Regulation of the Elevated T Cell Glycolysis May Alleviate Acute Graft-Versus-Host Disease Post-Allotransplant

Background: Acute graft-versus-host disease(aGVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). The pathogenesis of aGVHD is commonly considered to be caused by exaggerated and undesirable immune responses. Metabolism not only provide energy and substrates for T cell growth and survival, but also instruct effector functions, differentiation, and gene expression of T cells. In this regard, the metabolic profile of T cells was reported to play a critical role in the occurrence and development of many immunological disorders such as systemic lupus erythematosus and rheumatoid arthritis. Murine studies found that alloreactive T cells use aerobic glycolysis as the predominant metabolic process to meet activation and proliferation demand after allo-HSCT. However, the metabolic profile of T cells and the approach for regulating T cell metabolism in aGVHD patients remains to be elucidated.

Aims: To determine the metabolic state in T cells of patients with aGVHD. Moreover, to investigate the effect of the novel approach targeting the abnormal metabolism in T cells of aGVHD patients, which may provide a potential therapeutic target for aGVHD patients after allo-HSCT.

Methods: In this prospective case-control study, a total of 25 patients with aGVHD and 25 matched patients without aGVHD(non-aGVHD) after allo-HSCT were enrolled. T cell subsets were analyzed in aGVHD and non-aGVHD patients by flow cytometry. Th1, Th2, Th17, and Treg cells were identified as CD4⁺IFN-γ⁺, CD4⁺IL-4⁺, CD4⁺IL17A⁺, and CD4⁺CD25⁺Foxp3⁺, respectively. Tc1 and Tc2 cells were identified as CD8⁺IFN-γ⁺ and CD8⁺IL-4⁺, respectively. In order to determine the metabolic state in T cells of patients with aGVHD and non-aGVHD, the metabolic profile was determined using a Seahorse XF96 Analyzer. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. The mitochondrial mass, the mitochondrial membrane potential, the protein expressions for the lipid metabolism enzyme CTP1a and the glycolytic activator PFKFB3 were measured by flow cytometry. To further understand the metabolic state of T cells in aGVHD and non-aGVHD patients and investigate its mechanism, RNA sequencing (RNA-Seq) was performed to analyze the gene expression profiles of T cells. Subsequently, to explore the potential way of targeting the abnormal metabolism in T cells, the glycolysis inhibitor 3-PO was administrated to T cells from aGVHD patients.

Results: When compared with T cells in non-aGVHD patients, T cells in aGVHD patients were polarized towards pro-inflammatory T cells, characterized by an elevated proportion of Tc1, Th1 and Th17. Furthermore, T cells isolated from aGVHD patients exhibited higher extracellular acidification rate, as well as the increased glucose consumption rate and lactate production rate compared to those in non-aGVHD patients. Moreover, elevated expression of PFKFB3 was observed in T cells, especially in naïve T cells of aGVHD patients, but oxygen consumption rate, CPT1A, mitochondrial mass or membrane potential showed no significant differences in T cells between aGVHD and non-aGVHD patients. These results implied higher glycolytic activity of T cells in aGVHD patients when compared with those in non-aGVHD patients. Consistent with the increased glycolytic activity observed in T cells from aGVHD patients, the mRNA levels of genes involved in the glycolytic pathway were substantially elevated in T cells of aGVHD patients compared to those in non-aGVHD patients. Importantly, in vitro treatment with glycolysis inhibitor 3-PO improved the activity of T cells derived from aGVHD patients through down-regulating glycolytic activity of T cells.

Summary/Conclusion: The current study demonstrated that T cells in aGVHD patients preferentially depend on glycolysis to meet activation and proliferation demands. Furthermore, the activity of T cells from aGVHD patients could be ameliorated by glycolysis inhibitor 3-PO in vitro. Although further validation is required, T cell glycolysis promises to be a novel therapeutic target for aGVHD patients after allo-HSCT.