GSE107908
OX40 regulates double-negative regulatory T cells dependently of its effects on conversion and function
Methods: To better understand the role of OX40 in the regulation of double-negative regulatory T cells (DNT), OX40 deficiency and wild-type DNT converted from naïve CD4 T cells were compared in a transcriptome sequencing study. Total RNA was isolated from FACS-separated DNT. Transcriptome sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and sequenced on an Illumina Hiseq platform (Illumina, San Diego, CA). Sequences were aligned to the reference genome with TopHat and processed with Cufflinks, which quantifies each transcript in each sample using reference annotations produced by the University of California Santa Cruz UCSC. Differentially expressed genes with a fold change of >=2.0 and padj < 0.05 between OX40 deficiency and control DNT were submitted to GO and KEGG enrichment analysis, which uses unbiased methods to assess pathway enrichment. Results: Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that OX40 knockout are involved in regulatory function of DNT; including suppressive function, apoptosis, proliferation, and so on. Compared to control DNT, OX40 knockout DNT exhibited up-regulation of 52 genes and down-regulated of 46 genes. The most enriched GO term were Signal Transduction, Protein Binding, and Banding. Overall design: Double-negative regulatory T cells were converted from wild-type and OX40 knock mouse. mRNA profiles were generated and compared by deep sequencing in duplicate using Illumina HiSeq.