识别CD8 T细胞失活（Tex）的分子机制是改善癌症和其他疾病免疫治疗的关键目标。然而，对体内Tex进行高通量的探究可能成本高且效率低下。Tex的体外模型易于定制，并且能够快速产生高细胞产量，便于CRISPR筛选和其他高通量实验。我们建立了一种体外慢性刺激模型，并通过与真正的体内Tex进行关键表型、功能、转录和表观遗传特征的比较来进行基准测试。我们利用这种体外慢性刺激模型结合CRISPR筛选，鉴定出T细胞失活的转录调控因子，包括BHLHE40。体内和体外验证确定了BHLHE40在调控前体和中间Tex子群之间的关键分化检查点中的作用。通过开发和基准测试Tex的体外模型，然后应用高通量的CRISPR筛选技术，我们展示了以机制注释的体外模型在Tex中的有效性。

Identifying molecular mechanisms of exhausted CD8 T cells (Tex) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo Tex can be costly and inefficient. In vitro models of Tex 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 Tex. 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 Tex subsets. By developing and benchmarking an in vitro model of Tex, then applying high-throughput CRISPR screening, we demonstrate the utility of mechanistically annotated in vitro models of Tex.