biostudies-literatureXu XNIDDK NIH HHSNCI NIH HHSNIH HHS932-943.e8https://www.ebi.ac.uk/biostudies/studies/S-EPMC12118570biostudies-literatureUnknown31(5)Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate contributions to purine nucleotides from salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic or lymph node T cells) synthesize purines de novo. Shortage of 1C units for T cell purine synthesis is accordingly a potential bottleneck for anti-tumor immunity. Supplementing 1C units by infusing formate drives formate assimilation into purines in tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling kinetic control ofCell chemical biologyOne-carbon unit supplementation fuels purine synthesis in tumor-infiltrating T cells and augments checkpoint blockade.PMC12118570S10 OD028592DP1 DK113643R01 CA163591Rabinowitz JDChen ZXing XBartman CRXu XOlszewski KfalseOne-carbon unit supplementation fuels purine synthesis in tumor-infiltrating T cells and augments checkpoint blockade.Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate contributions to purine nucleotides from salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic or lymph node T cells) synthesize purines de novo. Shortage of 1C units for T cell purine synthesis is accordingly a potential bottleneck for anti-tumor immunity. Supplementing 1C units by infusing formate drives formate assimilation into purines in tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling kinetic control of2024-01-01T00:00:00Z2024 May2026-05-29T17:15:19.084Z2026-04-08T05:26:06.58ZS-EPMC121185703875961910.1016/j.chembiol.2024.04.007