show Abstracthide AbstractMR1T cells are a recently discovered class of T cells that recognize antigens presented by the MHC-I–related molecule MR1 in the absence of microbial infection. The nature of the self-antigens that stimulate MR1T cells remains unclear, hampering our understanding of their physiological role and therapeutic potential. By combining genetic, pharmacological, and biochemical approaches, we found that carbonyl stress and changes in nucleobase metabolism in target cells promote MR1T cell activation. Stimulatory compounds formed by carbonyl adducts of nucleobases were detected within MR1 molecules produced by tumor cells and their abundance and antigenicity were enhanced by drugs that induce carbonyl accumulation. Our data reveal carbonyl nucleobase adducts as MR1T cell antigens. Recognizing cells under carbonyl stress allows MR1T cells to monitor cellular metabolic changes with physiological and therapeutic implications. Overall design: A375-MR1-Cas9 cells were transduced by pooled part A and B GecKO v2 CRISPR library at an MOI of 0.3. Replicates of resultant APCs had a 64-fold representation of each guide within the library and underwent 4 consecutive rounds of killing by TC5A87 T cells at a E:T of 2:1, following which surviving cells were expanded for 24 hrs and genomic DNA extracted for PCR amplicon sequencing.