Cell state and transcription factor modulation during extended CD8+ T-cell expansion ex vivo

Adoptive cell therapy is fast becoming a cornerstone of modern tumour immunotherapy. It relies on the relatively long-term (>2 week) ex vivo expansion of T cells either in the form of tumour-infiltrating cells, or bulk cells modified with the expression of heterologous signaling proteins, e.g., chimeric antigen receptors. However, relatively little is known about the developmental trajectories of T cells under these conditions at the systems level, and whether entire genetic pathways could be manipulated for clinical advantage. Using bulk RNA-seq analysis of T cells expanded and rested over a 17-day period, we provide a resource of how gene expression changes as cells transition through distinct cellular states over the course of activation and ex-vivo expansion. We also performed experiments to investigate the role of FOSL1 in T-cell function. Overall design: We used naive CD8+ T cells as a starting population to reduce heterogeneity for bulk RNA-seq measurements, and because their use as a starting population (vs. unfractionated subsets of T cells) exhibits superior capacity to expand, persist and eliminate tumors. Naive cells were stimulated with anti-CD3/CD28 beads for seven days with RNA extracted at defined time-points (i.e., 30 mins, 3 hours, 12 hours, 24 hours, 48 hours, 72 hours, 7 days). Beads were then removed and RNA extracted at either 2, 7, or 10 days. For each time-point we extracted total RNA and performed bulk RNA-seq in three replicates. For the FOSL1 experiments, we generated FOSL1 overexpression and FOSL1 KO lines in immortalized human Jurkat T cells and compared them with the parental Jurkat cell lines using bulk RNA-seq. We also transduced three donor CD8+ T-cells with lentivirus encoding FOSL1 and performed bulk RNA-seq to assess the transcriptional changes in the cells overexpessing FOSL1 compared to the parental donors.