Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from mouse T cells after CLIP (Crosslinking immunoprecipitation) procedures (mRNA)

ABSTRACT: Molecular networks based on genome-wide mRNA and miRNA expression in activated T cells are interated by co-regulation with miRNA and protein-coding genes during immune responses, however, accurate prediction of miRs and their targets remains a challenge. Furthermore, the specific changes in the expression of miRNAs and/or mRNAs in allogeneic activated T cells during HCT would be distinct from those responding to non-specific stimulation. Recently, Ago-CLIP has been demonstrated to provide a robust platform for identification of precise miRNA-mRNA interactions by definitively distinguishing direct from indirect miRNA-target interactions. We utilized CLIP procedure followed by screening with standard microarray platforms for miRNA and mRNA transcripts to demonstrate the miR-mRNA landscape and identify novel molecular targets for modulating T cell responses. Mixed cell culture was performed with T cells in the presence of syngeneic 57BL/6, or allogeneic BALB/c dendritic cells or cultivated with anti-CD3e and anti-CD28 mAB9 for 24 h. Purified T cells were processde for CLIP procedures and followed by RNA isolation (TRIzol LS and miReasy Kit) and hybridization on Affymetrix microarrays and Exiqon 6th generation miRCURY LNA microRNA Arrays . We sought to obtain that the specific changes in the expression of miRNAs and/or mRNAs in allogeneic activated T cells during hematopoietic cells transplantation and that would be distinct from those responding to non-specific stimulation.

ORGANISM(S): Mus musculus

SUBMITTER: yaping sun

PROVIDER: E-GEOD-43635 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Introduction: Expansion of antigen (Ag)-specific natural occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloAg-specific nTregs. Methods: A highly enriched nTreg-fraction (CD4+CD25brightCD127- T cells) was alloAg-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDC) or peripheral blood mononuclear cells (PBMC). The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the TSDR of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4 and cytokines. In addition, the antigen-specific suppressive capacity of these expanded nTregs was tested. Results: Allogeneic mature moDC and skin-derived DC were superior in inducing nTreg-expansion compared to immature moDC or PBMC in an HLA-DR and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2, could facilitate optimal mature moDC-induced nTreg-expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloAg-induced proliferation without significant suppression of completely HLA-mismatched-Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the TSDR within the FOXP3 gene and highly expressed of FOXP3, HELIOS and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-g and TNF-a but very few IL-17 producing nTregs were found. Next generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Conclusions: Human allogeneic mature moDC are highly efficient stimulator cells, in presence of exogenous IL-15, for expansion of stable alloAg-specific nTregs with superior suppressive function. Four different batches of highly pure regulatory T cells (all from the same donor) were expanded in two different ways, and compared to non-expanded samples.

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Expression data from mouse T cells after CLIP (Crosslinking immunoprecipitation) procedures (mRNA)

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