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

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. CreERT2 expressing NR4a1flox/floxNR4a2flox/flox CD8+T cells were generated by transducing them with a retrovirus obtained from transfection with pMX-Cre-IRES-GFP retroviral vector and pCL-Eco packaging vectors into Plate-E cells 293T HEK cells . Naïve CD8+ T cells were isolated from the spleen, inguinal, axillary, and mesenteric lymph nodes of NR4a1flox/floxNR4a2flox/flox, OT-I, tdTomato-reporter mice and were activated by plate-coated with 2 μgml-1 of anti-CD3 monoclonal antibody and 1 μg ml-1 of soluble anti-CD28 monoclonal antibody, supplemented with 10 ngml-1 of IL-2, for 36-48 hr. The harvested cells were adoptively transferred into Rag2-/- recipient mice. On 21 days after tumor inoculation, CD8+PD1+TIM3+ TILs were isolated using a FACS AriaIII and secondary transferred into Rag2-/- mice. After one day those mice were subcutaneously injected with B16-OVA cells.