Early transcriptional and epigenetic regulation of CD 8 + T cell differentiation revealed by single-cell RNA sequencing

Heterogeneity of cell fate is a hallmark of the responses of T lymphocytes to microbial infection. During an immune response to a microbial infection, responding naive T lymphocytes give rise to terminal effector cells that mediate acute host defense and self-renewing memory cells that provide long-lived protection. Terminally differentiated effector T cells are characterized by high expression of the killer lectin-like receptor KLRG1 and low expression of the interleukin 7 receptor (IL-7R)1. Circulating memory T cells can be categorized into two subsets, central memory T cells (TCM cells) and effector memory T cells (TEM cells), distinguished by differences in their expression of homing and cytokine receptors such as L-selectin (CD62L) and CCR7, their proliferative capacity and their anatomical localization2. A third subset of memory cells, tissue-resident memory T cells (TRM cells), do not circulate but instead remain in peripheral tissues after pathogen clearance3. Transcriptional profiling approaches have greatly advanced understanding of the molecular regulation of the fate specification of T lymphocytes4,5. Through comparison of the gene expression of CD8+ T lymphocytes during the course of microbial infections, such studies have identified many transcription factors and pathways with a role in the specification of terminal differentiation versus long-lived memory6. However, most studies have been conducted on bulk populations of cells, which masks the potential heterogeneity among individual cells. Published work has sought to address these limitations through the use of single-cell quantitative RT-PCR analysis to investigate the gene-expression patterns of single CD8+ T lymphocytes responding to bacterial infection in vivo7. Although dynamic changes in gene expression in individual cells during differentiation have been identified, pre-selection of genes already known to encode products involved in differentiation for analysis precluded the discovery of as-yet unknown genes and molecular pathways. Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool that has substantially advanced the understanding of diverse biological processes, including development8, the pathogenicity of the TH17 subset of helper T cells9, and innate immune responses10. In our study here, we applied a scRNA-seq approach to analyze transcriptomewide changes in individual CD8+ T cells as they differentiated in vivo. We observed unexpected and substantial transcriptional heterogeneity among lymphocytes that had undergone their first division, which revealed two distinct subpopulations distinguished by their expression of hundreds of genes encoding products involved in diverse biological functions, including cell-cycle regulation, metabolism, effector function and fate specification. The expression of many transcription factors that have been linked to effector and memory cell differentiation, along with chromatin regulators, was markedly upregulated in cells differentiating along the terminal effector pathway and was extinguished by the peak of the adaptive immune response. That initial transcriptional program was subsequently refined by selective epigenetic repression of molecular determinants associated with memory-cell differentiation. In contrast, induction of the memory program seemed to be associated