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

TCR stimulation caused dynamic changes in the intracellular metabolism, including glucose, amino acid, intermediates of TCA cycle, and fatty acid metabolism to fuel biosynthetic processes

To evaluate the changes in metabolic pathways during T cell differentiation, we first analyzed the amounts of intracellular metabolites and expression of metabolic proteins using a mass-spectrum-based metabolomics approach and previously established proteogenomic database19 (Fig. 1a). Naïve CD4+ T cells were stimulated with immobilized the anti-TCR mAb and anti-CD28 mAb for 48 h under Th0, Th1, Th2, Th17, or induced regulatory T cell (iTregs) culture conditions. Dead cell removal was performed before sample preparation for omics analysis. As reported previously, our omics analysis showed that TCR stimulation elicited a rapid induction of glycolysis at the level of metabolites and proteins6,7 (Fig. 1b). The intermediate of glycolysis, G3P, was decreased, while the end product, lactate, displayed a substantial increase upon TCR stimulation (Fig. 1b). Proteomics data revealed significant increases in the expression of glucose transporters and glycolytic enzymes, including GLUT1, GLUT3, HK1, GPI, PKM, and LDHA. Furthermore, we observed that TCR stimulation enhanced the utilization of glucogenic amino acids synthesis, which was synthesized at lower levels in resting naïve CD4+ T cells (Fig. 1b lower panel, and Supplementary Fig. 1a). While the amounts of serine was reduced by 1.5-fold in Th0, Th1, and Th2 cells and increased by 1.5-fold in Th17 and iTreg cells, the amounts of glycine and cysteine was significantly elevated in each effector Th cell subsets (Fig. 1b, upper panel). In accordance with this result, protein expression of glucogenic amino acids synthase was highly upregulated. These amino acids serve as a primary source for the one carbon pathway, an essential component for the biosynthesis of both proteins and DNA. Thus, these findings suggest that activated Th cells acquire one carbon metabolism to support their rapid proliferation. Next, we focused on TCA cycle and its associated metabolites. Despite observed minimal variation in the intermediates of the TCA cycle, such as succinate, fumarate, malate, and citrate, between naïve CD4+ T cells and effector Th cell subsets, the amounts of glutamine were greatly reduced following TCR stimulation (Fig. 1c). This finding was supported by the decrease in expression of the enzyme glutamine synthase (GLUL) and the concurrent increase of degradative enzyme (GLS2) in effector Th cell subsets, demonstrating the validity of our multi-omics analysis in reflecting the activation of glutaminolysis in effector Th cells20. In addition, the level of glutamate was increased in Th17 cells, whereas no significant change was observed in the amounts of glutamate in the other effector Th cell subsets. Furthermore, there was a significant increase in the levels of several amino acids, including alanine, lysine leucine, threonine, aspartate, asparagine, phenylalanine, tyrosine, and proline. Correspondingly, we also noted an up-regulation in the expression of their synthase and transporters, indicating that amino acid biosynthesis and uptake were activated during T cell activation (Fig. 1b, c and Supplementary Fig. 1b). It is noteworthy that we observed a marked increase in the amounts of β-alanine, a crucial component for the production of acetyl-CoA, in effector Th cell subsets (Fig. 1d). Since acetyl-CoA was required for the fatty acid biosynthesis, we next investigated the effect of TCR-stimulation on fatty acid biosynthesis pathway. Initially, we detected minor alterations in the expression of enzymes associated with fatty acid elongation, such as ELOVL1, ELOVL5, ELOVL6, and ELOVL7 in Th cell subsets (Fig. 1e). In contrast, TCR stimulation resulted in a marked upregulation of proteins involved in fatty acid desaturation, including SCD1, SCD2, FADS1, and FADS2 (Fig. 1e). Subsequently, we sought to assess the levels of fatty acid saturation, focusing on saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA). In quiescent naïve CD4+ T cells, SFA accounted for 92.5% of total fatty acids, while MUFAs and PUFAs comprised relatively smaller proportions (MUFA: 2.68%, PUFA: 4.85%) (Fig. 1f). Activated Th cell exhibited changes in the degree of fatty acid saturation, with the MUFA composition of activated Th cell subsets increasing to 26-37% (Fig. 1f). These findings are reflected in the observation that while most fatty acids were largely increased by TCR-stimulation, the amounts of MUFAs increased the most (Fig. 1g). Taken together, these data indicate that TCR stimulation confers activated Th cells with an increased ability to engage in glycolysis and amino acid synthesis and generates a diversity of fatty acid composition.

