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Proteomic analyses on induced LGMN-deficient Tregs and WT Tregs

ABSTRACT: Mouse CD4 naïve T cells, isolated from spleens of WT or TLGMNKO mice, were activated with plate-bound anti-mouse CD3 and soluble anti-mouse CD28 for 3 days, and then characterized by 4D label-free proteomic analysis using nano-liquid chromatography-tandem mass spectrometry

ORGANISM(S): Mus Musculus

SUBMITTER: Shenghua Zhou

PROVIDER: PXD045679 | iProX | Tue Sep 26 00:00:00 BST 2023

REPOSITORIES: iProX

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Project description:In this work we present an analytical strategy to systematically identify early regulators by combining gene regulatory networks (GRN) with GWAS. We hypothesized that early regulators in T-cell associated diseases could be found by defining upstream transcription factors (TFs) in T-cell differentiation. Time series expression and DNA methylation profiling of T-cell differentiation identified several upstream TFs, of which TFs involved in Th1/2 differentiation were most enriched for disease associated SNPs identified by GWAS. NaÃ¯ve CD4+ T cells were isolated from buffy coat of four healthy donors using a naÃ¯ve CD4+ T cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). NaÃ¯ve CD4+ T cells were stimulated with plate-bound anti-CD3 (500 ng/mL), soluble anti-CD28 (500 ng/mL), in the presence of IL-12 (5 ng/mL), IL-2 (10 ng/mL) and antiâ??IL-4 (5 Âµg/mL) for Th1, IL-4 (10 ng/mL), IL-2 (10 ng/mL) and anti-IL-12 (5 Âµg/mL) and antiâ??IFN-g (5 Âµg/mL) for Th2. Cells were cultured for six days in Iscoveâ??s modified Dulbecco medium (IMDM) supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 10% heat-inactivated FCS (PAA Laboratories, Linz, Austria), 5 ÂµM Î²â??mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) and 50 ug/mL gentamicin (Sigma-Aldrich, St. Louis, Missouri, USA). Cells were cultured for 6 days and then re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of corresponding polarizing cytokines and antibodies for another 2 days (Zhang et al. 2013). RNA was extracted using a miRneasy Mini Kit (Qiagen). The RNA concentrations were analysed with NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). For the gene expression microarray analysis, The cRNA was prepared using a Low Input QuickAmp Labeling Kit. For Th1 and Th2 cells the gene expression microarray analysis was performed using SurePrint G3 Human Gene Expression 8x60K microarray kit, according to the manufactureâ??s instruction (Agilent Technologies).

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Proteomic analyses on induced LGMN-deficient Tregs and WT Tregs

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