Continuous expression of TOX safeguards exhausted CD8 T cell epigenetic fate

CD8 T cell exhaustion is a major barrier limiting anti-tumor therapy. Though checkpoint blockade temporarily improves exhausted CD8 T cell (Tex) function, the underlying epigenetic landscape of Tex remains largely unchanged, preventing their durable “reinvigoration.” Whereas the transcription factor (TF) TOX has been identified as a critical initiator of Tex epigenetic programming, it remains unclear whether TOX plays an ongoing role in preserving Tex biology after cells commit to exhaustion. Here, we decoupled the role of TOX in the initiation versus maintenance of CD8 T cell exhaustion by temporally deleting TOX in established Tex. Induced TOX ablation in committed Tex resulted in apoptotic-driven loss of Tex, reduced expression of inhibitory receptors including PD-1, and a pronounced decrease in terminally differentiated subsets of Tex cells. Simultaneous gene expression and epigenetic profiling revealed a critical role for TOX in ensuring ongoing chromatin accessibility and transcri..., Cells from inducible-Cre (Rosa26CreERT2/+Toxfl/fl P14) mice where TOX was temporally deleted from mature populations of LCMV-specific T exhausted cells after establishment of chronic LCMV infection 5 days post infection were subjected to scRNA and scATACseq coassay,naive cells and WT cells were used as controls.
Analysis pipeline developed by Josephine Giles and vignettes published by Satija and Stuart labs.Transcript count and peak accessibility matrices deposited in GSE255042,GSE255043.
Seurat/Signac was used to process the scRNA and scATACseq coassay data
The processed Seurat/Signac object above was subsequently used for downstream RNA and ATAC analyses as described below:
DEGs between TOX WT and iKO cells within each subset were identified using FindMarkers (Seurat, Signac), with a log2-fold-change threshold of 0, using the SCT assay. DACRs were identified using FindMarkers using the \"LR\" test, with a log2-fold-change threshold of 0.1, a min.pct of 0.05, and included the number of c..., , # Continuous expression of TOX safeguards exhausted CD8 T cell epigenetic fate

[https://doi.org/10.5061/dryad.8kprr4xx9](https://doi.org/10.5061/dryad.8kprr4xx9)

Seurat/Signac pipeline for multiomic scRNA-seq and scATAC-seq dataset, generated following inducible TOX deletion in LCMV-Cl13

Author

Yinghui Jane Huang

## Script information

**Purpose:** Generate and process Seurat/Signac object for downstream analyses
**Written:** Nov 2021 through Oct 2022
**Adapted from:** Analysis pipeline developed by Josephine Giles and vignettes published by Satija and Stuart labs
**Input dataset:** Transcript count and peak accessibility matrices deposited in GSE255042,GSE255043

# Signac Object Generation

1\) Create individual signac objects for each sample from the raw 10x cellranger output.

2\) Merge individual objects to create one seurat object.

3\) Add metadata to merged seurat object.

Following are the steps in the attached html file for analysis of the paired data (ATAC+RNA)

* Load fr...,