Fig. 1: Combination analysis of metabolomic and proteomic data showed T cell activation causes rewiring of the intracellular metabolism.
figure 1

a, Overview of the experimental design. b and c, Heatmap depict the metabolites or protein expression related to glycolysis (b) or TCA cycle (c) in naïve CD4+, Th0, Th1, Th2, Th17, and iTreg cells. d, The relative ratio of β-Alanine was shown here. e, Heatmap depict the protein expression related to FA biosynthesis in naïve CD4+, Th0, Th1, Th2, Th17, and iTregs cells. f, The ratio of fatty acid species detected by lipidomics analysis was shown. g, Heatmap depict the levels of fatty acid in naïve CD4+, Th0, Th1, Th2, Th17, and iTregs cells. Four biological replicate was prepared for metabolomics and proteomics analysis. The source data for the figures is provided in Supplementary Data 1 and Supplementary Data 2.

Effector Th cell subsets altered the composition of cellular lipids in comparison to naïve CD4+ T cells

Since activated Th cells dramatically altered fatty acid metabolism, we next analyzed lipidomic data to assess the changes in cellular lipid profiles after TCR stimulation. Our comprehensive cellular lipidomics analysis identified a total of 567 lipid species. These lipids were further classified based on their structure into cholesterol, free fatty acids, glycerolipids, lysophospholipids, phospholipids, and sphingolipids, with 27, 37, 132, 66, 196, and 109 species, respectively (Fig. 2a). After 48 h TCR stimulation, activated CD4+ T cells exhibited an almost 2-fold increase in cell size and 3-fold increase in the levels of cellular lipid droplets in comparison to the levels detected in naïve CD4+ T cells. (Supplementary Fig. 2a, b). Lipid droplets are composed of monoacylglycerol (MAG), diacylglycerol (DAG), triacylglycerol (TAG), and cholesteryl ester (ChE). When we normalized the lipidomics data based on cell size, the amounts of DAG and ChE were much lower in activated Th cell subsets than the levels in naïve CD4+ T cells. This is not consistent with our results of increased lipid droplets in activated Th cells and a previous study reporting that activated T cells increased intracellular cholesterol contents21 (Supplementary Fig. 2b). Furthermore, normalizing the data with cell size diminished the changes in the amount of free fatty acids between activated Th cell subsets and naïve T cells. This result is inconsistent with a previous reports suggesting that activated T cells exhibited augmented fatty acid uptake10. Considering with these issues, the lipidomics data were based on the cell number when comparing naïve CD4+ T cells and differentiated Th cell subsets. Accordingly, the metabolomics data were also normalized based on the cell number to compare naïve CD4+ T cells and differentiated Th cell subsets. We first observed that activated Th cell increased the overall quantity of these lipid species to over twice that of naïve CD4+ T cells (Fig. 2b). A deeper analysis revealed that the amounts of most lipid species was largely increased in Th cell subsets in comparison