Introduction: Effector lymphocytes activated through classical pathways require glycolysis for survival, differentiation, and effector functions. Activated T cells switch from OXPHOS to aerobic glycolysis, presenting a potential therapeutic target in diseases characterized by immune hyperactivation, such as aGVHD. 4-OI is a cell-permeable derivative of endogenous itaconate, associated with immunoregulation, oxidative stress, and lipid peroxidation. It can suppress the inflammatory response in macrophages by targeting GAPDH to reduce aerobic glycolysis. However, whether 4-OI could reverse the metabolic reprogramming in aGVHD by partial suppression of GAPDH while exhibiting selective inhibition glycolysis in stimulated lymphocyte has not been explored.
Method: Human primary T cells and the mouse splenocyte were isolated by negative selection of isolate kit. Cell proliferation was detected by Ki67 and CFSE assays. GAPDH activity was measured with a specific assay kit. T cells metabolism was examined by seahorse experiments. Cytokine secretion was detected by CBA and flow cytometry. An aGVHD mouse model was established to evaluated clinical and pathology score. BALB/c donor T cells expressing luciferase used to measure the expansion of T cells by BLI. The allo-HSCT model supplemented with EL4-luc for tumor tracking was utilized to evaluate the cytotoxic capacity of T cells.
Results: In vitro, There are no significant difference in the survival rate of CD4/CD8 T cells (92±2%) treated with various concentrations of 4-OI. Compared to resting cells, activated T cells treated with 4-OI showed reduced GAPDH activity.4-OI greatly suppressed proliferation (CD4 60% vs 9%, CD8 78% vs 20%) and significantly decreased glycolysis capacity, suggesting an alteration in T cell metabolic programming. Cytokine production decreased considerably in activated CD4/CD8 T cells treated with 4-OI compared to resting cells. CD4 T cells (IFN-γ 14% vs 0.4%, TNF 16779pg/ml vs 12095pg/ml, CD107A 56% vs 24%, Granzyme B 26% vs 16%), CD8 T cells (IFN-γ 13% vs 4%, TNF 2311pg/ml vs 880pg/ml, CD107A 53% vs 16%, Granzyme B 51% vs 28%). In aGVHD model mice, 4-OI reduced GVHD sign manifestations, clinical scores and improved survival rates. BLI imaging demonstrated a significant reduction in donor T cell proliferation following 4-OI treatment. The glycolytic rate of recipient mouse splenic T cells in the 4-OI treatment group was significantly reduced. Serum cytokine levels (TNF-α, IFN-γ, IL-10, MCP-1, IL-12p70, and IL-6) were significantly lower in the 4-OI group. Our BLI findings also demonstrate that 4-OI treatment does not compromise the GVL effect in allo-HSCT.
Conclusion: Our findings suggest that 4-OI can suppress the proliferation and function of activated T cells by reversing metabolic reprogramming, indicating its potential as a therapeutic agent for GVHD and autoimmune diseases.