Flexible and scalable control of T cell memory by a reversible epigenetic switch (RNA-seq)
In this study, we find that the Tcf7+ CD8 T cells present following acute challenge can form either from cells that maintain Tcf7 expression throughout activation or from cells that silence Tcf7 and reactivate it after stimulation removal. In this experiment, we asked whether both pathways can give rise to cells with genomic characteristics of memory. To do this, we activated naive CD8 T cells ex vivo for 2 days followed by 1 day of culture without TCR stimulation. On day 3, we sorted Tcf7-YFP high and low populations, all from a single CellTrace peak representing cells that had undergone the same number of divisions. The sorted populations were cultured for an additional 6 days and sorted again on day 9 by Tcf7-YFP levels. These samples, as well as control naive, antigen-experienced, and effector samples, were processed for RNA-seq analysis. Overall design: Control naïve samples were sorted from splenoyctes as CD8+CD44-CD62L+Tcf7-YFP+ (2 replicates); antigen-experienced samples were sorted from splenocytes as  CD8+CD44+CD62L+Tcf7-YFP+ (2 replicates); effector control samples were activated ex vivo for 3 days (2 replicates). Cells sorted as Tcf7-YFP high and low at day 3 were recultured and sorted as Tcf7-YFP high or low at day 9: Day9 Tcf7-YFP high, sorted Tcf7-YFP high at Day3 (2 replicates); Day9 Tcf7-YFP low, sorted Tcf7-YFP low at Day3 (2 replicates); Day9 Tcf7-YFP high, sorted Tcf7-YFP low at Day3 , 1 (2 replicates).