In vivo genome-scale enhancer screen decodes T cell fate decisions in the tumor

During chronic infection and cancer progression, T cells enter a dysfunctional state called exhaustion, posing a significant challenge to disease control and immunotherapy outcomes. Previously, we and others have shown that T cell exhaustion is a differentiation process regulated at the epigenetic level. Exhausted T cells share a conserved program of chromatin accessibility landscapes across chronic infections and tumors (core exhaustion program) that is imprinted in early T cell differentiation. However, it remains unclear whether and which open chromatin regions within the core exhaustion program play a causal role in driving the exhausted state. We hypothesize that the core exhaustion program contains transcriptional enhancers that regulate T cell persistence and differentiation in exhaustion, preventing reinvigoration of function. To test it, we leveraged our novel enhancer editing platform called Systematic Non-coding element Interrogation by Paired sgRNAs (SNIP-R), a pooled CRISPR-based enhancer deletion platform optimized for kilobase-scale perturbation in primary T cells. We performed the first in vivo genome-scale enhancer deletion screen on ova-specific CD8 + T cells (OT-1) in OVA-expressing tumors. We identified networks of regulatory elements in the core exhaustion program that regulate T cell persistence and subset formation in the tumor. Validation studies from the screen showed that deleting one such regulatory element 100kb upstream of the Klf6 gene ( Klf6-100kb ) in T cells improved tumor control. Klf6-100kb perturbed T cells strongly outcompeted control populations and had increased generation of effector-like T cells in the tumor compared to control perturbations. Together, we showed that the core exhaustion program contains causal regulatory elements underlying T cell exhaustion and pinpointed new targets for developing next-generation adoptive T cell therapies.