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

Abstract

AbstractIdentifying 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 ofin vivoTexcan be costly and inefficient.In vitromodels of Texare easily customizable and quickly generate high cellular yield, offering an opportunity to perform CRISPR screening and other high-throughput assays. We established anin vitromodel of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fidein vivoTex. We leveraged this model ofin vitrochronic stimulation in combination with pooled CRISPR screening to uncover transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40.In vitroandin vivovalidation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate subsets of Tex. By developing and benchmarking anin vitromodel of Tex, we demonstrate the utility of mechanistically annotatedin vitromodels of Tex, in combination with high-throughput approaches, as a discovery pipeline to uncover novel Texbiology.