Deletion of an exhaustion-specific PD-1 enhancer modulates CD8+ T cell fate and function

Abstract

Exhaustion is a state of CD8+ T cell dysfunction arising during chronic viral infection and cancer. Exhausted T cells are less proliferative and less cytotoxic, produce less cytokine, and express high levels of co-inhibitory receptors that dampen their activation. Blockade of the co-inhibitory receptor PD-1 improves exhausted CD8+ T cell effector function; initial work in mouse models has been translated to the clinic and is now a cornerstone of cancer immunotherapy. However, key questions remain as to how PD-1 expression is regulated in different contexts, and how different levels of PD-1 expression impact CD8+ T cell functionality. To examine the exhaustion-specific regulation of PD-1, we focused on an enhancer of PD-1 exclusively accessible in exhausted CD8+ T cells. Using CRISPR-Cas9, we created a PD-1 enhancer-deleted mouse model and demonstrated that deletion of this enhancer leads to lower levels of PD-1 expression in contexts where the enhancer is accessible, such as chronic infection and tumor. Importantly, in non-accessible contexts such as acute infection, normal PD-1 expression and effector/memory differentiation are preserved. To understand the effects of this decreased PD-1 expression, we compared PD-1 enhancer-deleted CD8+ T cells to wildtype and PD-1 knockout cells. In chronic infection, enhancer-deletion increased proliferation and decreased apoptosis compared to wildtype cells. Transcriptionally, genetic manipulation of PD-1 did not substantially alter the development of exhausted subsets, and enhancer-deleted cells were uniquely enriched for effector-related gene signatures. Compared to PD-1 KO cells, enhancer-deleted cells were less apoptotic and more functional, although at a numerical disadvantage. Notably, enhancer-deleted cells expressed lower levels of the exhaustion-related transcription factor Tox than both wildtype and PD-1 KO cells. Together, this work demonstrates that editing an enhancer can lead to changes in gene expression that are tightly linked to enhancer accessibility. Creating an intermediate level of PD-1 expression in chronic infection was sufficient to alter CD8+ T cell function including the generation of unique properties, such as lower apoptosis, that are not seen with wildtype nor absent PD-1 expression.