cells (Tregs) in the colon are key to promoting peaceful coexistence with symbiotic microbes. Differentiated in either thymic or peripheral locations, and modulated by microbes and other cellular influencers, colonic Treg subsets have been identified through key transcription factors (TFs; Helios, Rorγ, Gata3, and cMaf), but their interrelationships are unclear. Applying a multimodal array of immunologic, genomic, and microbiological assays, we find more overlap than expected between populations. The key TFs (Rorγ, Helios, Gata3, and cMaf) play different roles, some essential for subset identity, others driving functional gene signatures. Functional divergence was clearest under challenge. Single-cell genomics revealed a spectrum of phenotypes between the Helios+ and Rorγ+ poles, different Treg-inducing bacteria inducing the same Treg phenotypes to varying degrees, not distinct populations. TCR repertoires in monocolonized mice revealed that Helios+ and Rorγ+ Tregs are related and cannot be uniquely equated to tTreg and pTreg. Comparison of spleen and colon repertoires revealed that 2 to 5% of clonotypes are shared between the locations. We propose that rather than the origin of their differentiation, tissue-specific cues dictate the spectrum of colonic Treg phenotypes.
Assuntos
Linfócitos T Reguladores , Fatores de Transcrição , Camundongos , Animais , Fatores de Transcrição/genética , Diferenciação Celular/genética , Timo , Colo , Fatores de Transcrição Forkhead/genéticaRESUMO
Mutations of the transcription factor FoxP3 in patients with "IPEX" (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome) disrupt regulatory T cells (Treg), causing an array of multiorgan autoimmunity. To understand the functional impact of mutations across FoxP3 domains, without genetic and environmental confounders, six human FOXP3 missense mutations are engineered into mice. Two classes of mutations emerge from combined immunologic and genomic analyses. A mutation in the DNA-binding domain shows the same lymphoproliferation and multiorgan infiltration as complete FoxP3 knockouts but delayed by months. Tregs expressing this mutant FoxP3 are destabilized by normal Tregs in heterozygous females compared with hemizygous males. Mutations in other domains affect chromatin opening differently, involving different cofactors and provoking more specific autoimmune pathology (dermatitis, colitis, diabetes), unmasked by immunological challenges or incrossing NOD autoimmune-susceptibility alleles. This work establishes that IPEX disease heterogeneity results from the actual mutations, combined with genetic and environmental perturbations, explaining then the intra-familial variation in IPEX.
Assuntos
Fatores de Transcrição Forkhead , Linfócitos T Reguladores , Animais , Feminino , Humanos , Masculino , Camundongos , Alelos , Fatores de Transcrição Forkhead/genética , Camundongos Endogâmicos NOD , Mutação/genéticaRESUMO
Foxp3 + regulatory T cells (Tregs) in the colon are key to promoting peaceful co-existence with symbiotic microbes. Differentiated in either thymic or peripheral locations, and modulated by microbes and other cellular influencers, colonic Treg subsets have been identified through key transcription factors (TF; Helios, Rorg, Gata3, cMaf), but their inter-relationships are unclear. Applying a multimodal array of immunologic, genomic, and microbiological assays, we find more overlap than expected between populations. The key TFs play different roles, some essential for subset identity, others driving functional gene signatures. Functional divergence was clearest under challenge. Single-cell genomics revealed a spectrum of phenotypes between the Helios+ and Rorγ+ poles, different Treg-inducing bacteria inducing the same Treg phenotypes to varying degrees, not distinct populations. TCR clonotypes in monocolonized mice revealed that Helios+ and Rorγ+ Tregs are related, and cannot be uniquely equated to tTreg and pTreg. We propose that rather than the origin of their differentiation, tissue-specific cues dictate the spectrum of colonic Treg phenotypes.
RESUMO
Regulatory T cells (Treg cells) are key players in ensuring a peaceful coexistence with microorganisms and food antigens at intestinal borders. Startling new information has appeared in recent years on their diversity, the importance of the transcription factor FOXP3, how T cell receptors influence their fate and the unexpected and varied cellular partners that influence Treg cell homeostatic setpoints. We also revisit some tenets, maintained by the echo chambers of Reviews, that rest on uncertain foundations or are a subject of debate.
