Tumor-specific T-cells that have differentiated into a terminal dysfunctional state exist in the tumor microenvironment. A systematic understanding of the requirements of immunotherapeutic rescue of these cells is critically needed to improve clinical results in patients. Mouse models of chronic infection and cancer have been studied to elucidate biologic mechanisms of persistent antigen stimulation resulting in T-cell dysfunction, or “exhaustion.” Recently, chromatin accessibility imprinting has been associated with T-cells falling back into the dysfunctional state after temporary rescue by checkpoint blockade, suggesting epigenetic mechanisms in control of T-cell dysfunction. However, comprehensive characterization of T-cell dysfunction across models based on their epigenetic and transcriptional profiles is lacking.We collected 106 chromatin accessibility (ATAC-seq) samples and 87 gene expression (RNA-seq) samples from seven recent publications. We analyzed these data by first applying batch effect correction using generalized linear modeling. This enabled mapping profiles of chromatin accessibility peaks in gene promoters and enhancers from different studies into the same space. We observed that epigenetic profiles of dysfunctional tumor-infiltrating T-cells and dysfunctional T-cells in chronic viral infection were, surprisingly, extremely similar. Furthermore, a recently characterized discrete distinction between epigenetic profiles of early (day 7-8) and late (day 28-35) dysfunction in the tumor was recapitulated in the model of chronic infection. Overall we observed across mouse models that T-cells committed to becoming dysfunctional early after an immune challenge, rather than first mounting and then loosing an effector response. These observations were also largely recapitulated in gene expression analysis. Differentially expressed genes with massive differential accessibility of their promoter and enhancer peaks during development of dysfunction, observed consistently across models, including transcription factors (TF) well studied in immunity such as Tcf7, Lef1, Satb1, Ikzf2, Tox, are good candidates for further targeted analysis.We then turned to TF binding analysis. We associated absolute levels of chromatin accessibility in peaks of each sample with TF binding (predicted by motif analysis) using regularized negative binomial regression with cross-validation. We estimated the effect of each TF in each sample, which allowed us to map chromatin accessibility profiles into the TF activity space of much lower dimensionality. This mapping largely preserved the hierarchy of relative similarities between samples. We identified key TFs whose binding was associated with open or closed chromatin in functional and dysfunctional cell states. For example, not surprisingly, binding of well known effector factors Eomes and Batf was associated with closed chromatin in naive cells and open chromatin in effector cells. Strikingly, the strongest association with closing chromatin in dysfunction, consistently across mouse models, was observed for Tcf7/Lef1 binding, further suggesting the role of these TFs in establishing the terminal CD8 T-cell dysfunctional state. Notably, we found two large groups of TFs anti-correlated to each other whose predicted binding sites had higher accessibility in either functional or dysfunctional cells. This suggested that coordinated activity of a broad range of TFs (not necessarily binding at the same sites) might be responsible for establishing and maintaining T-cell functional states, and focusing on one or a handful of TFs is not sufficient to explain them. This analysis provides a better systematic understanding of cell-intrinsic mechanisms driving different functional states of CD8 T-cells, and the developed computational methods are broadly applicable in other experimental setups where diverse cell states are profiled by high-throughput genomic assays.
Citation Format: Yuri Pritykin, Christina Leslie. A unified genome-wide analysis of dysfunctional T-cell states in cancer and chronic viral infection [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B038.