RNAseq of control OT-I cells and Eomes-overexpressing OT-I cells

High amount of Eomes might drive T cell exhaustion. In order to understand how Eomes contributes to exhaustion of CD8+ T cell in the TME, we conducted transcriptional analysis of control OT-I cells and Eomes-overexpressing OT-I cells Overall design: Eomes was cloned into a retroviral expression vector (RVKM) which also encodes an IRES-hCD2 cassette. This vector was transfected into Pheonix to package retrovirus. The empty vector was used as a control. CD8+ T cells were isolated from spleen and lymph nodes of OT-I mice using Dynabeads Flowcomp mouse CD8 kit (Invitrogen). Then the cells were stimulated with SIINFEKL peptide (OVA257-264) at 2.5 ng/mL in the presence of 10U/mL IL-2 for 24 hr. Retroviral supernatants were harvested, filtered, and supplemented with 6µg/mL polybrene. OT-I T cell cultures were spinduced with the retroviral supernatant for 90 min at 1800 rpm, 32ºC. 48 hr later, hCD2+ cells were sorted prior to re-stimulation. hCD2+ OT-I cells were plated at 40,000 cells/well in 96-well plates and re-stimulated with 2.5ng/mL OVA with 10U/mL IL-2 for 3 days before harvested for RNAseq analysis. Total RNA was extracted from re-stimulated control or Eomes-overexpressing OT-I cells and sent to BGI Genomics for library construction. The library products were sequenced via Illumina Hiseq4000 by BGI Genomics. The sequencing reads were filtered by SOAPnuke without quality problems. Genome mapping was done by HISAT. Clean reads were mapped to the mm10 reference genome using Bowtie2, and gene expression indicated by RPKM (Reads Per Kilobases per Million reads) was calculated by RSEM. Differentially expressed genes (DEG) were detected with PoissonDis by at least 1.5 fold change and FDR lower than 0.01.

2

Li J, He Y, Hao J, Ni L, Dong C