Gamma-Glutamyl Cysteine Ligase Activity as a Proxy for Human T Cell Function and Drug-Induced Immunosuppression

Abstract

T cell effector functions are critical for immune defense, but theirdysregulation can cause diseases like immune exhaustion in cancer and lossof tolerance in autoimmunity. Curtailing these functions is essential intherapies such as chimeric antigen receptor T-cell (CAR-T) therapies or organtransplantation to avoid hyperactivation and rejection. A major challenge inthe field is the precise, live measurement of T cell function at the single-celllevel, limiting the prediction of immune responses, the development ofeffective immunotherapies, and optimization of immunosuppressiveregimens. Gamma-Glutamyl Cysteine Ligase (GCL), the rate-limiting enzymein glutathione (GSH) synthesis, is essential for T cell function in mice, but itsrole in human T cells is underexplored. GLed, a novel reversiblelanthanide-based GSH sensor is introduced that enables real-time,quantitative measurements of GCL activity at single-cell resolution. The GLedapproach distinguishes GSH contributions from GCL and GSR, linking GCLactivity directly to human T cell effector functions. Additionally, this revealspreviously unknown modulation of GCL activity by immunosuppressivedrugs, underscoring GCL as a critical player in T cell function and a potentialtherapeutic target in immune-related diseases.