ABSTRACT Cytotoxic T cells dynamically rewire their metabolism during the course of an immune response. While T-cell metabolism has been extensively studied at phenotypic endpoints of activation and differentiation, the underlying dynamics remain largely elusive. Here, we leverage on single-cell RNA-sequencing (scRNA-seq) measurements of in vitro activated and differentiated CD8 + T cells cultured in physiological media to resolve these metabolic dynamics. We find that our scRNA-seq analysis identifies most metabolic changes previously defined in in vivo experiments, such as a rewiring from an oxidative to an anabolism-promoting metabolic program during activation to an effector state, which is later reverted upon memory polarization. Importantly, our scRNA-seq data further provide a dynamic description of these changes. In this sense, our data predict a differential time-dependent reliance of CD8 + T cells on the synthesis versus uptake of various non-essential amino acids during T-cell activation, which we corroborate with additional functional in vitro experiments. We further exploit our scRNA-seq data to identify metabolic genes that could potentially dictate the outcome of T-cell differentiation, by ranking them based on their expression dynamics. Among the highest-ranked hits, we find asparagine synthetase ( Asns ), whose expression sharply peaks for effector CD8 + T cells and further decays towards memory polarization. We then confirm that these in vitro Asns expression dynamics are representative of an in vivo situation in a mouse model of viral infection. Moreover, we find that disrupting these expression dynamics in vitro , by depleting asparagine from the culture media, delays central-memory polarization. Accordingly, we find that preventing the decay of ASNS by stable overexpression at the protein level in vivo leads to a significant increase in effector CD8 + T-cell expansion, and a concomitant decrease in central-memory formation, in a mouse model of viral infection. This shows that ASNS expression dynamics dictate the fate of CD8 + T-cell differentiation. In conclusion, we provide a resource of dynamic expression changes during CD8 + T-cell activation and differentiation that is expected to increase our understanding of the dynamic metabolic requirements of T cells progressing along the immune response cascade.

CD8<sup>+</sup> T-Cell Metabolic Rewiring Defined by Single-Cell RNA-Sequencing Identifies a Critical Role of ASNS Expression Dynamics in T-Cell Differentiation

CD8+ T-Cell Metabolic Rewiring Defined by Single-Cell RNA-Sequencing Identifies a Critical Role of ASNS Expression Dynamics in T-Cell Differentiation