1024 - Impaired mitochondrial metabolism drives T-cell dysfunction in chronic lymphocytic leukemia

Background: Chronic lymphocytic leukemia (CLL) remains incurable despite implementation of novel targeted therapies. Successful autologous cell-based anti-cancer therapies require functionality and longevity of effector cells, features that highly depend on complex metabolic processes. Our group previously demonstrated a disease-specific dysfunction in CLL T-cells including signs of impaired metabolic plasticity (Van Bruggen, 2019). However, in-depth analysis of the metabolic phenotype of CLL T cells and mechanisms underlying the dysfunction are lacking, preventing development of therapeutic strategies aiming to overcome dysfunction by metabolic reprogramming.

Methods: In this study, we assessed mitochondrial and glycolytic activity of T cells from CLL patients and age-matched healthy controls (HD) upon T cell receptor stimulation in-vitro (αCD3/28) by extracellular flux analysis. Concurrently, we measured the expression of key mitochondrial regulators and markers of T cell function by flow cytometry along time after two stimulations separated by a period of 15 days.

Results: Extracellular flux analyses revealed impaired mitochondrial activity in T cells from CLL patients, including reduced spare respiratory capacity, an important indicator of mitochondrial and T cell fitness. The response in glycolysis, a key pathway for early T-cell activation, varied between patients and reflected activation levels (Figure 1). These findings pointed towards mitochondrial dysfunction rather than overall metabolic
impairment at the basis of T-cell dysfunction in CLL. 13C-glucose and glutamine isotope tracing revealed that glutamine rather than glucose is the dominant fuel for mitochondrial metabolism. Although metabolic fluxes were conserved in CLL T-cells, metabolomics analyses showed significantly decreased tricarboxylic acid cycle (TCA) intermediates and nucleotides in stimulated T-cells from CLL patients, as well as a high ratio of oxidized/reduced glutathione, indicator of impaired mitochondrial activity and enhanced production of reactive oxygen species (Figure 2). In line with this, we observed a significant increase of mitochondrial depolarization in CLL T cells, which has been previously associated with mitochondrial damage and loss of T-cell effector function. Accordingly, CLL T cells showed delayed expression of activation markers, impaired proliferation and were almost completely unresponsive to re-stimulation. Mitochondrial function is primarily regulated by the expression of Peroxisome proliferator-activated receptor Gamma Coactivator 1-alpha (PGC1α) and Mitochondrial Transcription factor A (TFAM). Upregulation of both PGC1a and TFAM upon stimulation was delayed in CLL T cells compared to healthy T cells, and especially impaired after the second stimulation.

Conclusion: These results pinpoint to defects at the core of mitochondrial regulation as potential causes of T cell dysfunction in CLL. Specifically, we identified impaired expression of mitochondrial regulators in these cells, providing possible targets to improve T-cell based therapies in CLL