Following viral infection, naïve CD8+ (“killer”) T cells undergo a program of proliferation and differentiation resulting in a large pool of effector cells secreting inflammatory cytokines and cytotoxic (effector) molecules required for viral clearance. Importantly, a long-lived pool of virus-specific memory T cells remain after clearance that produce effector molecules more rapidly following re-infection, providing the basis of T cell immunity. While it is known that the different functional capacities of naïve, effector and memory T cells are underscored by unique transcriptomes, how these transcriptomes arise and are maintained is poorly understood.

Differentiation-specific transcriptomes are established through binding of activating and repressive transcription factors (TFs) to regulatory sequences including promoters and transcriptional enhancers (TEs). In particular, cell-type specific TF binding to TEs is an essential determinant of differentiation. TEs occur upstream, downstream, or on different chromosomes from genes that they regulate, and while their positions can be determined using techniques including ChIP-Seq, these approaches can’t discern which gene is regulated by which TE. We are using “HI-C” to determine how modulated TE engagement regulates influenza-specific CD8+ T cell differentiation. Briefly, because TEs physically interact with the promoter of genes that they regulate, restriction digestion and proximity-mediated ligation generates chimeric promoter-enhancer DNA fragments; high-throughput sequencing of these chimeras then allows genome-wide mapping of TE-promoter interactions.

Combining HI-C with matched transcriptomic data, we find that the promoters of many signature effector genes (including interferon gamma and granzyme B) are in physical contact with their cognate TEs within naïve cells. Moreover, characterisation of the chromatin landscape shows those TEs to have a “poised” chromatin state, which is activated upon effector differentiation. Importantly, looping of CD8-lineage specific genes is absent in CD4+ T cells, and from precursor, double-positive thymocytes. Taken together, these data suggest preconfigured chromatin loops dictate T cell lineage fate potential.