Adenosine impairs T cell function via activation of purine salvage pathway and AMP-induced inhibition of pyrimidine nucleotide biosynthesis

Extracellular adenosine (ADO) is a well-established immune checkpoint mediator suppressing T cells via activation of A2A receptors, yet intracellular mechanisms of ADO action remain less understood. Here, we show that activated human T cells transport extracellular ADO through equilibrative nucleoside transporter 1 (ENT1) and metabolize it through ATP salvage pathway. The generated ADO metabolite AMP inhibits UMP synthase (UMPS), the rate-limiting enzyme of de novo pyrimidine biosynthesis, leading to diminished ATP production via conventional bioenergetic pathways, increased apoptosis, and impaired effector functions of T cells. Flow cytometric and single-cell transcriptomic analyses further revealed that ADO restrains the exit of naive T cells from quiescence. All effects of ADO were phenocopied by treating T cells with the inhibitors of UMPS and reversed after pharmacological blockage of ENT1 or uridine supplementation. Additional molecular docking, molecular dynamics simulations, and free-energy analyses indicate that AMP can occupy the orotidine 5'-monophosphate (OMP) decarboxylase catalytic site of UMPS with the same anchoring contacts as substrate OMP and product UMP, providing a structural rationale for direct AMP-mediated inhibition of pyrimidine biosynthesis. These findings demonstrate that immunosuppressive effects of ADO extend beyond canonical adenosinergic signaling and involve cellular ADO uptake and modulation of purine and pyrimidine metabolism.