to naive CD4+ T cells, the numbers of which were 504, 504, 487, 522, and 511 in Th0, Th1, Th2, Th17, and iTreg cells, respectively (Fig. 2c, d). A principal-component analysis (PCA) demonstrated that TCR-mediated activation had a significant impact on cellular lipid profiles (Fig. 2e). Additionally, Th17 and iTreg cells formed a cluster that was distinct from other activated Th cell clusters including Th0, Th1, and Th2 cells. The stimulation of TCR not only led to the upregulation of lipid biosynthesis, but also resulted in a change in cellular lipid composition as well as free fatty acid levels as shown in Fig. 1g. The TCR stimulation increased the ratio of glycerolipids with values of 15.4% for Naïve CD4+ T cells, 28% for Th0 cells, 28.3% for Th1 cells, 27.1% for Th2 cells, 23.9% for Th17 cells, and 24.7% for iTreg cells (Fig. 2f). In a group of glycerolipid, effector Th cell subsets increased the proportion of TAG, which are incorporated into lipid droplet as energy storage with values of 26.2% for Naïve CD4+ T cells, 54.4% for Th0 cells, 52.3% for Th1 cells, 57.1% for Th2 cells, 44.9% for Th17 cells, and 51.0% for iTreg cell (Fig. 2g). The stimulation of TCR has only a minor impact on the composition of ChE (as depicted in Fig. 2h, with the percentages of 91.2% for Naïve CD4+ T cells, 83.1% for Th0 cells, 84.0% for Th1 cells, 88.3% for Th2 cells, 88.3% for Th17 cells, and 86.0% for iTreg cells). The percentage of phosphatidylcholine (PC) was nearly comparable across each T cell subset (Fig. 2i, with the percentages of 37.5% for Naïve CD4+ T cells, 46.0% for Th0 cells, 46.6% for Th1 cells, 45.8% for Th2 cells, 46.2% for Th17 cells, and 46.3% for iTreg cells). In comparison to naive CD4+ T cells, activated Th cells were found to alter the composition of phosphatidylethanolamine (PE) and phosphatidylserine (PS) from 27.0% to 14-16% or from 11.1% to 6–9%, respectively (Fig. 2i). We also observed substantial changes in the category of lysophospholipids and sphingolipids. Naïve CD4+ T cells exhibited the greatest proportion of LPC, yet TCR stimulation resulted in the most prominent lipid shifting from LPC to LPI. In naïve CD4+ T cells, LPC and LPI comprised 53.0% and 7.83%, respectively (Fig. 2j). In activated Th cell subsets, LPC and LPI occupied 23-34% or 27-39% of total lysophospholipids, respectively. TCR stimulation also largely changed the composition of cellular sphingolipids, with the most abundant lipid shifting from sphingomyelin (SM) to ceramide (Cer). In naïve CD4+ T cells, SM and Cer comprised 58.0% and 8.34%, respectively, while in activated Th cell subsets, SM and Cer occupied 14-20% or 34-43% of total sphingolipids, respectively (Fig. 2k). In addition, TCR-stimulation led to the production of sphingoglycolipids, including ganglioside GM1 (GM1) and ganglioside GD1 (GD1; Fig. 2k). These findings demonstrate that T-cell activation dynamically changes the quantity and composition of cellular lipids, which suggests the establishment of a metabolic environment favorable to the regulation of T cell homeostasis.

