RNA sequencing analysis of gene expression profiles in WT and Etv5-deficient TFH cells
Purpose: The aim of this study is compare the gene expression profiles between wild-type (WT) and Etv5-deficient follicular helper T (TFH) cells to identify the molecular mechanism of how ETV5 promotes the TFH cell differentiation. Methods: TFH cells (CD4+CD44+CXCR5+PD-1+) from the spleen of WT and Etv5f/f;Cd4-Cre mice immunized with OVA in alum for 7 days were sorted by a MoFlo-XDP (Beckman Coulter). Total RNA was extracted using TRIzol (GeneAll) according to the manufacturer's instruction. The library for RNA sequencing was generated using the Truseq stranded mRNA library prep kit (Illumina), and sequencing was performed with Novaseq 6000 (Illumina). Results: Reads were mapped to the mouse reference genome (mm10) with Tophat (v2.0.13), and the aligned results were analyzed with Cuffdiff (v2.2.0) for differentially expressed genes (DEGs). A total of 92 genes were differentially expressed in Etv5-deficient TFH cells (46 genes upregulated and 46 genes downregulated) when compare with WT TFH cells (fold change > 2 and P-value < 0.05). Conclusions: Our study presents the first comparative gene expression analysis of TFH cells from control and T cell-specific Etv5-deficient mice. Since ETV5 usually acts as a transcription activator, we focused on the downregulated genes in Etv5-deficient TFH cells. Through this study, we identified the candidate genes that are targeted by ETV5 for TFH cell differentiation. Overall design: mRNA profiles of TFH cells from the spleen of wild type (WT, Etv5f/f) and Etv5f/f;Cd4-Cre mice immunized with OVA in alum for 7 days. Two replicates for each genotypes were anaylzed.