Introduction: Classically activated effector lymphocytes require glycolysis for survival, differentiation, and effector functions. Activated T cells switch from OXPHOS to aerobic glycolysis, presenting a potential therapeutic target in immune over activation disease such as aGVHD. 4-OI is a cell-permeable derivative of endogenous itaconate, has been implicated in 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. The proliferation ability was detected by Ki67 and CFSE. The GAPDH activity assay kit was used to detect GAPDH activity. T cells metabolism was examined by seahorse. The secretion of cytokines was detected by CBA and flow cytometry. We established the aGVHD model in mice to evaluated clinical score and pathology score. BALB/c donor Tcells expressing luciferase used to measure the expansion of Tcells by BLI. The allo-HSCT model supplemented with EL4-luc for tumor tracking were used for evaluating the killing ability of T cells.
Results: In vitro, we did not find differences in survival rate of CD4/CD8 T cells (92±2%) treated with different 4-OI concentrations. GAPDH activity in activated T cells treated with 4-OI was lower compared to resting cells. 4-OI greatly suppressed proliferation (CD4 60% vs 9%, CD8 78% vs 20%). 4-OI greatly lowered glycolysis capacity. It suggests that it can alter T cells' metabolic patterns. 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 and clinical scores and improved survival rates. BLI imaging showed that 4-OI treatment resulted in a significant decrease in proliferation of donor T cells. In the 4-OI treatment group, the glycolysis rate of T cells from spleen of recipient mice was significantly decreased. And In the cytokines of serum, the levels of TNF-α, IFN-γ, IL-10, MCP-1, IL-12p70 and IL-6 from 4-OI group were greatly reduced. Our BLI findings also demonstrate that 4-OI treatment does not compromise the GVL effect in allo-HSCT.
Conclusion: Our results demonstrated that 4-OI could inhibit the proliferation and function of activated T cells by reversing metabolic reprogramming which suggested 4-OI a potential therapeutic agent in GVHD and autoimmune diseases.