Chromatin State Dynamics Underlying CD8 T Cell Differentiation and Dysfunction in Cancer
Authors: Philip Mary , Fairchild Lauren , Sun Liping , Viale Agnes , Merghoub Taha , Wolchok Jedd D. , Leslie Christina S. , Schietinger Andrea 
Published: October 2019

Abstract
T cells recognizing tumor-specific antigens are detected in cancer patients but are dysfunctional. Upon antigen encounter, T cells differentiate into discrete phenotypic and functional states. Cellular differentiation is driven by epigenetic remodeling, however, it is not known whether and how epigenetic programming establishes and regulates tumor-specific T cell (TST) dysfunction and determines a T cell's ability to respond to therapeutic interventions such as immune checkpoint blockade (PD-1 and CTLA-4). Here for the first time, we (1) identify chromatin dynamics underlying T cell differentiation to the dysfunctional state in mouse and human tumors and (2) provide insights into the epigenetic and transcriptional regulatory mechanisms determining T cell susceptibility to therapeutic reprogramming.
Using a genetic cancer mouse model, we previously showed that CD8 TST become unresponsive early during carcinogenesis at the pre-malignant stage, even before the emergence of a pathologically-defined malignant tumor. While T cell dysfunction was initially reversible, it ultimately became a fixed state that could not be rescued by therapeutic interventions such as PD1 checkpoint blockade.
To identify the hierarchical changes in chromatin states resulting in "dysfunction imprinting," we used the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) to map the genome-wide changes in chromatin accessibility in TST cells over the course of cancer development. In parallel, we carried out RNA-Seq to determine the interplay between chromatin remodeling and transcriptional networks. Substantial chromatin remodeling occurred during early T cell activation in the pre-malignant lesion (days 5-7) followed by a second wave of chromatin accessibility changes between days 7 and 14. Strikingly, after the second wave, no further CD8 T cell chromatin remodeling occurred during carcinogenesis, even after progression to an advanced late-stage tumor with an immunosuppressive microenvironment. Interestingly, these 2 distinct chromatin accessibility patterns in TST correlated temporally with the plastic and fixed dysfunctional states and susceptibility to therapeutic reprogramming in vivo. To understand the transition from plastic to fixed dysfunction, we analyzed the differential expression of transcription factors (TF) in conjunction with changes in peak accessibility at TF-binding motifs genome-wide. We identified a network including CD8 T cell regulatory TF such as TCF1, LEF1, BLIMP1, and BACH2 as well as less-well-characterized TF (NR4A2, TOX) potentially controlling differentiation to the dysfunctional state. Moreover, ATAC-Seq analysis of human tumor-infiltrating CD8 T cells revealed similar tumor-associated changes in peak accessibility, and studies are ongoing to assess the associated TF networks.
In this study, we have defined discrete chromatin states and associated transcriptional networks underlying plastic and fixed dysfunction in TST, thus providing new insights into the genomic control circuitry of T cell differentiation/dysfunction that may point to new strategies for cellular reprogramming of T cells for cancer immunotherapy.

Disclosures
No relevant conflicts of interest to declare.