In Vitro Modeling of CD8 T Cell Exhaustion Enables CRISPR Screening to Reveal a Role for BHLHE40

Identifying novel 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, offering an opportunity to perform 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 pooled CRISPR screening to uncover 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 subsets of Tex. By developing and benchmarking an in vitro model of Tex, we demonstrate the utility of mechanistically annotated in vitro models of Tex, in combination with high-throughput approaches, as a discovery pipeline to uncover novel Tex biology. ### Competing Interest Statement EJW is a member of the Parker Institute for Cancer Immunotherapy that supports research in the Wherry lab. EJW is an advisor for Danger Bio, Marengo, Janssen, NewLimit, Pluto Immunotherapeutics Related Sciences, Santa Ana Bio, Synthekine, and Surface Oncology. EJW is a founder of and holds stock in Surface Oncology, Danger Bio, and Arsenal Biosciences. OK holds equity in Arsenal Biosciences and is an employee of Orange Grove Bio. RPS is an employee of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.