Integrated single-cell profiling dissects cell-state-specific enhancer landscapes of human tumor-infiltrating CD8⁺...

Despite extensive studies on the chromatin landscape of exhausted T cells, the transcriptional wiring underlying the heterogeneous functional and dysfunctional states of human tumor-infiltrating lymphocytes (TILs) is incompletely understood. Here, we identify gene-regulatory landscapes in a wide breadth of functional and dysfunctional CD8⁺ TIL states covering four cancer entities using single-cell chromatin profiling. We map enhancer-promoter interactions in human TILs by integrating single-cell chromatin accessibility with single-cell RNA-seq data from tumor-entity-matching samples and prioritize cell-state-specific genes by super-enhancer analysis. Besides revealing entity-specific chromatin remodeling in exhausted TILs, our analyses identify a common chromatin trajectory to TIL dysfunction and determine key enhancers, transcriptional regulators, and deregulated genes involved in this process. Finally, we validate enhancer regulation at immunotherapeutically relevant loci by targeting non-coding regulatory elements with potent CRISPR activators and repressors. In summary, our study provides a framework for understanding and manipulating cell-state-specific gene-regulatory cues from human tumor-infiltrating lymphocytes.