ABSTRACT: Objective:mRNA sequencing of sorted naive CD4+ T cells from the spleens of PIK3CD GOF mice and WT littermate mice. Methods:Naive CD4+ T cells were negatively isolated ex vivo(Stemcell Technologies).Total RNA from isolated naive CD4+ T cells was extracted using RNeasy Plus Mini Kit (Qiagen, GmBH, Germany) according to the manufacture’s instruction and checked for RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, US). Qualified total RNA was further purified by RNeasy micro kit (QIAGEN) and RNase-Free DNase Set (QIAGEN). Paired-end libraries were constructed using the TruSeq® RNA Sample Preparation Kit (Illumina, USA) according to the manufacturer’s instructions. The products are purified, enriched, quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer to confirm insert size and calculate the mole concentration. Cluster was generated using cBot with the library diluted to 10 pM and then were sequenced on the Illumina HiSeq 2500 (Illumina, USA). The library construction and sequencing was performed at Shanghai Biotechnology Corporation (Shanghai, China). Results:Compared to the expression profiles for the WT cells, those for the mutant TNaive showed 2,307 genes with differential expression, of which 1,749 were upregulated and 558 were downregulated. Gene ontology analysis showed that PIK3CD GOF TNaive upregulated the expression of genes encoding molecules involved in the cell cycle and mitosis, including E2f1, E2f2, cyclin A2, cyclin B2, Cdk1, Hells and Nuf2, all of which might synergistically promote entry into the cycling phase. In addition, the genes encoding T cell activation, cytokines and cytokines receptors, chemokines and chemokine receptors, transcription factors, and metabolic regulators were differentially regulated in PIK3CD GOF mice compared to WT mice. Kyoto Encyclopedia of Genes and Genomes analysis showed that the altered genes in TNaive from PIK3CD GOF mice displayed significant enrichment of several sets associated with infection, inflammation and autoimmune disease Conlusion:PIK3CD GOF results in a loss of quiescence-associated gene expression patterns in naive T cells by collectively coupling the cell cycle, nutrient metabolism, cell trafficking and signal transduction.
