Gallic Acid Enhances GVL Effects of T Cells without Exacerbating Gvhd after Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for acute myeloid leukemia (AML), predominantly due to its potent graft-versus-leukemia (GVL) effect. However, leukemia relapse and the risk of graft-versus-host disease (GVHD) pose significant challenges to the successful outcomes of allo-HSCT. Here, we propose gallic acid (GA), a dietary polyphenolic compound found in fruits, vegetables, gallnuts, sumac, tea leaves, and herbal medicines, can enhance T-cell-mediated GVL effects without exacerbating GVHD. GA has garnered considerable attention for its antitumor effects, especially in inducing cancer cell apoptosis and enhancing immune responses. However, the modulatory effects of GA during the antileukemia process in HSCT are still poorly understood. In this study, we demonstrated that GA can enhance T-cell-mediated GVL effects both in vitro and in vivo. Firstly, we pre-cultured peripheral blood mononuclear cells (PBMCs) overnight and then treated the cells with GA for 24 h. Subsequently, we co-cultured PBMCs with THP-1 AML cells for 4 h at an effector-to-target ratio of 10:1. We found that GA increased the ratio of CD3⁺ T cells and CD3⁺CD8⁺ T cells and elevate the expression level of activation marker CD25 in the GA group compared with that in Vehicle group. We further analyzed the cytotoxic cytokine secretion of T cells and found that higher level of TNF-α and IFN-γ secreted by CD3⁺CD4⁺ T cells in the presence of GA. Moreover, compared with control group, GA significantly increased percentages of CD4⁺ CD107a⁺ and CD8⁺ CD107a⁺ cytotoxic T cells. Together, these results suggest that the GA could bolster T cell cytotoxic function, which in turn promotes the anti-leukemia efficacy against AML cell. Notably, unlike the direct inhibition of leukemia cells reported in previous study, our findings suggest that GA administration is more likely to exert a cytotoxic effect on tumor cells by enhancing T-cell activation rather than directly promoting apoptosis in AML tumor cells. Furthermore, we employed a non-irradiated leukemia mouse model created by transferring AML cells with the human fusion gene AML-ETO into BALB/c mice. The results showed that GA treatment prolonged the survival of leukemic mice and reduced leukemia cell infiltration. we also observed higher percentages of CD3⁺ T cells and an increase in CD25⁺ T cells among CD4⁺ T cells following treatment with GA. Furthermore, treatment with GA significantly increased the secretion level of TNF-α in CD4⁺ T cells and CD8⁺ T cells. Moreover, we detected the apoptosis levels of T cells under the influence of GA across different groups and the results showed that CD8⁺ T cells from spleen in GA group exhibited lower levels of apoptosis compared with that in PBS group. To further investigate whether GA treatment exacerbates GVHD-induced damage, we used a murine GVHD model. The results showed that there was no significant difference in GVHD scores or survival between the groups. Moreover, the GA group showed increased weight in the late stage of GVHD compared to the PBS group. These results suggest that GA enhances the GVL effects of T cells while minimizing GVHD-induced damage. To explore the mechanism by which GA enhances T cell cytotoxicity against leukemia cells, we performed RNA-seq and proteomic analysis of purified CD4⁺ and CD8⁺ T cells from the spleens of AML model mice. The results indicated that GA augments T-cell-mediated GVL effects through the activation of the MAPK and NF-κB pathways. Subsequently, we blocked MAPK/NF-κB signaling individually while simultaneously administering GA in leukemia mice. Our results showed that, compared with AML mice in the PBS group, leukemic mice treated with GA exhibited significantly prolonged survival. However, this prolonged survival was abolished after the addition of MAPK and NF-κB pathways inhibitors. This indicates that blocking these pathways individually weakened the protective effect of GA in AML mice.

Overall, we demonstrated that GA elevates T-cell anti-leukemia activity without exacerbating GVHD. Notably, GA, as a novel dietary component, typically derives from food or other natural sources, and its low dosage and easy availability make it feasible for patients to benefit from daily consumption. The availability and safety of GA make it a potential therapeutic agent for improving the prognosis of leukemia after HSCT.