NR4A1 transcriptionally regulates the differentiation of stem-like CD8+ T cells in the tumor microenvironment (ChIP-Seq)

CD8+ T cells are rendered exhausted in tumor and chronic infection. Among heterogeneous exhausted T cells, a subpopulation of progenitor-like (Tpex) cells have been found important for long-term tumor or pathogen control, and are also the main responders in immunotherapy. Using a new reporter mouse for the orphan nuclear receptor NR4A1, originally characterized critical in T cell dysfunction, we discover that the reporter is highly expressed in Tpex cells in tumor and chronic infection. Enforced expression of Nr4a1 promotes Tpex cell accumulation, whereas tumor control is improved after Nr4a1 deletion, associated with increased effector function but decreased long-term maintenance of CD8+ T cells. Integrating ChIP-seq and RNA-seq analysis, NR4A1 is found to bind and promote the expression of Tpex-related genes, and suppresses terminal differentiation-associated genes. This study therefore has identified a key role of NR4A1 in Tpex regulation and provides a promising target for immunotherapy. Overall design: NR4A1-OV/ RV-GFP virus infected CD8+ T cells re-stimulated with PMA/Ionomycin were used for RNA extraction. NR4A1 WT/KO naïve CD8+ T cells were activated with plate-bound anti-CD3/CD28 (2µg/mL) and re-stimulated with PMA/Ionomycin for RNA extraction. The tumor-infiltrating OT-I cells were isolated on day 21 post tumor cell inoculation, and the Nr4a1RFP+ and Tim-3+ OT-I cells were sorted with BD FACS Aria Cell Sorter. Tumor infiltrated Nr4a1-/- and Nr4a1+/+ Ly108+ OT-I cells were sorted out for RNA harvest. Total RNA was extracted reverse transcribed into cDNA and built library for sequencing using BGI-Hiseq500 (BGI Genomics) or Smartseq2. For ChIP-seq samples, Nr4a1-HA infected CD8+ T cells were purified and re-stimulated with PMA/Ionomycin, the the cells were fixed and digested with MNase cocktail (Active Motif). Antibodies against HA tag (3724, clone C29F4; Cell Signaling Technology) were used to precipitate the NR4A1 bounded chromatin. The immunoprecipitated DNA was purified and sent to BGI Genomics company for library building and sequencing with Illumina HiSeq 2500.