GSTP1 Improves CAR-T Cell Proliferation and Cytotoxicity to Combat Lymphoma

Guangsong Xu¹Jiani Wang¹Yuliang Qu¹Jing Ning²Yanting Zhang²Guangxian Xu³Yunxia Shi¹Ying Li¹Le Guo^1*Xuebo Han^1*Hongxia Wang^1*

• ¹Ningxia Medical University, Yinchuan, China
• ²General Hospital of Ningxia Medical University, Yinchuan, China
• ³Guangdong Medical University, Dongguan, China

Introduction The exhaustion of CAR-T cells hampers the efficacy of CAR-T cell therapy. Persistent antigen stimulation in T cells results in a surge of intracellular reactive oxygen species (ROS). ROS, as mitochondrial metabolites, alter the integrity of the mitochondrial membrane, promoting T-cell exhaustion. Glutathione S-transferase Pi-1 (GSTP1), a member of the glutathione S-transferase family, is an important enzyme in the intracellular clearance of ROS. Overexpression of GSTP1 can potentially enhance the anti-tumor capability of CAR-T cells. Methods The correlations between GSTP1 and genes related to T-cell exhaustion were analyzed using the TIMER database. Peripheral blood mononuclear cell (PBMCs) were collected from patients with hematologic malignancies (n=61) and healthy donors (n=45) to test GSTP1, BLIMP1, and PD-1 expression by qRT-PCR. A T-cell exhaustion model was built to test GSTP1 expression by western blotting. The dual-luciferase assay and ChIP-qPCR were used to determine whether the transcription factor BLIMP1 negatively regulated the activity of the GSTP1 promoter. CD19 CAR-T, GSTP1 CAR-T, and shGSTP1CAR-T cells were generated to evaluate their anti-tumor capacity. Results GSTP1 expression was downregulated when BLIMP1 and PD-1 were upregulated in PBMCs of cancer patients and in the in vitro T-cell exhaustion model. Meanwhile, ROS levels in the T-cell exhaustion model increased. Mechanistically, the BLIMP1 transcription factor negatively regulated the activity of the GSTP1 promoter. Based on the above findings, we engineered GSTP1 CAR-T cells, which exhibited improved functionality. GSTP1 CAR-T increased the TEMRA population, improved proliferation, cytotoxicity, elevated antioxidant capacity, increased IL-2 and IFN-γ secretion, reduced the expression of immune checkpoints and decreased apoptosis. In vivo, the residual levels of GSTP1 CAR-T cells were higher than those of CD19 CAR-T cells and shGSTP1 CAR-T cells, indicating that GSTP1 CAR-T cells exhibited a good anti-tumor capacity. Conclusion BLIMP1 directly suppressed GSTP1 transcription, while overexpression of GSTP1 enhanced the anti-tumor capacity of CAR-T cells and maintained redox homeostasis, offering a novel therapeutic strategy to improve CAR-T cell immunotherapy.