Summary Memory T cells provide rapid and long-term protection against infection and tumors. The memory CD8 + T cell repertoire contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. While these T cell populations have been well characterized in terms of differentiation potential and function, the epigenetic changes underlying memory T cell fate determination and tissue-residency remain largely unexplored. Here, we examined the single-cell chromatin landscape of CD8 + T cells over the course of acute viral infection. We reveal an early bifurcation of memory precursors displaying distinct chromatin accessibility and define epigenetic trajectories that lead to a circulating (T CIRC ) or tissue-resident memory T (T RM ) cell fate. While T RM cells displayed a conserved epigenetic signature across organs, we demonstrate that these cells exhibit tissue-specific signatures and identify transcription factors that regulate T RM cell populations in a site-specific manner. Moreover, we demonstrate that T RM cells and exhausted T (T EX ) cells are distinct epigenetic lineages that are distinguishable early in their differentiation. Together, these findings show that T RM cell development is accompanied by dynamic alterations in chromatin accessibility that direct a unique transcriptional program resulting in a tissue-adapted and functionally distinct T cell state. Graphical Abstract Highlights scATAC atlas reveals the epigenetic variance of memory CD8 + T cell subsets over the course of acute infection Early bifurcation of memory precursors leads to circulating versus tissue-resident cell fates Integrating transcriptional and epigenetic analyses identified organ-specific T RM cell regulators including HIC1 and BACH2 Epigenetic distinction of T RM cells and T EX cell subsets

A unique epigenomic landscape defines CD8+ tissue-resident memory T cells