ATAC-seq of primary human T Follicular Helper (TFH) cells and naive CD4-positive helper T cells from tonsils of healthy volunteers
ABSTRACT: Genome Wide Association Studies (GWAS) have been successful in yielding >60 loci for Systemic Lupus Erythematosus (SLE). However, it is known that GWAS just reports genomic signals and not necessarily the precise localization of culprit genes, with eQTL efforts only able to infer causality to a minority of such loci. Thus, we sought to carry out physical and direct ‘variant to gene mapping’ by integrating results from high-throughput chromatin conformation capture and ATAC-seq assays. This experiment refers to the ATAC-seq part of our work. To determine informative proxy SNPs for each of the SLE GWAS sentinel loci, we generated ATAC-seq open chromatin maps for primary human T Follicular Helper (TFH) cells from tonsils of healthy volunteers (3 biological replicates), a model relevant to SLE as TFH operate upstream of the activation of pathogenic autoantibody-producing B cells during the disease. We also generated open chromatin maps for naive CD4-positive helper T cells (3 biological replicates).
Project description:Genome Wide Association Studies (GWAS) have been successful in yielding >60 loci for Systemic Lupus Erythematosus (SLE). However, it is known that GWAS just reports genomic signals and not necessarily the precise localization of culprit genes, with eQTL efforts only able to infer causality to a minority of such loci. Thus, we sought to carry out physical and direct ‘variant to gene mapping’ by integrating results from high-throughput chromatin conformation capture and ATAC-seq assays. This experiment refers to the chromatin conformation capture part of our work. Detecting contacts between distant regions of the genome offers a powerful opportunity to understand GWAS signals that principally reside in non-coding regions, and thus likely act as regulatory elements for neighboring genes. To move beyond analyzing one locus at a time and to improve on the low resolution of available Hi-C data, we employed a massively parallel, high resolution Capture-C based method to simultaneously characterize the genome-wide interactions of all human promoters in any cell type. We applied this approach to study the promoter ‘interactome’ of primary human T Follicular Helper (TFH) cells from tonsils of healthy volunteers (3 biological replicates), a model relevant to SLE as TFH operate upstream of the activation of pathogenic autoantibody-producing B cells during the disease. We also analyzed the promoter interactome of naive CD4-positive helper T cells (3 biological replicates). We designed a custom Agilent SureSelect library targeting both ends of DpnII restriction fragments that overlap promoters of protein-coding, noncoding, antisense, snRNA, miRNA, snoRNA and lincRNA transcripts. Each library was sequenced on 8 lanes of an Illumina HiSeq 4000.
Project description:In this experiment we generated Affymetrix gene expression data for T Follicular Helper (TFH) cells from tonsils of healthy volunteers (4 biological replicates) and naive CD4-positive helper T cells (2 biological replicates). TFH cells provide a model relevant to SLE as TFH operate upstream of the activation of pathogenic autoantibody-producing B cells during the disease. This experiment accompanies promoter capture-C and ATAC-seq experiments on the same cell types.
Project description:Convincing lines of evidence in both mice and humans show that exaggerated T follicular helper (Tfh) responses is pathogenic in autoimmune diseases. However, the cause of exaggerated Tfh response in humans is still much less clear than in mouse models where genetic factors can be manipulated for in vivo testing. Nonetheless, recent advances in our understanding on the mechanisms of human Tfh differentiation and identification of multiple risk loci in genome-wide association studies have revealed several pathways potentially associated with exaggerated Tfh response in human autoimmune diseases. In this review, we will first briefly summarize the differentiation mechanisms of Tfh cells in humans. We describe the features of "Tfh-like" cells recently identified in inflamed tissues of human autoimmune diseases. Then we will discuss how risk loci identified in GWAS are potentially involved in exaggerated Tfh response in human autoimmune diseases.
Project description:T follicular helper (Tfh) cells aid effector B cells, and augment autoimmunity, whereas the role of Tfh cells on regulatory B (Breg) cells in systemic lupus erythematosus (SLE) is not known. The aim of this study is to investigate the percentage of Breg cells in SLE, and the role of Tfh cells on Breg cells. First, we demonstrated the presence of Breg cells in SLE peripheral blood mononuclear cells and in involved skins. Both the percentage of circulating Breg cells and the ability to produce interleukin-10 (IL-10) were elevated in SLE patients. The percentage of Breg cells increased during SLE flares and decreased following disease remission. Second, Tfh cell expansion was not only related to autoantibody production but also correlated with the increased percentage of Breg cells. Third, in vitro studies revealed that Tfh cell-derived IL-21 could promote IL-10 production and Breg cell differentiation. In conclusions, these data imply that SLE flares may be linked to the expansion of Tfh cells and that Breg cells are increased in a regulatory feedback manner. Thus, SLE development may be associated with the complex regulation of Tfh cells and diverse B cell subsets.
Project description:Increased activity of T follicular helper (Tfh) cells plays a major pathogenic role in systemic lupus erythematosus (SLE). However, the mechanisms that cause aberrant Tfh cell responses in SLE remain elusive. Here we showed the OX40 ligand (OX40L)-OX40 axis contributes to the aberrant Tfh response in SLE. OX40L was expressed by myeloid antigen-presenting cells (APCs), but not B cells, in blood and in inflamed tissues in adult and pediatric SLE patients. The frequency of circulating OX40L-expressing myeloid APCs positively correlated with disease activity and the frequency of ICOS(+) blood Tfh cells in SLE. OX40 signals promoted naive and memory CD4(+) T cells to express multiple Tfh cell molecules and were sufficient to induce them to become functional B cell helpers. Immune complexes containing RNA induced OX40L expression on myeloid APCs via TLR7 activation. Our study provides a rationale to target the OX40L-OX40 axis as a therapeutic modality for SLE.
