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Abstract

The field of immunometabolism, which explores how metabolism regulates the function of immune cells, has emerged over the past decade. The available metabolomic methods require a large amount of biological material and only provide bulk measurements that miss details like cell heterogeneity or rare populations. The recent advances in the single-cell omics field aim to provide a response to these drawbacks. HT-SpaceM is a high-throughput single-cell metabolomics method that combines light microscopy with MALDI-Imaging Mass Spectrometry. This dissertation aims to characterize the metabolic requirements of human primary CD4+T cells in vitro with HT-SpaceM. CD4+T cells are a heterogeneous population that plays a key role in regulating and coordinating the adaptive immune response. The activation of these cells, which is triggered by antigen presentation, is the starting point for proper immune function and is heavily supported by a metabolic shift. Naïve CD4+T cells exhibit a quiescent oxidative metabolic profile that, upon activation, is remodeled in favor of glycolysis to support proliferation and differentiation. The metabolic profiles of CD4+T cells after activation and modulation with several compounds were measured at the single-cell level with HT-SpaceM. The detection of unique metabolites in cells modulated with different drugs helped unravel the mechanism of action of these compounds. Activated CD4+T cells exhibited higher levels of asparagine, proline, and glutaminolysis metabolites than the naïve state. The high intensities of orotic acid and L-dihydroorotic acid in cells treated with antimycin A revealed a potential effect of this complex III inhibitor on the pyrimidine de novo biosynthesis. The increased detection of phosphoribosyl pyrophosphate and ribose 5-phosphate in cells treated with the anti-inflammatory drug methotrexate resulted from the impairment of the purine de novo biosynthesis caused by this drug. The metabolic modulation of activated CD4+T cells also enabled the generation of a single-cell atlas of metabolic states that covered key metabolic pathways such as glycolysis and respiration. The projection of activated cells treated with Janus kinase inhibitors on the atlas revealed that these drugs have a heterogeneous effect and affect the purine synthesis pathway. The results of this thesis help advance the fields of single-cell metabolomics and immunometabolism by describing a method for characterizing the metabolic states of CD4+T cells at the single-cell level and providing insights regarding the metabolic mode of action of different inhibitors.