Assuntos
Microbioma Gastrointestinal , Linfócitos T Reguladores , Humanos , Intestinos , Antígenos , Receptores de Antígenos de Linfócitos T , Fatores de Transcrição ForkheadRESUMO
Specific combinations of transcription factors (TFs) control the gene expression programs that underlie specialized immune responses. Previous models of TF function in immunocytes had restricted each TF to a single functional categorization [e.g., lineage-defining (LDTFs) vs. signal-dependent TFs (SDTFs)] within one cell type. Synthesizing recent results, we instead propose a variegated model of immunological TF function, whereby many TFs have flexible and different roles across distinct cell states, contributing to cell phenotypic diversity. We discuss evidence in support of this variegated model, describe contextual inputs that enable TF diversification, and look to the future to imagine warranted experimental and computational tools to build quantitative and predictive models of immunocyte gene regulatory networks.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Redes Reguladoras de Genes , Sistema Imunitário/metabolismoRESUMO
Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organoid or animal studies. While protocols for these analyses are typically developed with biosafety level 2 (BSL-2) considerations in mind, such analyses are equally useful for the study of viruses that require higher biosafety containment levels. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories ensuring virus inactivation and must therefore be modified. Here we show that TCL buffer (Qiagen), which was developed for bulk sequencing of small numbers of cells and also facilitates scRNA-seq, inactivates both Ebola virus (EBOV) and SARS-CoV-2, BSL-4 and BSL-3 viruses, respectively. We show that additional heat treatment, necessary for the more stringent biosafety concerns for BSL-4-derived samples, was additionally sufficient to inactivate EBOV-containing samples. Critically, this heat treatment had minimal effects on extracted RNA quality and downstream sequencing results.
RESUMO
Gene expression programs are specified by higher-order chromatin structure and enhancer-promoter loops (EPLs). T regulatory cell (Treg) identity is dominantly specified by the transcription factor (TF) FoxP3, whose mechanism of action is unclear. We applied chromatin conformation capture with immunoprecipitation (HiChIP) in Treg and closely related conventional CD4+ T cells (Tconv). EPLs identified by H3K27Ac HiChIP showed a range of connection intensity, with some superconnected genes. TF-specific HiChIP showed that FoxP3 interacts with EPLs at a large number of genes, including some not differentially expressed in Treg versus Tconv, but enriched at the core Treg signature loci that it up-regulates. FoxP3 association correlated with heightened H3K27Ac looping, as ascertained by analysis of FoxP3-deficient Treg-like cells. There was marked asymmetry in the loci where FoxP3 associated at the enhancer- or the promoter-side of EPLs, with enrichment for different transcriptional cofactors. FoxP3 EPL intensity distinguished gene clusters identified by single-cell ATAC-seq as covarying between individual Tregs, supporting a direct transactivation model for FoxP3 in determining Treg identity.
Assuntos
Fatores de Transcrição Forkhead/imunologia , Regiões Promotoras Genéticas/genética , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.
Assuntos
COVID-19/imunologia , Células Epiteliais/imunologia , Monócitos/imunologia , SARS-CoV-2/patogenicidade , Adulto , Linfócitos B/imunologia , COVID-19/patologia , Criança , Técnicas de Cocultura , Ebolavirus/patogenicidade , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Inflamação , Vírus da Influenza A/patogenicidade , Pulmão/imunologia , Células Mieloides/imunologia , Especificidade da Espécie , Proteínas Virais/imunologiaRESUMO
The hallmark of severe COVID-19 is an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We hypothesized that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, correlating with poor outcomes. These Tregs showed a distinct transcriptional signature, with overexpression of several suppressive effectors, but also proinflammatory molecules like interleukin (IL)-32, and a striking similarity to tumor-infiltrating Tregs that suppress antitumor responses. Most marked during acute severe disease, these traits persisted somewhat in convalescent patients. A screen for candidate agents revealed that IL-6 and IL-18 may individually contribute different facets of these COVID-19-linked perturbations. These results suggest that Tregs may play nefarious roles in COVID-19, by suppressing antiviral T cell responses during the severe phase of the disease, and by a direct proinflammatory role.
