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Next Generation Sequencing unveils the impact of CD4+ regulatory T cell (Treg)-derived extracellular vesicles (EVs) on the transcriptome of CD4+ T cells.

ABSTRACT: Purpose: The goal of this study was the transcriptome high-throughput data analysis of CD4+ T cells isolated from peripheral blood of healthy subjects and T cell receptor (TCR)-stimulated in vitro in the presence of Treg cell-derived EVs, compared with Tconv cell-derived EVs, or with control (mock) EVs (particles isolated from unconditioned medium). Methods: total RNA profile of human CD4+ T cells from healthy subjects. Libraries were prepared for sequencing on NextSeq 500 (llumina technology). Results: Paired differential expression and pathway enrichment analysis showed that CD4+ T cells treated with Treg cell-derived EVs are characterized by lower expression level of a plethora of transcripts linked to protein catabolism and cell growth. Conclusions: Several transcripts are differently regulated between CD4+ T cells treated with Treg cell-derived EVs and the same cells treated with either Tconv cell-derived EVs or mock EV controls.

ORGANISM(S): Homo sapiens

PROVIDER: GSE208570 | GEO | 2025/03/20

REPOSITORIES: GEO

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Project description:We have previously developed an approach that fractionates genomic DNA fragments depending on their CpG density (methyl-CpG-immunoprecipitation, MCIp), and adapted this approach to identify regions that are differentially methylated in the two closely related regulatory T-cells (Treg cells) and conventional T-cells (Tconv cells). Because Treg cells naturally occur at a relatively low frequency, we used a previously established protocol to expand Treg cells from a stable naÃ¯ve Treg population that is characterized by the co-expression of CD4, CD25 and CD45RA. We separated gDNA of both expanded T cell lineages (Tregexp and Tconvexp) into unmethylated (CpG) and methylated pools (mCpG) using MCIp and compared cell type-specific differences in DNA methylation by co-hybridization of the two umethylated or the two methylated DNA subpopulations of Treg and Tconv, respectively, to these locus-wide custom tiling arrays. As enriched DNA-fragments from a cell type in the methylated fraction should be depleted in the unmethylated fraction, the signal intensities in CpG pool and mCpG pool hybridizations should complement themselves (âMirror-Imageâ approach) and thereby allow the identification of differentially methylated regions (DMR). Because we expected to find lineage-specific methylation differences with greater probability in regions associated with differential transcriptional activity, we limited our analysis to gene loci that showed cell type-specific gene expression in Treg versus Tconv cells plus a handful of control regions that were equally expressed in both cell types. The microarray used in this study covered 12 megabases of the human genome and contained 69 regions (with a median size of 100.000 kb) and 128 proximal promoter regions and 181 genes, which included a number of well known and functionally relevant genes like CD40LG, IFNG, FOXP3, IL2RA and CTLA4. Keywords: MCIp-on-chip; comparative genomic hybridization With MCIp gDNA from Treg or Tconv cells was separated into hypo- and hypermethylated pools. On each array, wheather the two hypomethylated fractions- one from Treg, the other from Tconv cells- or the two hypermethylated fractions were cohybridized. Two biological replicates.

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Next Generation Sequencing unveils the impact of CD4+ regulatory T cell (Treg)-derived extracellular vesicles (EVs) on the transcriptome of CD4+ T cells.

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