Fig. 2: The intracellular lipid metabolism was largely changed by TCR stimulation.
figure 2

a, The number of detected lipid species by lipidomics analysis was shown. b, Spider chart depicts the changes in total amounts of each lipid group compared to naïve CD4+ T cells. c, Heatmap depict the levels of each lipid species in Th0, Th1, Th2, Th17, and iTregs cells compared to naïve CD4+ T cells. d, The number of differentially expressed lipid species was shown. e, PCA depicts lipid profiles of naïve CD4+, Th0, Th1, Th2, Th17, and iTregs cells. f-k Bar plots showed that the ratio of total amounts of each lipid group (f), cholesterol (g), glycerol lipid (h), phospholipid (i), lysophospholipid (j), or sphingolipid (k). Four biological replicate was prepared for metabolomics analysis. The source data for the figures is provided in Supplementary Data 1 and Supplementary Data 2.

The activation of a specific Th cell subset resulted in a significant change of metabolites and proteins involved in the biosynthesis pathway of sphingolipids and glycosphingolipids

Subsequently, we seek to determine the species of lipids that influence the generation of effector Th cell subsets. The activated Th cell subsets differ in cell size; however, the differences in cell size were comparable under our experimental conditions (Supplementary Fig. 2a, b). Thus, to accurately compare lipid contents, the data were normalized by the signal intensity of total lipid species when comparing the lipidomics data of each Th cell subset to Th0 cells (Formula: the signal value of each lipid metabolite multiplied by 2000000/total signal value of lipid metabolites). To this end, we conducted an initial evaluation of the expression of 269 proteins involved in lipid metabolism in naïve CD4+ T cells, Th0, Th1, Th2, Th17, and iTreg cells. Our proteomic analysis revealed that protein expression levels related to lipid metabolism underwent dynamic changes following TCR stimulation (Fig. 3a, b). As indicated by the PCA plots of lipid metabolites (Fig. 2e), Th0, Th1, and Th2 cells displayed similar protein profiles, whereas Th17 and iTreg cells exhibited greater similarity to each other than to the other cell states within the PCA space. We then sought to identify proteins that were commonly changed among the various T cell subsets. Our observations indicated that the expression of diacylglycerol kinase (DGKA, DGKB, DGKZ), phosphatidylinositol kinase (PIK3CB, PIK3CD, PIP4K2A, PIP4K2B), and phospholipase (PLBD1, PLCB2, PLCB3, PLCL1) was decreased in a common manner among effector Th cell subsets (Supplementary Fig. 2c). Each Th cell subset commonly upregulated proteins that are essential for the biosynthesis of fatty acids (ACC1, FASN), elongation of fatty acids (ELOVL1, ELOVL6), desaturation of fatty acids (FADS1, FADS2, SCD2), fatty acid binding protein (FABP5), and cholesterol synthesis (HMGCS1 and HMGCR) (Fig. 3c). Especially, the proteins predominantly associated with the biosynthesis of lipids, primarily sphingolipids, that were commonly upregulated include CERS5, DEGS1, and B4GALT5 (Fig. 3c). In accordance with upregulation of these proteins, the quantity of ceramides (Cer) was considerably increased in effector T cell subsets (Fig. 3d). Lipidomics analysis further revealed substantial elevations in the amounts of cardiolipin (CL) and phosphatidylglycerol (PG) in glycerophospholipid, bis monoacylglycero phosphate (BMP), LPI, and LPG in lysophospholipid, TAG in glycerolipid (Supplementary Fig. 2df). Nevertheless, the synthases of these lipids were not among the commonly altered proteins in effector Th cell subsets except for LPGAT1. Thus, we next focused on the sphingolipid synthesis pathway for the following reasons: Proteomic analysis indicated that proteins related to the sphingolipid synthesis pathway were upregulated in a general manner (Supplementary Fig. 2g), and lipidomic analysis showed substantial increase in the quantity of Cer and significant changes in the composition of sphingolipids (Fig. 2k). Subsequently, we conducted a detailed evaluation of the changes in sphingolipids during T-cell activation. We first observed that the amounts of ceramide lipids and their precursor, sphinganine, was greatly increased (Fig. 3d). Cer serves as the hub of sphingolipid metabolism and is responsible for the generation of sphingomyelin, sphingosine, and glycosphingolipids. Although the amounts of SM remained largely unchanged, there were increases in the amounts of hexosyl Cer (HexCer) and di-hexosyl Cer (DiHexCer), which are types of glycosphingolipids (Fig. 3d). In addition, although many gangliosides, which are metabolites of these glycosphingolipids, were barely detectable in naive CD4+ T cells, effector Th cell subsets increased the levels of ganglioside, such as GM2, GM3, and GT1a (Fig. 3e). Taken together, these data suggest that TCR activation enhances the utilization of the sphingolipid synthesis pathway and confers the regulation of glycosphingolipid metabolism.

Fig. 3: Activated Th cells enhanced sphingolipid biosynthesis pathway among lipid biosynthesis.
figure 3

a, Heatmap depict the expression levels of protein related to lipid metabolism. b, PCA depicts profiles of protein related to lipid metabolism in naïve CD4+, Th0, Th1, Th2, Th17, and iTregs cells. c, Venn diagram showed overlaps and differences between 2.0-fold increased proteins related to lipid metabolism in Th0, Th1, Th2, Th17, or iTregs cells relative to naive CD4+T cells. d and e, The lipidomics analysis shows the relative contents of lipid species related to ceramide lipids (d) or glycosphingolipid (e) in Th0, Th1, Th2, Th17, and iTreg cells compared to naïve CD4+ T cells. Four biological replicate was prepared for metabolomics and proteomics analysis. The source data for the figures is provided in Supplementary Data 1 and Supplementary Data 2.

