Caryophylli Cortex Suppress PD-L1 Expression in Cancer Cells and Potentiates Anti-Tumor Immunity in a Humanized PD-1/PD-L1 Knock-In MC-38 Colon Cancer Mouse Model

Immune checkpoints are essential for regulating excessive autoimmune responses and maintaining immune homeostasis. However, in the tumor microenvironment, these checkpoints can lead to cytotoxic T cell exhaustion, allowing cancer cells to evade immune surveillance and promote tumor progression. The expression of programmed death-ligand 1 (PD-L1) in cancer cells is associated with poor prognoses, reduced survival rates, and lower responses to therapies. Consequently, downregulating PD-L1 expression has become a key strategy in developing immune checkpoint inhibitors (ICIs). Caryophylli cortex (CC), derived from the bark of the clove tree Syzygium aromaticum, possesses antioxidant and cytotoxic properties against cancer cells, yet its potential as an ICI remains unclear. Therefore, we investigated whether CC extract modulates PD-L1 expression in cancer cells and assessed its ability to activate T cell immunity in both in vitro co-culture and in vivo animal models. The results indicated that CC extract significantly downregulated both constitutive and inducible PD-L1 expression at non-toxic concentrations for cancer cells while enhancing cancer cell mortality and T cell activity in co-culture. Additionally, administering CC extract to hPD-L1/MC-38 tumor-bearing mice resulted in over a 70% reduction in tumor growth and increased CD8+ T cell infiltration in the tumor microenvironment. Principal component analysis identified bergenin, chlorogenic acid, and ellagic acid as active ICIs. These findings suggest that CC extract exerts a potent antitumor effect as an immune checkpoint blocker by inhibiting PD-L1 expression in cancer cells and disrupting the PD-1/PD-L1 interaction.