In vitro modeling of CD8+ T cell exhaustion enables CRISPR screening to reveal a role for BHLHE40.

Identifying molecular mechanisms of exhausted CD8 T cells (T_ex) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo T_ex can be costly and inefficient. In vitro models of T_ex 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_ex. 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_ex subsets. By developing and benchmarking an in vitro model of T_ex, then applying high-throughput CRISPR screening, we demonstrate the utility of mechanistically annotated in vitro models of T_ex. Editor's summary: T cell exhaustion represents a distinct cellular state that contributes to immune dysfunction during chronic viral infections and cancer. To overcome limitations of in vivo models, Wu et al. developed and benchmarked an in vitro platform of T cell exhaustion. CD8⁺ T cells were cultured with dendritic cells repeatedly pulsed with cognate peptide and IL-2. This system reproduced the functional, transcriptomic, and epigenetic features of exhausted T cells generated during chronic lymphocytic choriomeningitis virus infection in mice. Using a pooled CRISPR screen targeting 120 transcription factors, they identified BHLHE40 as a regulator of T cell exhaustion, and genetic silencing of BHLHE40 prevented the development of more differentiated exhausted T cell subsets. Together, these findings provide a scalable and controlled platform for interrogating the biology underlying T cell exhaustion. —Claire Olingy [ABSTRACT FROM AUTHOR]