SRA

During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants controlling CD8+ T lymphocyte fate specification, including Ezh2, the catalytic component of the Polycomb Repressive Complex 2. Our data provide a revised model of terminal effector cell differentiation initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes. These findings provide unexpected insights into tightly coupled transcriptional and epigenetic mechanisms underlying CD8+ T lymphocyte fate specification and highlight the power and necessity of single-cell approaches. Overall design: Gene expression profiles for key stages of T cell differentiation were derived from 256 single-cell RNAseq libraries prepared on Fluidigm C1 and sequenced on Illumina HiSeq2500 (with 224 unique sequencing samples and 64 duplicates). Epigenetic profiles of Ezh2 binding, its associated H3K27me3 mark, and PolII occupancy in invitro-activated T cells were derived from ChIP libraries prepared with NEB Next ChIP (4 Ezh2 fl/fl and 4 WT samples, each with a biological duplicate). These ChIPseq libraries and their matched IGG input DNA controls were sequenced on Illumina HiSeq 4000 together with bulk RNAseq libraries from T cells invivo-activated in either C57BL/6 wild-type or Ezh2-knockout mice (each with a biological duplicate).