RNA-seq analysis of human CD8 CAR-EGFR T cells in expansion and repeated hypoxic coculture

Human CD8 EGFR CART cells during expansion and repeated hypoxic coculture were collected at different time points and subject to RNA-seq to identify key players in T cell differentiation and hypoxia-induced exhaustion. The RNA-seq data together with other functional analysis identified P4HA1 as an important enzyme that regulates T cell early differentiation and terminal exhaustion. To investigate the mechanism, P4HA1 inhibitor DPCA was used to treat the human CD8 cells during early expansion and repeated coculture and the cells were harvested for RNA-seq analysis. Part 1) Naïve CD8 cells were isolated from PBMC of healthy donors and stimulated with CD3/CD28 and infected with CAR-virus. The CD3/CD28 antibodies and CAR virus were washed away on Day 5 and expanded in medium with IL7/15 for another 7 days before being subjected to repeated coculture under normoxia/hypoxia. CD8 CAR T cells were collected at indicated time points for RNA-seq. Part 2) Human CD8 CART cells were expanded or cocultured as described above. Cells were treated with either P4HA1 inhibitor DPCA or DMSO as vehicle control and collected at different time points for RNA-seq analysis.