Dataset Information

The DNA methylation signature of human TCR??+CD4-CD8- double negative T cells reveals CG demethylation and a unique epigenetic architecture permissive to a broad stimulatory immune response.

ABSTRACT: T cell receptor (TCR) ??+CD4-CD8- double negative T cells represent a rare T cell subset implicated in the pathogenesis of several autoimmune diseases. We investigated the DNA methylation signature of double negative T cells to gain insight into the epigenetic architecture of peripheral blood primary human double negative T cells compared to autologous CD4+ and CD8+ T cells. We identified 2984 CG sites across the genome with unique loss of DNA methylation in double negative T cells, and showed significant reduction in mRNA expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. DNA methylation was increased in CD8A/CD8B and CD4 consistent with epigenetic repression of both the CD8 and CD4 genetic loci in double negative T cells. We show a consistent increase in non-CG methylation in double negative T cells, a finding suggestive of pluripotency. Network analyses indicate a strong relationship between double negative T cells and functions related to cell-cell interaction, cell adhesion, and cell activation pathways. Our data also suggest a robust pro-inflammatory epigenetic signature in double negative T cells, consistent with a transcriptional permissiveness in key inflammatory cytokines including IFN?, IL-17F, IL-12B, IL-5, IL-18, TNFSF11 (RANKL), and TNFSF13B (BLYS or BAFF). These findings highlight an epigenetic basis for a role of double negative T cells in autoimmunity.

SUBMITTER: Renauer PA

PROVIDER: S-EPMC4278938 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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The DNA methylation signature of human TCRαβ+CD4-CD8- double negative T cells reveals CG demethylation and a unique epigenetic architecture permissive to a broad stimulatory immune response.

Renauer Paul A PA Coit Patrick P Sawalha Amr H AH

Clinical immunology (Orlando, Fla.) 20141029 1

T cell receptor (TCR) αβ+CD4-CD8- double negative T cells represent a rare T cell subset implicated in the pathogenesis of several autoimmune diseases. We investigated the DNA methylation signature of double negative T cells to gain insight into the epigenetic architecture of peripheral blood primary human double negative T cells compared to autologous CD4+ and CD8+ T cells. We identified 2984 CG sites across the genome with unique loss of DNA methylation in double negative T cells, and showed s ...[more]

PMID: 25451162

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Project description:T cell receptor (TCR) αβ+ CD4- CD8- double negative T cells represent a rare T cell subset implicated in the pathogenesis of several autoimmune diseases. In this study, we investigated the DNA methylation signature of double negative T cells to gain insight into the epigenetic architecture and transcriptional capacity of peripheral blood primary human double negative T cells compared to autologous CD4+ and CD8+ T cells. We identified 2,984 CG sites across the genome with unique loss of DNA methylation in double negative T cells, and showed significant reduction in mRNA expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. DNA methylation was increased in CD8A/CD8B and CD4 consistent with epigenetic repression of both the CD8 and CD4 genetic loci in double negative T cells. Curiously, we show consistent increase in non-CG methylation in double negative T cells, a finding suggestive of pluripotency and previously only known to occur in embryonic stem cells and developing brain tissue. Network analyses of genes hypomethylated in double negative compared to CD4+ and CD8+ T cells indicate a strong relationship between double negative T cells and functions related to cell-cell interaction, cell adhesion, and cell activation pathways. Our data also suggest a robust pro-inflammatory epigenetic signature in double negative T cells, consistent with a transcriptional permissiveness to produce key inflammatory cytokines including IFNγ, IL-17F, IL-12B, IL-5, IL-18, TNFSF11 (RANKL), and TNFSF13B (BLYS or BAFF). These findings highlight an epigenetic basis for a role of double negative T cells in autoimmunity and extend the potential pathogenic transcriptional capacity of this unique T cell subset. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples of 7 unrelated healthy women (age range 25-60) (Supplementary Table 1) by Ficoll-gradient centrifugation (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Subsequently, T cells were negatively-selected using the Human Pan T Cell Isolation II kit (Miltenyi Biotec, Cambridge, MA). T cells were then analyzed and sorted into TCRαβ+ CD4+ T cells, TCRαβ+ CD8+ T cells, and TCRαβ+ DN T cell subsets by flow cytometry. DNA was isolated from CD4+, CD8+, and DN T cell subsets using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA), then bisulfite-converted using the EZ DNA Methylation kit (Zymo Research, Irvine, CA) for DNA methylation studies.

Project description:The role of conventional TCR??+CD4+ or TCR??+CD8?+ single-positive (sp) T lymphocytes in adaptive immunity is well-recognized. However, non-conventional T cells expressing TCR?? or TCR?? but lacking CD4 and CD8? expression [i.e., CD4-CD8?- double-negative (dn) T cells] are thought to play a role at the interface between the innate and adaptive immune system. Dn T cells are frequent in swine, cattle or sheep and predominantly express TCR??. In contrast, TCR??+ T cells are rare in dogs. In this study, we identified a high proportion of canine dn T cells in the TCR??+ T cell population of PBMC, lymphatic and non-lymphatic organs. In PBMC, the frequency of this T cell subpopulation made up one third of the frequency of TCR??+CD4+ sp, and almost half of the frequency of TCR??+CD8?+ sp T cells (i.e., ~15% of all TCR??+ T cells). Among TCR??+CD4-CD8?- dn T cells of PBMC and tissues, FoxP3+ cells were identified indicating regulatory potential of this T cell subset. 80% of peripheral blood FoxP3+TCR??+CD4-CD8?- dn T cells co-expressed CD25, and, interestingly, also the FoxP3-negative TCR??+CD4-CD8?- dn T cells comprised ~34% CD25+ cells. Some of the FoxP3-positive TCR??+CD4-CD8?- dn T cells co-expressed GATA-3 suggesting stable function of regulatory T cells. The frequency of GATA-3 expression by FoxP3-TCR??+CD4-CD8?- dn T cells was even higher as compared with TCR??+CD4+ sp T cells (20.6% vs. 11.9%). Albeit lacking FoxP3 and CD25 expression, TCR??+CD4-CD8?- dn T cells also expressed substantial proportions of GATA-3. In addition, TCR??+CD4-CD8?- dn T cells produced IFN-? and IL-17A upon stimulation. T-bet and granzyme B were only weakly expressed by both dn T cell subsets. In conclusion, this study identifies two dn T cell subsets in the dog: (i) a large (~7.5% in Peyer's patches, ~15% in lung) population of TCR??+CD4-CD8?- dn T cells with subpopulations thereof showing an activated phenotype, high expression of FoxP3 or GATA-3 as well as production of IFN-? or IL-17A and (ii) a small TCR??+CD4-CD8?- dn T cell subset also expressing GATA-3 without production of IFN-? or IL-17A. It will be exciting to unravel the function of each subset during immune homeostasis and diseases of dogs.

Dataset Information

The DNA methylation signature of human TCR??+CD4-CD8- double negative T cells reveals CG demethylation and a unique epigenetic architecture permissive to a broad stimulatory immune response.

Publications

The DNA methylation signature of human TCRαβ+CD4-CD8- double negative T cells reveals CG demethylation and a unique epigenetic architecture permissive to a broad stimulatory immune response.

Similar Datasets

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