Attenuation of CD8 T Cell Exhaustion via miR-379-5p-Mediated Memory Promotion and Immune Checkpoint Inhibition

MicroRNAs are epigenetic regulators of T cell maturation and exhaustion. However, the specific mechanisms by which miRNAs modulate T cell functions during tumor development are not well understood. Here, we demonstrate that miR-379-5p counteracts the exhaustion phenotype induced by chronic T cell stimulation, enhancing anti-tumor effector functions. MiR-379-5p is downregulated in exhausted T cells, negatively associated with exhausted tumor-infiltrating lymphocytes in advanced tumors, and positively correlated with favorable survival in breast cancer and other cancer types. MiR-379-5p directly targets the 3' untranslated region (3’-UTR) of TIM3 and TIGIT, suppressing their expression in CD8 T cells. Ectopic miR-379-5p expression directs differentiation to memory-like effector cells, enhancing cancer cell killing activity. Conversely, the nuclear receptor NR4A1 negatively regulates miR-379 in T cells, restoring immune checkpoint gene expression and mitigating the cancer-killing ability. OT-1 T cells expressing miR-379-5p have elevated cytotoxic killing against B16F10-OVA tumors in NOD-SCID mice. Importantly, autologous T cells isolated from breast cancer patients introduced with miR-379-5p significantly increase their killing activity against tumor organoids derived from the matched patients. Our findings identify miR-379-5p as an epigenetic tumor suppressor that promotes CD8+ T cell effector functions, offering promise for novel cancer immunotherapy strategies. Whole blood was collected from healthy donors, and native CD8 T cells were purified from the isolated peripheral blood mononuclear cells (PBMCs). These CD8 T cells were then subjected to chronic stimulation with anti-CD3/anti-CD28 antibodies and IL-2 for 12 days to induce exhaustion. To evaluate miRNA expression, small RNAs were isolated from both naïve and exhausted T cells and analyzed using next-generation sequencing (miRNA-seq). A comparative miRNA-seq analysis was performed on exhausted versus unstimulated T cells. To evaluate gene expression, total RNA was isolated from both naïve and exhausted T cells and analyzed using next-generation sequencing (RNA-seq). A comparative RNA-seq analysis was performed on exhausted versus unstimulated T cells. CD8 T cells were subjected to chronic stimulation with anti-CD3/anti-CD28 antibodies and IL-2 for 12 days to induce exhaustion. Then, the cells were transfected with miR-Scr or miR-379-5p, and cultured for 48 hours. To evaluate gene expression, total RNA was isolated and analyzed using next-generation sequencing (RNA-seq). A comparative RNA-seq analysis was performed on miR-379-5p versus miR-Scr treated T cells.