Activated sphingolipid metabolism is required for the proper generation of Th17 and iTreg cells

Next, to explore the influence of sphingolipid biosynthesis on the differentiation of effector Th cells, we inhibited sphingolipid production through the utilization of myriocin, a molecule known to inhibit the initial steps of sphingolipid biosynthesis. Inhibition of sphingolipid biosynthesis by myriocin resulted in a moderate decrease in the number of Th17 and iTreg cells (Fig. 4a). FACS analysis also showed that myriocin-treated Th17 and iTreg cells showed impaired TCR-induced cell division with slight changes in the cell death rate (Supplementary Fig. 3ad). We also observed that the myriocin-treated Th17 and iTreg cells substantially reduced the amounts of Cer and its downstream glycosphingolipids, including HexCer, DiHexCer, GD1, and AcGD1 (Fig. 4b, c and Supplementary Fig. 3e, f). Subsequently, the effect of myriocin on cell cycle progression was analyzed via the evaluation of BrdU incorporation in Th17 and iTreg cells. As a consequence, myriocin treatment prevented Th17 and iTreg cells from entering the S-phase following TCR stimulation (Fig. 4d, e). No significant changes were observed in the number and cytokine production of Th0, Th1, and Th2 cells (Supplementary Fig. 3gi). Since TGFβ is required to induce Th17 and iTreg differentiation, we next tested whether TGFβ changes the sensitivity to myriocin in Th1 and Th2 cells. We first found that in the presence of TGFβ, myriocin treatment caused a decrease in the number of Th1 and Th2 cells (Supplementary Fig. 3j). However, there were no significant changes in the production of IFNγ and IL-9, which were produced by TGFβ-treated Th2 cells defined as Th9 cells (Supplementary Fig. 3k, l). In addition to impaired cell division, treatment of myriocin resulted in the inhibition of the cytokine production of Th17 cell (Fig. 4f, g and Supplementary Fig. 4a, b). We also addressed whether myriocin treatment suppresses the expression levels of FOXP3, IL-2, and IL-10. Our findings also indicated that the inhibition of sphingolipid biosynthesis led to a decrease in PD-1 expression and IL-2 production, but there were no significant changes in the expression of FOXP3 and IL-10 at either the mRNA or protein level (Fig. 4h, i and Supplementary Fig. 4bd). Although treatment with myriocin from 48 h after T cell activation failed to suppress differentiation of Th17 or iTreg cells, treatment with myriocin 24 h after TCR stimulation suppressed IL-17A production or the PD-1 expression (Supplementary Fig. 4eh). Consistent with decreased PD-1 expression and IL-2 production, myriocin-treated iTreg cells showed impaired suppressive capacity (Supplementary Fig. 4i, j). Next, we investigated whether supplementation with sphingolipid restored the effect of myriocin on Th17 and iTreg cell differentiation. Although the number of myriocin-treated iTreg cells was almost unchanged by SM, Sph, Cer (d18:1/16:0), and Cer (d18:1/18:0) treatment (Supplementary Fig. 4k, l), Sph and Cer (d18:1/16:0) moderately recovered the PD1 expression (Fig. 4j, k). We also found that the supplementation with SM, Sph, Cer (d18:1/16:0), and Cer (d18:1/18:0) moderately restored cell number of myriocin-treated Th17 cells. Supplementation with these lipid species did not affect IL-17A and IL-17F production (Fig. 4l, m and Supplementary Fig. 4m, p).

Fig. 4: Inhibition of ceramide biosynthesis resulted in the suppression of Th17 and iTreg differentiation.
figure 4

