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. 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: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: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 distinct CD4+ helper T (Th) cell subsets, such as Th1, Th2, Th17, and regulatory T cells (Treg), play a pivotal role in the host-defense against pathogen invasion and the pathogenesis of inflammatory disorders. In this project, DIA-MS-based proteome analysis was performed on naïve CD4+ T, Th0, Th1, Th2, Th17 and iTreg cells using Q Exactive HF-X (Thermo Fisher Scientific) to search for proteins that differ among the cell subsets.
Project description:The aim of this study was to identify differentially-expressed genes in CCR4hi/CXCR3- and CCR4lo CXCR3+ CCR6+ human Th17 cell subsets Human CD45RO+ memory T cells isolated from the peripheral blood of healthy adult donors were sorted into 4 predominant CCR7lo CD25- effector memory subsets: (1) Th1 - CCR6- CCR4lo CXCR3+; (2) Th2 - CCR6- CCR4hi CXCR3+; (3) Th17 - CCR6+ CCR4hi CXCR3-; (4) Th17.1 - CCR6+ CCR4lo CXCR3-. Sorted cells were cultured in media and activated via anti-CD3/anti-CD28 beads for 36 hours. All subsets were then harvested and used for RNA extraction and microarray experiments. Th1 vs Th2; Th1 vs Th17; Th1 vs Th17.1; Th2 vs Th17; Th2 vs Th17.1; Th17 vs Th17.1
Project description:IRF4 is critical for differentiation of various CD4+ effector T cells, such as T helper 1 (Th1), Th2, and Th17 subsets, through interaction with BATF-containing AP-1 heterodimers. A major BATF heterodimeric partner, JunB, regulates Th17 differentiation, but the role of JunB in other CD4+ effector T subsets is not fully understood. Here we demonstrate that JunB is essential for accumulation of Th1 and Th2 cells, as well as Th17 cells, both in vitro and in vivo. In mice immunized with lipopolysaccharide (LPS), papain, or complete Freund’s adjuvant (CFA), that induce predominantly Th1, Th2 and Th17 cells, respectively, accumulation of antigen-primed, Junb-deficient CD4+ T cells is significantly impaired. Loss of JunB decreases viability of cells activated under Th1-, Th2-, and Th17-polarizing conditions. RNA-sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) reveal that JunB directly regulates expression of various genes that are commonly induced in priming of naïve CD4+ T cells, including a pro-apoptotic gene Bcl2l11 (encoding Bim), and genes that are specifically induced in Th1, Th2, and Th17 cells. Furthermore, JunB colocalizes with BATF and IRF4 at genomic regions for approximately half of JunB direct target genes. Taken together, JunB, in collaboration with BATF and IRF4, serves a critical function in differentiation of diverse CD4+ T cells by controlling common and lineage-specific gene expression.
Project description:In this study, we examined differential gene expression in naïve human CD4+ T cells, as well as in effector Th1, Th17-negative and Th17-enriched CD4- T cell subsets. We observed a marked enrichment for increased gene expression in effector CD4+ T cells compared to naive CD4+ among immune-mediated disease oci genes. Within effector T cells, expression of disease-associated genes was increased in Th17-enriched compared to Th17-negative cells. We used microarray to examine the gene expresssion profile and level of human naïve, Th1 and effector T cell subsets. Human PBMCs were isolated and sorted to naïve, CD161-CCR6- and CD161+CCR6+ memory T cells. Naïve T cells were differentiatied to Th1 cells, and CD161-CCR6- and CD161+CCR6+ memory T cells were in vitro expanded for Th17-negative and Th17-enriched effector T cells. The gene profile was compared among naive, Th1, Th17-negative, and Th17-enriched cell subsets.
Project description:A generic genome-scale metabolic model (GEMs) of human CD4+ T-cells. Several cell-specific GEMs for CD4+ T-cell subsets such as Th1, Th2, Th17 and iTreg cells derived from "HTimmR" are included as additional files. The model formats are compatible with RAVEN v.2.0 toolbox.
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:In this study, we examined differential gene expression in naïve human CD4+ T cells, as well as in effector Th1, Th17-negative and Th17-enriched CD4- T cell subsets. We observed a marked enrichment for increased gene expression in effector CD4+ T cells compared to naive CD4+ among immune-mediated disease oci genes. Within effector T cells, expression of disease-associated genes was increased in Th17-enriched compared to Th17-negative cells. We used microarray to examine the gene expresssion profile and level of human naïve, Th1 and effector T cell subsets.
Project description:Multipotential naïve CD4+ T cells differentiate into distinct lineages including T helper 1 (Th1), Th2, Th17, and inducible T regulatory (iTreg) cells. The remarkable diversity of CD4+ T cells begs the question whether the observed changes reflect terminal differentiation with heritable epigenetic modifications or plasticity in T cell responses. We generated genome-wide histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) trimethylation maps in naïve, Th1, Th2, Th17, iTreg, and natural (n)Treg cells. We found that although modifications of signature cytokine genes (Ifng, Il4, and Il17) partially conform to the expectation of lineage commitment, critical transcription factors such as Tbx21 exhibit a broad spectrum of epigenetic states, consistent with our demonstration of T-bet and IFN-? induction in nTreg cells. Our data suggest an epigenetic mechanism underlying the specificity and plasticity of effector and regulatory T cells and also provide a framework for understanding complexity of CD4+ T helper cell differentiation. Experiment Overall Design: Different T helper subsets are profiled for mRNA expression.