NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment[scRNA-seq_1]

Cytotoxic T cells (CTLs) play a crucial role in the elimination of cancer cells. However, within the intra-tumor microenvironment, these cells eventually undergo cell death or enter a state of dysfunction known as "exhaustion". T cell exhaustion not only impairs the efficacy of tumor immunity but also contributes to resistance against immune-checkpoint inhibitors. Our previous investigations have demonstrated that the NR4a family of nuclear receptors, induced by chronic TCR stimulation, plays a pivotal role in driving exhaustion. These receptors enhance the transcription of inhibitory molecules, such as PD1, while suppressing the expression of functional molecules, including cytokines. However, it remains unclear at which stage of T cell differentiation NR4a exerts its influence and whether the loss of NR4a can "rejuvenate" T cell fate. In the present study, we confirmed that simultaneous deletion of NR4a1 and NR4a2 in CD8+ T cells led to a less exhausted phenotype characterized by reduced expression of PD1, Tim3, and Tox, as well as increased oxidative phosphorylation (OXPHOS) and glycolysis activities, resulting in potent suppression of tumor growth. Interestingly, the transfer of activated NR4a1-/-NR4a2-/-CD8+ T cells into tumor-bearing mice gave rise to TCF1+ (Tcf7+) early memory T cells. Wild-type PD1+TIM3+CD8+ tumor-infiltrating lymphocytes (TILs) diminished in the secondary transfer to tumor-bearing mice, while NR4a-deficient CD8+ TILs expanded even during the secondary transfer, indicating a strong stemness of memory T cells due to NR4a loss. We discovered that NR4a directly repressed the expression of Tcf7 by binding to the enhancer region of the Tcf7 gene. Collectively, these findings indicate that inhibiting NR4a in tumors represents a potent strategy for immuno-oncotherapy by enhancing stemness and reducing exhaustion of memory T cells. Fourteen days after B16 tumor (1x10^6 cells/mouse) transplantation, intratumor CD3⁺ cells were isolated using an autoMACS Pro Separator by positive selection after Percoll enrichment. To obtain scRNA-seq data, we used an BD Rhapsody system (BD Biosciences) and TAS-Seq. A single-cell suspension of ~20,000 cells was captured from all isolated cells, without selection, on an array of >200,000 microwells through a limited dilution approach. Beads with oligonucleotide barcodes were added to saturation so that one bead was paired with each cell in a microwell. After exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription and Exonuclease treatment. cDNA amplification was performed by using TAS-Seq approach. Sequencing libraries were prepared by using NEBNext Ultra II FS library prep kit for Illumina (New England Biolabs). The sequencing analysis were performed by ImmunoGeneTeqs, Inc (Chiba, Japan) by a Novaseq 6000 sequencer (Illumina, San Diego, CA, USA) and a Novaseq S4 200 cycles v1.5 kit (Illumina).