Assuntos
COVID-19/etiologia , Linfócitos T Reguladores/fisiologia , Adulto , Idoso , Linfócitos T CD4-Positivos/virologia , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Inflamação/virologia , Interleucina-18/genética , Interleucina-18/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Linfócitos do Interstício Tumoral/fisiologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/virologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
CD4+ effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic Teff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (TH) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as TH markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ.
Assuntos
Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/parasitologia , Colo/microbiologia , Colo/parasitologia , Microbioma Gastrointestinal , Heligmosomatoidea/patogenicidade , Enteropatias Parasitárias/parasitologia , Animais , Bactérias/imunologia , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Citrobacter rodentium/imunologia , Citrobacter rodentium/patogenicidade , Colo/imunologia , Colo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Heligmosomatoidea/imunologia , Interações Hospedeiro-Patógeno , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Enteropatias Parasitárias/genética , Enteropatias Parasitárias/imunologia , Enteropatias Parasitárias/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nematospiroides dubius/imunologia , Nematospiroides dubius/patogenicidade , Nippostrongylus/imunologia , Nippostrongylus/patogenicidade , Fenótipo , Salmonella enterica/imunologia , Salmonella enterica/patogenicidade , Análise de Célula Única , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , TranscriptomaRESUMO
The hallmark of severe COVID-19 disease has been an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We explored the hypothesis that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in both Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, which correlated with poor outcomes. Accordingly, these Tregs over-expressed a range of suppressive effectors, but also pro-inflammatory molecules like IL32. Most strikingly, they acquired similarity to tumor-infiltrating Tregs, known to suppress local anti-tumor responses. These traits were most marked in acute patients with severe disease, but persisted somewhat in convalescent patients. These results suggest that Tregs may play nefarious roles in COVID-19, via suppressing anti-viral T cell responses during the severe phase of the disease, and/or via a direct pro-inflammatory role.
RESUMO
Human erythropoiesis serves as a paradigm of physiologic cellular differentiation. This process is also of considerable interest for better understanding anemias and identifying new therapies. Here, we apply deep transcriptomic and accessible chromatin profiling to characterize a faithful ex vivo human erythroid differentiation system from hematopoietic stem and progenitor cells. We reveal stage-specific transcriptional states and chromatin accessibility during various stages of erythropoiesis, including 14,260 differentially expressed genes and 63,659 variably accessible chromatin peaks. Our analysis suggests differentiation stage-predominant roles for specific master regulators, including GATA1 and KLF1. We integrate chromatin profiles with common and rare genetic variants associated with erythroid cell traits and diseases, finding that variants regulating different erythroid phenotypes likely act at variable points during differentiation. In addition, we identify a regulator of terminal erythropoiesis, TMCC2, more broadly illustrating the value of this comprehensive analysis to improve our understanding of erythropoiesis in health and disease.
Assuntos
Montagem e Desmontagem da Cromatina , Eritropoese , Transcriptoma , Idoso , Células Cultivadas , Cromatina/química , Cromatina/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Feminino , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
A hexanucleotide (GGGGCC) repeat expansion in C9ORF72 is the most common genetic contributor to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Reduced expression of the C9ORF72 gene product has been proposed as a potential contributor to disease pathogenesis. Additionally, repetitive RNAs and dipeptide repeat proteins (DPRs), such as poly-GR, can be produced by this hexanucleotide expansion that disrupt a number of cellular processes, potentially contributing to neural degeneration. To better discern which of these mechanisms leads to disease-associated changes in patient brains, we analyzed gene expression data generated from the cortex and cerebellum. We found that transcripts encoding heat shock proteins (HSPs) regulated by the HSF1 transcription factor were significantly induced in C9ORF72-ALS/FTLD patients relative to both sporadic ALS/FTLD cases and controls. Treatment of human neurons with chemically synthesized DPRs was sufficient to activate a similar transcriptional response. Expression of GGGGCC repeats and also poly-GR in the brains of Drosophila lead to the upregulation of HSF1 and the same highly-conserved HSPs. Additionally, HSF1 was a modifier of poly-GR toxicity in Drosophila. Our results suggest that the expression of DPRs are associated with upregulation of HSF1 and activation of a heat shock response in C9ORF72-ALS/FTLD.