P595: DEFECTIVE MITOCHONDRIAL METABOLISM IS AT THE BASIS OF T-CELL DYSFUNCTION IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

Background: Chronic lymphocytic leukemia (CLL) remains incurable despite availability of targeted therapies. Successful autologous cell-based anti-cancer therapies require functionality and longevity of effector cells, features that highly depend on complex metabolic processes. A key feature of CLL is the suppression of T-cell function, but the underlying mechanism is still poorly understood. We previously found signs of impaired metabolic plasticity upon stimulation of T cells from CLL patients (van Bruggen, Blood 2019). However, in-depth analysis of the metabolic signature of CLL T cells, and how it differs from healthy T cells, is lacking. Aims: With this study, we aim to analyze mitochondrial and glycolytic metabolism of T cells from healthy individuals and CLL patients in real-time, and characterize their metabolic signatures and fuel dependencies upon T cell receptor (TCR) engagement. Methods: Peripheral blood mononuclear cells (PBMCs) of age-matched healthy donors (HD) and untreated CLL patients were analyzed by flow cytometry, extracellular flux analysis, LC-MS metabolomics and C13 fractional labelling upon stimulation with αCD3/αCD28 antibodies. Results: Upon stimulation, T cells from CLL patients showed decreased activation levels and reduced increase in mitochondrial mass, as compared to HD T cells. Accordingly, extracellular flux analysis revealed an impairment to upregulate mitochondrial activity upon stimulation in T cells from all patients analyzed, including reduced spare respiratory capacity, an important indicator of mitochondrial and (CAR)-T cell fitness. The response in glycolysis, a key pathway for early T-cell activation, varied between patients. These findings pointed towards mitochondrial dysfunction rather than overall metabolic impairment at the basis of T-cell dysfunction in CLL. Metabolomics analyses on stimulated T-cells from CLL and HD indicated a global decrease in metabolite abundance in CLL, with several intermediates of the tricarboxylic acid cycle (TCA) as significant hits. This deficient mitochondrial metabolism in CLL T cells could be caused by hindered activity of mitochondrial enzymes or decreased fuel utilization. Glucose and glutamine are major fuels of the TCA cycle and, together with fatty acids, are used for biomolecule synthesis and energy production. Fractional labelling showed that mitochondrial enzymes are functional in CLL T cells and identified glutamine as the key contributor to the TCA cycle in stimulated T cells derived from both HD and CLL. These observations, together with our recent finding that activated CLL cells predominantly depend on glutamine (Chen, Blood 2022), hint at a compromised availability of glutamine for the surrounding T cells in CLL. Counterintuitively, activation and proliferation studies revealed that HD T cells are more susceptible to glucose and glutamine deprivation than CLL-derived T cells, which might be due to the fact that HD T cells rely on highly active metabolism sustained by these fuels, whereas the metabolic state of CLL T cells is already lower in non-restricted nutrient conditions. Summary/Conclusion: CLL T cells have a distinct metabolic profile upon stimulation, which is characterized by defective mitochondrial content and activity. Despite finding glutamine as the main fuel for the TCA cycle in both HD and CLL T cells, the metabolic rate of CLL T cells is lower, which ultimately contributes to T-cell dysfunction.Keywords: Mitochondria, Chronic lymphocytic leukemia