Abstract PR09: Identifying the epigenetic code of tumor-specific CD8 T cell dysfunction and therapeutic reprogramming
Authors: Philip Mary , Fairchild Lauren , Sun Liping , Viale Agnes , Camara Steven , Horste Ellen , Merghoub Taha , Wolchok Jedd D. , Leslie Christina S. , Schietinger Andrea 
Published: November 2016

Abstract
Mutant neoantigens are commonly expressed in human solid tumors, and CD8 T cells specific for such antigens are detected in cancer patients. However, we know that these tumor-specific T cells are non-functional because despite their presence, tumors progress and often eventually cause death. Distinct T cell differentiation states are associated with specific epigenetic states that define the T cell's functional and phenotypic properties. It is currently not known what epigenetic changes establish and regulate tumor-specific CD8 T cell dysfunction and whether specific epigenetic modifications in dysfunctional T cells determine the ability to respond to therapeutic interventions such as checkpoint blockade (PD-1 and CTLA-4). Here, for the first time, we (1) characterize the progressive chromatin remodeling 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 in which tamoxifen treatment induces expression of an oncogenic driver neoantigen, SV40 large T antigen (Tag), we previously showed that tumor-specific CD8 T cells (TCRSV40-I) become unresponsive early during tumorigenesis, even before the emergence of a pathologically-defined malignant tumor (pre-malignant phase). While CD8 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 TCRSV40-I cells over the course of tumor development. In parallel, we carried our RNA-Seq on all samples to determine the interplay between chromatin remodeling and transcriptional networks. Substantial chromatin remodeling occurred during early T cell activation in the pre-malignant lesion (day 5), followed by a second wave of chromatin accessibility changes between day 7 and 14. Strikingly, after the second wave, no further CD8 T cell chromatin remodeling occurred during tumorigenesis, even after progression to an advanced late-stage tumor with an immunosuppressive microenvironment. Interestingly, these 2 distinct chromatin accessibility patterns (states 1 and 2) in dysfunctional TCRSV40-I correlated temporally with the plastic and fixed dysfunctional states and susceptibility to therapeutic reprogramming in vivo. To understand the transition from plastic to imprinted 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 tumor-specific T cells, 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.
Citation Format: Mary Philip, Lauren Fairchild, Liping Sun, Agnes Viale, Steven Camara, Ellen Horste, Taha Merghoub, Jedd D. Wolchok, Christina S. Leslie, Andrea Schietinger. Identifying the epigenetic code of tumor-specific CD8 T cell dysfunction and therapeutic reprogramming [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR09.