Profiling of human CD4+ T subsets identifies a Th2-specific non-coding RNA GATA3-AS1
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ABSTRACT: Host defense against diverse pathogens involves the recruitment and differentiation of CD4+ T effector subsets including T helper 1 (Th1), Th2, Th17 and induced regulatory T (Treg) cells. Surface phenotype studies have revealed subset-specific surface markers for the identification and purification of human primary CD4+ T effector subsets. In the present study, we aimed to characterize the mRNA and large intergenic non-coding RNA (lincRNA) expression differences between human primary CD4+ T effector subsets and identify potential subset-specific genes. To achieve this goal, mRNA and lincRNA microarray profiling of flow cytometry-sorted human primary Th1, Th2, Th17 and Treg cells was performed. Principal component and pathway analyses revealed subset-specific gene expression patterns. A Th2-specific lincRNA, GATA3-AS1, also termed FLJ45983, was identified in primary immune cells and tissues, as well as in in vitro polarized CD4+ T effector subsets. Further analysis showed that GATA3-AS1 was a potential diagnostic marker in allergy, a Th2-associated disease. This first systematic genome-wide analysis of gene expression differences between primary CD4+ T effector subsets may help to identify novel regulatory protein-coding genes and lincRNAs regulating CD4+ T cell subset differentiation, as well as potential diagnostic markers. As an example, we identified a GATA3-associated Th2-specific marker lincRNA GATA3-AS1.
Project description:Host defense against diverse pathogens involves the recruitment and differentiation of CD4+ T effector subsets including T helper 1 (Th1), Th2, Th17 and induced regulatory T (Treg) cells. Surface phenotype studies have revealed subset-specific surface markers for the identification and purification of human primary CD4+ T effector subsets. In the present study, we aimed to characterize the mRNA and large intergenic non-coding RNA (lincRNA) expression differences between human primary CD4+ T effector subsets and identify potential subset-specific genes. To achieve this goal, mRNA and lincRNA microarray profiling of flow cytometry-sorted human primary Th1, Th2, Th17 and Treg cells was performed. Principal component and pathway analyses revealed subset-specific gene expression patterns. A Th2-specific lincRNA, GATA3-AS1, also termed FLJ45983, was identified in primary immune cells and tissues, as well as in in vitro polarized CD4+ T effector subsets. Further analysis showed that GATA3-AS1 was a potential diagnostic marker in allergy, a Th2-associated disease. This first systematic genome-wide analysis of gene expression differences between primary CD4+ T effector subsets may help to identify novel regulatory protein-coding genes and lincRNAs regulating CD4+ T cell subset differentiation, as well as potential diagnostic markers. As an example, we identified a GATA3-associated Th2-specific marker lincRNA GATA3-AS1. Gene expression microarray analysis of flow-cytometry sorted human primary naïve CD4+ T cells, CD4+ T central memory cells, Th1, Th2, Th17 and Treg cells from buffy coat of four healthy controls Gene expression microarray analysis was performed using SurePrint G3 Human Gene Expression 8X60K microarray.
Project description:Although lincRNAs are implicated in regulating gene expression in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identify 1,524 lincRNAs in 42 T cell samples from early T cell progenitors to terminally differentiated T helper subsets. Our analysis revealed highly dynamic and cell-specific expression patterns of lincRNAs during T cell differentiation. Importantly, these lincRNAs are located in genomic regions enriched for protein-coding genes with immune-regulatory functions. Many of these transcripts are bound and regulated by the key T cell transcription factors, T-bet, GATA3, STAT4 and STAT6. We demonstrate that the lincRNA LincR-Ccr2-5'AS, together with GATA3, is an essential component of a regulatory circuit in Th2-specific gene expression. To obtain comprehensive profiles of lincRNA expression during the development and differentiation of T cell lineages, we purified CD4-CD8 double negative (DN) cells (DN1, DN2, DN3 and DN4), double positive (DP) cells (CD4+CD8+CD3low and CD4+CD8intCD69+), single positive (SP) CD4 and CD8 cells, and thymic-derived regulatory T cells (tTreg) from thymi of C57BL/6 mice. Additionally, we obtained Th1, Th2, Th17 and iTreg cells by in vitro differentiation of naM-CM-/ve CD4 T cells for a various length of time in culture (4 hrs, 8 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs, 1 week, 2weeks). Total and/or polyadenylated RNAs from these cells was analyzed using RNA-Seq. To understand the regulation of lincRNAs by T cell master regulator T-bet, we compared the transcriptiomes between T-bet deficient Th1 cells and control Th1 cells. We did similar experiments and data analysis for STAT4 (Th1), GATA3 (Th2) and STAT6 (Th2). Finally, to address the funcation of a Th2-specifically expressed lincRNA, lincR-Ccr2-5'AS, we compared the transcriptomes between lincR-Ccr2-5'AS knockdown Th2 cells and control Th2 cells.
