3D genome of CD8+ T cells reveals IRF8-mediated exhaustion in cancer [RNA-seq]

Exhausted CD8+ T cell differentiation is accompanied by extensive changes in epigenome. However, whether and how higher-order chromatin organization is involved in exhausted CD8+ T cell differentiation is unclear. Here, we showed extensive changes in the three-dimensional genome during exhausted CD8+ T cell differentiation, associated with changes in gene transcription. Moreover, we identified Interferon regulatory factor 8 (IRF8) as an essential transcription factor in re-organization of spatial chromosomal interactions. Irf8 deficiency inhibits the development of terminal exhausted CD8+ T cells and promotes tumor growth. Mechanistically, IRF8 bound to genes associated with exhausted CD8+ T cells differentiation and promoted the formation of chromosomal loops. At the loop anchor regions, IRF8 recruited CTCF to form active chromosomal structure to regulated exhaustion-associated genes. These results thus identify a critical role of IRF8-dependent chromatin topology in gene transcription during exhausted CD8+ T cell differentiation. Overall design: To assess transcriptional changes associated with IRF8 deficiency in CD8? T cells, 2.5?×?106 FACS-sorted naïve CD45.1/2 wild-type (WT) and CD45.2 Irf8?/? P14 CD8? T cells were mixed at a 1:1 ratio and intravenously transferred into CD45.1 congenic recipient mice one day prior to infection with LCMV clone 13. On day 25 post-infection, splenic CD8? T cells were isolated by FACS based on CD45 allelic markers. Total RNA was extracted using TRIzol reagent, and RNA integrity was verified using a Bioanalyzer (Agilent). RNA-seq libraries were prepared using a stranded mRNA-seq kit (e.g., NEBNext Ultra II) and sequenced on an Illumina platform to a depth of ~30 million paired-end reads per sample.