Identifying molecular mechanisms of exhausted CD8 T cells (T) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo T can be costly and inefficient. In vitro models of T are easily customizable and quickly generate high cellular yield, enabling CRISPR screening and other high-throughput assays. We established an in vitro model of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fide in vivo T. We leveraged this model of in vitro chronic stimulation in combination with CRISPR screening to identify transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40. In vitro and in vivo validation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate T subsets. By developing and benchmarking an in vitro model of T, then applying high-throughput CRISPR screening, we demonstrate the utility of mechanistically annotated in vitro models of T.
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