Project description:T follicular helper (Tfh) cells are indispensable for the formation of germinal center (GC) reactions, whereas T follicular regulatory (Tfr) cells inhibit Tfh-mediated GC responses. Aberrant activation of Tfh cells contributes substantially to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE). Nonetheless, the molecular mechanisms mitigating excessive Tfh cell differentiation are not fully understood. Herein we demonstrate that the adenovirus E4 promoter-binding protein (E4BP4) mediates a feedback loop and acts as a transcriptional brake to inhibit Tfh cell differentiation. Furthermore, we show that such an immunological mechanism is compromised in patients with SLE. Establishing mice with either conditional knockout (cKO) or knockin (cKI) of the E4bp4 gene in T cells reveals that E4BP4 strongly inhibits Tfh cell differentiation. Mechanistically, E4BP4 regulates Bcl6 transcription by recruiting the repressive epigenetic modifiers HDAC1 and EZH2. E4BP4 phosphorylation site mutants have limited capability with regard to inhibiting Tfh cell differentiation. In SLE, we detected impaired phosphorylation of E4BP4, finding that this compromised transcription factor is positively correlated with disease activity. These findings unveiled molecular mechanisms by which E4BP4 restrains Tfh cell differentiation, whose compromised function is associated with uncontrolled autoimmune reactions in SLE.
Project description:Helping B cells and antibody responses is a major function of CD4+T helper cells. Follicular helper T (Tfh) cells are identified as a subset of CD4+T helper cells, which is specialized in helping B cells in the germinal center reaction. Tfh cells express high levels of CXCR5, PD-1, IL-21, and other characteristic markers. Accumulating evidence has demonstrated that the dysregulation of Tfh cells is involved in infectious, inflammatory, and autoimmune diseases, including lymphocytic choriomeningitis virus (LCMV) infection, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), IgG4-related disease (IgG4-RD), Sjögren syndrome (SS), and type 1 diabetes (T1D). Activation of subset-specific transcription factors is the essential step for Tfh cell differentiation. The differentiation of Tfh cells is regulated by a complicated network of transcription factors, including positive factors (Bcl6, ATF-3, Batf, IRF4, c-Maf, and so on) and negative factors (Blimp-1, STAT5, IRF8, Bach2, and so on). The current knowledge underlying the molecular mechanisms of Tfh cell differentiation at the transcriptional level is summarized in this paper, which will provide many perspectives to explore the pathogenesis and treatment of the relevant immune diseases.
Project description:To assess circulating follicular helper T (Tfh)-like CD4+ T cells in patients with systemic lupus erythematosus (SLE) and determine their relationship to disease activity.Blood samples from patients with SLE, as well as blood samples from patients with Behçet's disease (BD) and healthy individuals as controls, were analyzed. In all samples, circulating Tfh-like cells were enumerated by flow cytometry, using, as markers, expression of CXCR5, inducible T cell costimulator (ICOS), and programmed death 1 (PD-1) protein, as well as secretion of interleukin-21 (IL-21). The frequency of circulating Tfh-like cells was compared to that of circulating plasmablasts (CD19+IgD-CD38+). In addition, the possible association of circulating Tfh-like cells with the SLE Disease Activity Index (SLEDAI) was evaluated.The subset of circulating Tfh-like T cells, identified as CXCR5(high) ICOS(high) PD-1(high) , was expanded in the blood of SLE patients compared to controls. Circulating Tfh-like cells were found to produce IL-21 and had lower expression of CCR7 as compared to that in circulating CXCR5(high) central memory T cells, thereby enabling their distinction. Expression of PD-1, but not ICOS or CXCR5, was significantly elevated in circulating Tfh-like cells from SLE patients compared to controls. PD-1 expression among CXCR5(high) circulating Tfh-like cells correlated with the SLEDAI, frequency of circulating plasmablasts, and anti-double-stranded DNA antibody positivity, but not with disease duration or past organ injury; rather, this cell profile appeared to be a reflection of current active disease.Circulating Tfh-like cells are associated with disease activity in SLE, suggesting that their presence indicates abnormal homeostasis of T cell-B cell collaboration, with a causal relationship that is central to disease pathogenesis. These findings also suggest that circulating Tfh-like cells provide a surrogate for aberrant germinal center activity in SLE, and that their PD-1 expression offers a tool for measuring disease activity and monitoring the response to therapies.
Project description:Neuropsychiatric symptoms in systemic lupus erythematosus (SLE) are not uncommon, yet the mechanisms underlying disease initiation and progression in the brain are incompletely understood. Although the role of T cells in other lupus target organs such as the kidney is well defined, which T cells contribute to the pathogenesis of neuropsychiatric SLE is not known. The present study was aimed at characterizing the CD4 T cell populations that are present in the choroid plexus (CP) of MRL/MpJ-faslpr mice, the primary site of brain infiltration in this classic lupus mouse model which exhibits a prominent neurobehavioral phenotype. T cells infiltrating the CP of MRL/MpJ-faslpr mice were characterized and subset identification was done by multiparameter flow cytometry. We found that the infiltrating CD4 T cells are activated and have an effector phenotype. Importantly, CD4 T cells have a T follicular helper cell (TFH) like phenotype, as evidenced by their surface markers and signature cytokine, IL-21. In addition, CD4 TFH cells also secrete significant levels of IFN-? and express Bcl-6, thereby conforming to a potentially pathogenic T helper population that can drive the disease progression. Interestingly, the regulatory axis comprising CD4 T regulatory cells is diminished. These results suggest that accumulation of CD4 TFH in the brain of MRL/MpJ-faslpr mice may contribute to the neuropsychiatric manifestations of SLE, and point to this T cell subset as a possible novel therapeutic candidate.