Immunometabolic Profiling Reveals Mitochondrial Dysfunction as a Driver of T Cell Impairment in Chronic Lymphocytic Leukemia
Autologous T-cell therapies, including chimeric antigen receptor (CAR)-T cells, show limited efficacy in chronic lymphocytic leukemia (CLL) due to disease-specific T-cell dysfunction. This dysfunction manifests as a skewing towards an effector phenotype, compromising T-cell memory formation. Effective T-cell activation and memory development require a balance between glycolysis, which rapidly provides energy and biosynthetic precursors for effector function, and a shift towards mitochondrial metabolism for long-term survival and memory formation. Our group previously identified signs of impaired metabolic plasticity in CLL T cells (Van Bruggen, Blood 2019 & Blood Adv 2022). However, in-depth analysis of the mechanisms underlying this metabolic dysfunction was lacking.
A comprehensive metabolic analysis of T cells from treatment-naive CLL patients and age-matched healthy controls (HD) was conducted following in-vitro αCD3/28 stimulation. Techniques included Flow Cytometry-based single-cell metabolic profiling, which measures protein synthesis inhibition as a proxy for metabolic activity (CENCAT; Vrieling, Cell Reports Methods 2024), alongside extracellular flux analysis, and 13C-glucose and glutamine tracing. Metabolic reprogramming was tested by treating CLL T cells with the PI3Kδ inhibitor idelalisib during stimulation.
CENCAT analysis revealed increased glucose dependence and glycolytic capacity in T cells from CLL patients upon early stimulation. However, unlike HD, CLL T cells showed reduced mitochondrial dependence and capacity during late activation, indicating an inability to switch from glycolytic to mitochondrial metabolism. In line with this, extracellular flux analysis revealed reduced spare respiratory capacity in CLL T cells, an important indicator of mitochondrial flexibility and memory development. To investigate the cause of impaired mitochondrial activity, we examined fuel utilization, as mitochondrial function relies on both substrate availability and intrinsic activity. 13C-glucose and glutamine tracing indicated correct fueling in CLL T cells. However, extracellular flux analysis on permeabilized cells, where mitochondria have access to readily available substrates, revealed persistently reduced mitochondrial activity. This indicated that mitochondrial dysfunction in CLL T cells arises from intrinsic limitations rather than lack of metabolic fuels. During late activation, CLL T cells exhibited elevated mTOR (mammalian target of rapamycin) signaling and reduced AMPK (AMP-activated protein kinase) activity, an imbalance that likely contributes to mitochondrial dysfunction due to the critical role of the AMPK/mTOR balance in regulating the switch to oxidative metabolism. This imbalance was confirmed by PI3K-mTOR inhibition during T cell activation, which resulted in mitochondrial activity and memory phenotype.
Our study reveals that mitochondrial dysfunction, characterized by intrinsic limitations rather than merely inadequate substrate availability, drives T cell impairment in T cells from patients with CLL. Targeting metabolic pathways, particularly through mTOR inhibition, enhances mitochondrial activity and promotes memory formation in CLL T cells, offering promising therapeutic strategies for improving autologous T-cell therapies.