CD4+ T cell response to IL12 in vitro
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE84567 | GEO | 2016/07/20
SECONDARY ACCESSION(S): PRJNA330590
REPOSITORIES: GEO
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Project description:Here we analyzed the transcriptional response to IL12 in activated CD4+ T cells in vitro. Splenic CD4+ T cells were isolated by negative selection from 6wk old male C57BL/6 mice. These were activated with CD3/28 beads for 48hrs and thereafter cultured with IL12 (100U/ml) for 2hrs or 8hrs in vitro. Cells were then transferred directly into TriZol. RNA was prepared in Trizol for gene expression profiling by Affymetrix Mouse Gene 1.0 ST Arrays.
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Project description:CD4+ T cells respond robustly to type 1 interferons which signal through JAK1 and TYK2. Here we analyzed the effects of a panel of JAK inhibitors on the IFNa transcriptional response in activated CD4+ T cells in vitro.
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Project description:To activate primary T cells, lymph nodes were removed and disaggregated. Cells were cultured in RPMI 1640 containing L-glutamine, 10% FBS, 50 μM β-mercaptoethanol and penicillin/streptomycin. Cells were stimulated with 1 μg/ml of the CD3 monoclonal antibody (2C11) and 2 μg/ml anti-CD28 (37.51) in the presence of cytokines IL12 (10 ng/ml) and 20 ng/ml IL2 (20 ng/ml). Cells were stimulated for 24 hours. Live TCR activated CD4+ cells were sorted for CD4+ and CD45.1+ expression and DAP1 exclusion prior to collection for proteomics processing. Mice were “wild-type” (ie non-TCR transgenic) from OT-2 CD45.1 expressing C57bl6 background maintained in-house line.
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Project description:CD4+ T cells respond robustly to type 1 interferons which signal through JAK1 and TYK2. Here we analyzed the effects of a panel of JAK inhibitors on the IFNa transcriptional response in activated CD4+ T cells in vitro. Splenic CD4+ T cells were isolated by negative selection from 6wk old male C57BL/6 mice. These were activated with CD3/28 beads for 48hrs and thereafter cultured with IFNa (100U/ml) in the presence or absence of JAK inhibitors at IC80 for 2hrs or 8hrs in vitro. Cells were then transferred directly into TriZol. RNA was prepared in Trizol for gene expression profiling by Affymetrix Mouse Gene 1.0 ST Arrays.
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Project description:Purpose: FURIN is a member of the proprotein convertase subtilisin/kexin (PCSK) family of serine endoproteases important in converting immature proproteins into their functional form. While FURIN is important in various aspects of the immune response such as in CD4+ T cells, FURIN´s role in CD8+ T cells is unclear. Here, we isolated naive CD8+ T cells from conditional T cell specific Furin KO (Cd4-Cre-Furinflox/flox) and WT (Furinflox/flox) mice and studied the genome-wide gene expression pattern upon in vitro anti-CD3/anti-CD28 activation (plate-bound, 5µg/ml each) using RNA sequencing. We also used recombinant TGFB1 (0.5ng/ml) or IL12 (10ng/ml) in the same experimental setting to study their impact on the CD8+ T cell transcriptome. Methods: Total RNA was isolated from the stimulated CD8+ T cells and the RNA-sequencing data was produced by the Functional Genomics Unit of the HiLIFE Genome Analysis Infrastructure (University of Helsinki, Helsinki, Finland) using Illumina NextSeq High Output 1x75 bp. Data processing pipeline was built on Snakemake wrappers, and used STAR to quantify features from quality and adapter trimmed reads in gene level. Prior the normalisation and differential gene expression analysis with DESeq2, the matrix of raw gene counts was prefiltered to keep only rows that have at least 10 reads in total. Result tables were annotated with biomaRt. Results: Snakemake preprocessing pipeline measured 55573 genes against GRCm38, ENSEMBL release 97. 17714 of them was used for statistical testing. Full result tables for each treatment were ranked by p-value and the normalized DESeq2 abundances in samples were included. The genes with BH-adjusted p-value <0.05 was considered differentially expressed. Conclusions: Our study revealed that the abscence of FURIN influences the expression of several genes in activated CD8+ T cells. Additionally, although FURIN-deficient CD8+ T cells can respond to exogenous TGFB1 and IL12, administrating these cytokines further increases the number of differentially expressed genes between Furin KO and WT CD8+ T cells.
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| PRJNA685792 | ENA