The integration of metabolic and proteomic data uncovers an augmentation of the sphingolipid biosynthesis pathway during T-cell differentiation.

Recent studies have highlighted the significance of cellular metabolism in the initiation of clonal expansion and effector differentiation of T cells. Upon exposure to antigens, naïve CD4+ T cells undergo metabolic reprogramming to meet their metabolic requirements. However, only few studies have simultaneously evaluated the changes in protein and metabolite levels during T cell differentiation. Our research seeks to fill the gap by conducting a comprehensive analysis of changes in levels of metabolites, including sugars, amino acids, intermediates of the TCA cycle, fatty acids, and lipids. By integrating metabolomics and proteomics data, we discovered that the quantity and composition of cellular lipids underwent significant changes in different effector Th cell subsets. Especially, we found that the sphingolipid biosynthesis pathway was commonly activated in Th1, Th2, Th17, and iTreg cells and that inhibition of this pathway led to the suppression of Th17 and iTreg cells differentiation. Additionally, we discovered that Th17 and iTreg cells enhance glycosphingolipid metabolism, and inhibition of this pathway also results in the suppression of Th17 and iTreg cell generation. These findings demonstrate that the utility of our combined metabolomics and proteomics analysis in furthering the understanding of metabolic transition during Th cell differentiation.

Characterization of proteogenomic signatures of differentiation of CD4+ T cell subsets.

Functionally distinct CD4+ helper T (Th) cell subsets, including Th1, Th2, Th17, and regulatory T cells (Treg), play a pivotal role in the regulation of acquired immunity. Although the key proteins involved in the regulation of Th cell differentiation have already been identified how the proteogenomic landscape changes during the Th cell activation remains unclear. To address this issue, we characterized proteogenomic signatures of differentiation to each Th cell subsets by RNA sequencing and liquid chromatography-assisted mass spectrometry, which enabled us to simultaneously quantify more than 10,000 protein-coding transcripts and 8,000 proteins in a single-shot. The results indicated that T cell receptor activation affected almost half of the transcript and protein levels in a low correlative and gene-specific manner, and specific cytokine treatments modified the transcript and protein profiles in a manner specific to each Th cell subsets: Th17 and Tregs particularly exhibited unique proteogenomic signatures compared to other Th cell subsets. Interestingly, the in-depth proteome data revealed that mRNA profiles alone were not enough to delineate functional changes during Th cell activation, suggesting that the proteogenomic dataset obtained in this study serves as a unique and indispensable data resource for understanding the comprehensive molecular mechanisms underlying effector Th cell differentiation.