Project description:Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in in vivo generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity. Mock-transfected and GATA3 siRNA-transfected Th2 and Th2-4th cells are profiled for mRNA expression
Project description:Differentiation of naive CD4 T cells into type 2 helper (Th2) cells is accompanied by chromatin remodeling and increased expression of a set of Th2-specific genes including those encoding Th2 cytokines. IL-4-mediated STAT6 activation induces high levels of transcription of GATA3, a master regulator of Th2 cell differentiation, and enforced expression of GATA3 induces Th2 cytokine expression. However, it remains unclear whether the expression of other Th2-specific genes is induced directly by GATA3. A genome-wide unbiased ChIP-seq analysis revealed that GATA3 bound to 1,279 genes selectively in Th2 cells, and 101 genes in both Th1 and Th2 cells. Simultaneously, we identified 26 highly Th2-specific STAT6-dependent inducible genes by a DNA microarray analysis-based three-step selection processes, and among them 17 genes showed GATA3 binding. We assessed dependency on GATA3 for the transcription of these 26 Th2-specific genes, and 10 genes showed increased transcription in a GATA3-dependent manner while 16 genes showed no significant responses. The transcription of the 16 GATA3-nonresponding genes was clearly increased by the introduction of an active form of STAT6, STAT6VT. Therefore, although GATA3 has been recognized as a master regulator of Th2 cell differentiation, many Th2-specific genes are not regulated by GATA3 itself but in collaboration with STAT6. Th1 and Th2 subsets are profiled for mRNA expression Examination of GATA3-binding and 3 different histone modifications in Th1 and Th2 cells.
Project description:The type 2 helper effector program is driven by the master transcription factor GATA3 and can be expressed by subsets of both innate lymphoid cells (ILCs) and adaptive CD4+ T helper (Th) cells. While ILC2s and Th2 cells acquire their type 2 differentiation program under very different contexts, the distinct regulatory mechanisms governing this common program are only partially understood. Here we show that the differentiation of ILC2s, and their concomitant high level of GATA3 expression, are controlled by a Gata3 enhancer, Gata3 +674/762, that plays only a minimal role in Th2 cell differentiation. Mice lacking this enhancer exhibited defects in several but not all type 2 inflammatory responses, depending on the respective degree of ILC2 and Th2 cell involvement. Our study provides molecular insights into the different gene regulatory pathways leading to the acquisition of the GATA3-driven type 2 helper effector program in innate and adaptive lymphocytes.
Project description:Unraveling the diversity of Th2 effector cells subsets may help us to understand their pathogenic role in Type-2 associated inflammatory disorders, including allergic diseases and helminth infections. To characterize the heterogeneity and function of these effector Th2 cells, we analyzed 47 subjects’ PBMCs [23 filarial-infected (Fil+) with or without coincident HDM sensitization (Fil+HDM+ n=12; Fil+HDM-, n=11) and 24 subjects without filarial infection (Fil-) (Fil-HDM+ n=12; and Fil-HDM-, n=12) using multiparameter flow cytometry and two-level FlowSOM analysis. The frequency of 3 memory CD4+ T cell clusters, including CCR4+CCR6+CRTH2- (subset 1), CCR4+CCR6-CRTH2+ (subset 2), and CCR6+CCR4+CRTH2+ (subset 3) were markedly enriched among Fil+ subjects. The functional characterization indicated subset 2 and 3 as distinct Th2 cytokine producers, which together were responsible for the majority of IL-4, IL-5, or IL-13 produced among the Fil+ subjects. These subsets were sorted and analyzed by multiomic single cell RNA profiling. Indeed, both subset 2 and subset 3 presented features of pathogenic Th2 effector cells based on their single cell molecular signature, including downregulation of cd27 and high expression levels of itga4, il-17rb, hpgds, klrb1, ptgdr2, il-9r, il-4, il5 and il-13 genes when compared with conventional Th2 cells from healthy controls. When the Fil+ subjects were divided based on allergic status, the Fil+HDM+ subjects had an expansion of both subset 2 (3.19% vs 1.28%, p=0.001) and subset 3 (0.39% vs 0.15%, p=0.002) Th2 cells when compared with Fil+HDM-. Gene expression analysis further demonstrated that concomitant HDM sensitization in the presence of filarial infection reshaped the molecular program of these pathogenic effector Th2 subsets by even further upregulating the expression of gata3, il17rb, clrf2, and klrb1. Notably, Fil+HDM+ patients’ cells showed higher responsiveness to filarial or HDM antigenic stimulation, producing even higher levels of IL-4, IL-5 and IL-13 when compared with Fil+HDM- patients. This distinct molecular and functional program of Th2 effector cells subsets sheds new light on the Th2 cell plasticity and their contribution to immune regulation in helminth infection and allergic diseases.
Project description:Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in in vivo generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity.
Project description:How CD4+-lineage gene expression is initiated in differentiating thymocytes remains poorly understood. Here, we show that the paralog transcription factors Zfp281 and Zfp148 control both this process and cytokine expression by Th2 effector cells. Genetic, single-cell, and spatial transcriptomic analyses show that these factors promote the intrathymic CD4+ T cell differentiation of MHC II-restricted thymocytes, including expression of the CD4+ lineage-committing factor Thpok. In peripheral T cells, Zfp281 and Zfp148 promote chromatin opening at and expression of Th2 cytokine genes, but not of the Th2 lineage determining transcription factor Gata3. Zfp281 interacts with Gata3, and binds Gata3 genomic binding sites at loci encoding Thpok and Th2 cytokines. Thus, Zfp148 and Zfp281 collaborate with Gata3 to promote CD4+ T cell development and Th2 cell responses.
Project description:Although lincRNAs are implicated in regulating gene expression in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identify 1,524 lincRNAs in 42 T cell samples from early T cell progenitors to terminally differentiated T helper subsets. Our analysis revealed highly dynamic and cell-specific expression patterns of lincRNAs during T cell differentiation. Importantly, these lincRNAs are located in genomic regions enriched for protein-coding genes with immune-regulatory functions. Many of these transcripts are bound and regulated by the key T cell transcription factors, T-bet, GATA3, STAT4 and STAT6. We demonstrate that the lincRNA LincR-Ccr2-5'AS, together with GATA3, is an essential component of a regulatory circuit in Th2-specific gene expression.