Technical approach:
To model immunological memory in T cells, CD8+ naive and effector memory T cells were isolated from the peripheral blood of healthy human volunteers. Cells were cultured for 24h in the presence or absence of beads coated with antibodies against CD3/CD28 to mimic natural activation via the T cell receptor and coreceptor. From these CD8+ T cell populations, we profiled the transcriptome using RNA-sequencing (RNA-Seq; n = 3 or 4 independent biological replicates), chromatin accessibility by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq; n = 4 independent biological replicates) and 3D chromatin architecture by chromosome conformation capture coupled to high-throughput sequencing (Hi-C; n = 4 independent biological replicates). We used a combined single cell RNA-Seq/ATAC-Seq protocol (10X Genomics Multiome technology; n = 3 independent biological replicates) to jointly capture transcriptional/epigenomic heterogeneity and to dissect the crosstalk between transcriptome and epigenome dynamics in individual CD8+ T cells. CRISPR/Cas9-mediated gene perturbation followed by downstream analyses using flow cytometry, RNA-Seq and ATAC-Seq was performed for functional validation, aiming to gain mechanistic insight into the molecular underpinnings that maintain epigenetic priming in memory CD8+ T cells.
Outcomes:
Using this multidimensional epigenomics approach, we identified regions of transcription-permissive accessible chromatin specifically enriched in memory CD8+ T cells compared to naive CD8+ T cells, often residing near genes associated with memory recall. Joint gene expression and epigenome profiling of CD8+ T cells at single cell resolution using 10X Multiome technology showed that memory CD8+ T cells exhibit precisely regulated accessible chromatin dynamics underlying unique transcriptional signatures, and revealed that chromatin priming mechanisms are concentrated in a specific subset of effector memory CD8+ T cells. We pinpointed sets of accessible chromatin sites specifically overrepresented in memory CD8+ T cell already at baseline (i.e. prior to activation), which are enriched for 3D regulatory interactions and specific transcription factor binding sites. Compared to naive CD8+ T cells, memory CD8+ T cells selectively upregulated three key members of these transcription factor families – already at the resting state. Combined CRISPR/Cas9-mediated knock-out of this trio of transcription factors in memory CD8+ T cells caused a loss of epigenetic priming, culminating in an impaired capacity to produce inflammatory molecules essential for CD8+ T cell-mediated immunity. Together, our findings show that memory CD8+ T cells maintain a transcription-permissive chromatin landscape, which is imprinted by cooperating transcription factors to enable the rapid reactivation of inflammatory genes crucial for protective immune responses.
