Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Impact of gamma chain cytokines on the differentiation of recently antigen-activated CD8 T cells

ABSTRACT: Analysis of how different gamma chain family cytokines influence CD8 T cell differentiation. Naïve CD8 T cells were isolated from the spleens OT-I Thy1.1 TCR Tg mice. Whole splenocytes from wild-type C57BL/6 mice were used as stimulator cells. Purified naïve wild-type OT-I (1×106/well) were stimulated with OVA peptide (SIINFEKL) pulsed (5 µg/ml) and irradiated (2,000 rads) syngeneic splenocytes (6×106/well) in 24-well plates. Forty-eight hours later, activated OT-I T cells were harvested and viable cells were enriched over a Ficoll-paque gradient and washed with cRPMI prior to being reseeded in cRPMI (5×105 cells/ml) and treated with the media supplemented with various γc cytokines (IL-2, IL-4, IL-7, IL-15 and IL-21). Experimental samples were treated with 100 ng/ml of their respective gamma chain cytokine and incubated at 37ºC for 24 hours. RNA was isolated using either the RNeasy Mini kit, or a combination of TRIzol reagent and the Direct-zol RNA miniprep kit, all following manufacturer protocols. Two biological replicates for mRNA analysis were prepped using the RNeasy kit. The third replicate was prepped using the Trizol/Direct-zol approach. The quality and quantity of RNA samples was further analyzed on the Bioanalyzer. Labeled target cDNA was prepared from total RNA samples using the Ambion MessageAmp Premier protocol (3’IVT assay). Each sample target was hybridized to a Mouse 430 2.0 GeneChip array. Image processing and expression analysis were performed using Affymetrix GeneChip Command Console (AGCC) v. 3.1.1 and Affymetrix Expression Console v.1.1 software, respectively. Data from all biological replicates and conditions was imported into the Affymetrix Expression Console and normalized (RMA). RNA processing and microarray hybridization were performed by the Oregon Health & Science University Gene Microarray Shared Resource core facility in Portland, Oregon.

ORGANISM(S): Mus musculus

SUBMITTER: Michael McNamara

PROVIDER: E-GEOD-58262 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Several versions of SWI/SNF complexes, BAF and PBAF, have been described based on unique subunit composition. We find that T cell development in the thymus and lymphoid periphery is largely normal in the absence of the PBAF-specific component BAF180. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus. These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription. mouse primary T helper lymphocytes, naïve and Th2 cells, resting and stimulated, comparison of wild-type and BAF180/Pbrm1 deficient cells RNA from T helper cells was compared in the presence (WT) and absence (KO) of the Pbrm1/BAF180 gene. Comparison was made in 2 T helper subsets (Naive and Th2), each under 2 condtions (resting and stimulated), in triplicate (A,B,C). Resting naïve CD4+ T cells (N) were purified to 95% purity from the spleens and lymph nodes of 4-6 week old Balb/c mice (WT, or BAF180 cKO) using CD4+CD62L+ T cell Purification Kit II per manufacturer’s instructions (Miltenyi). Stimulated Naive cells (S) were prepared from resting naive cells by stimulation for 24 hours on anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml. Resting Th2 cells (2U) were prepared from resting naive cells by plating onto anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml in the presence of 10ng/ml IL-4, 10ug/ml anti-IFN-gamma. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture. Stimulated Th2 cells (2S) were prepared from resting Th2 cells, stimulated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 1.5 hours. RNA was harvested from 3 replicates of the primary Th cells under each condition. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) Biotin labeled cRNA was hybridized to Illumina’s Sentrix MouseRef-8,v2 Expression BeadChips.

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Impact of gamma chain cytokines on the differentiation of recently antigen-activated CD8 T cells

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