Highlights d Checkpoint blockade induces transcriptional changes in PD-1 À CD8 + and PD-1 + CD8 + TILs d PD-1 À CD8 + TILs contain naive-, memory-precursor-, and effector-like subsets d Memory-precursor-and effector-like PD-1 À CD8 + TILs expand upon checkpoint blockade d Tcf7 is required for memory-precursor-like cells and efficacy of immunotherapies

Graphical Abstract Highlights d Somatic mtDNA mutations can track cellular relationships and hierarchies in vitro d Single-cell genomic assays faithfully detect mtDNA mutations d Lineage inference can be combined with gene expression or chromatin state profiles d mtDNA mutations enable studies of clonal architecture in human health and disease

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Natural mitochondrial DNA (mtDNA) mutations enable the inference of clonal relationships among cells. mtDNA can be profiled along with measures of cell state, but has not yet been combined with the massively parallel approaches needed to tackle the complexity of human tissue. Here, we introduce a high-throughput, droplet-based mitochondrial single-cell Assay for Transposase Accessible Chromatin with sequencing (mtscATAC-seq), a method that combines high-confidence mtDNA mutation calling in thousands of single cells with their concomitant high-quality accessible chromatin profile. This enables the inference of mtDNA heteroplasmy, clonal relationships, cell state, and accessible chromatin variation in individual cells. We reveal single-cell variation in heteroplasmy of a pathologic mtDNA variant, which we associate with intra-individual chromatin variability and clonal evolution. We clonally trace thousands of cells from cancers, linking epigenomic variability to subclonal evolution and infer cellular dynamics of differentiating hematopoietic cells in vitro and in vivo . Taken together, our approach enables the study of cellular population dynamics and clonal properties in vivo .