a, Cell number of Th17 or iTreg cells treated with DMSO or 6.25 μM myriocin was shown. b,c Heatmap depict the levels of ceramide in myriocin-treated Th17 (b) and iTreg cells (c). d, BrdU incorporation by Th17 or iTreg cells treated with myriocin was examined by flow cytometry. e, Summary data of cell cycle related to (d) was shown. f, Intracellular staining of IL-17A and IL-17F in Th17 cells treated with myriocin was shown. g, Summary data of IL-17A and IL-17F expression related to (f) was shown. h, Surface staining of PD-1 in iTreg cells treated with myriocin was shown. i, Summary data of PD-1 expression related to iTreg cells was shown. j, k, FACS analysis shows PD-1 expression of iTreg cells treated with 2 μM SM or 2 μM Sph (j), 0.2 μM Cer(d18:1/16:0) or 0.2 μM Cer(d18:1/18:0) (k) in the presence of 6.25 μM myriocin. l, m, FACS analysis shows the cell number of Th17 cells treated with 2 μM SM or 2 μM Sph (l), 0.2 μM Cer(d18:1/16:0) or 0.2 μM Cer(d18:1/18:0) (m) in the presence of 6.25 μM myriocin. n, Intracellular staining of IL-17A and IL-17F in sgSptlc1/sgSptlc2 Th17 cells was shown. o, Summary data of IL-17A and IL-17F expression related to (n) was shown. p, Surface staining of PD-1 in sgSptlc1/sgSptlc2 iTreg cells treated was shown. q, Summary data of PD-1 expression related to (p) was shown. Three independent experiments were performed and showed similar results. Error bar indicates SD. The source data for the figures is provided in Supplementary Data 1 and Supplementary Data 2.

We next performed gene edition of Sptlc1 and Sptlc2, which are key targets of myriocin, to elucidate the importance of sphingolipid metabolism for Th17 and iTreg differentiation (Supplementary Fig. 5a). The genetic deletion of Sptlc1 and Sptlc2 moderately suppressed the differentiation of Th17 cells and the expression of PD-1 and IL-2 in iTreg cells (Fig. 4n–q and Supplementary Fig. 5be). Genetic deletion of Sptlc1 and Sptlc2 caused impaired production of sphingolipid and glycosphingolipid in Th17 and iTreg cells (Supplementary Fig. 5fi). Collectively, our combined analysis demonstrates that effector Th cell subsets exhibit enhanced sphingolipid and glycosphingolipid metabolism. Additionally, the generation of both Th17 and iTreg cells was inhibited by the ceramide biosynthesis inhibitor.

Glycosphingolipid metabolism was facilitated in Th17 and iTreg cells in comparison to Th0, Th1, and Th2 cells

The inhibition of sphingolipid biosynthesis selectively suppressed the generation of Th17 and iTreg cells, suggesting characteristic differences in cellular lipid metabolism between these effector Th cell subsets and Th0, Th1, and Th2 cells. To further investigate this cellar metabolic profile, we examined global lipidomic profiling and analyzed protein expression related to lipid metabolism among the various effector Th cell subsets. Results from PCA revealed that Th0, Th1, and Th2 cells showed similar lipid and protein profiles. While Th17 and iTreg cells were more alike to each other than to the other cell states in PCA space (Fig. 5a, b). The analysis also revealed elevated amounts of sphingolipids in Th17 and iTreg cells in comparison to non-polarizing Th0 cells, as well as decreased levels of 3-ketosphinganine, an intermediate in sphingolipid synthesis, in Th17 and iTreg cells (Fig. 5c, d). These results led us to focus on sphingolipids and their downstream metabolic pathways, where we observed increased levels of HexCer, DiHexCer, GD1, and GD1[O-acetyl NeuAc] (AcGD1) (Fig. 5e). Although total amounts of ceramide lipid levels did not significantly differ in Th17 and iTreg cells in comparison to Th0 cells, a closer examination revealed that the proportion of individual ceramide species in the most of HexCer, DiHexCer, GD1, and AcGD1 was altered in Th17 and iTreg cells (Fig. 5f–h). Furthermore, inhibition of glycosphingolipid through pharmacological inhibitor, GENZ-123346, suppressed the number of Th17 and iTreg cells as well as myriocin treatment (Fig. 5i–k and Supplementary Fig. 6a, b). In accordance with these results, GENZ-123346 inhibited the cytokine production of Th17 cells and PD-1 expression on Treg cells, further demonstrating the importance of the glycospingolipid biosynthesis pathway in the differentiation of Th17 and iTreg cells (Fig. 5l, m and Supplementary Fig. 6c, d). GENZ-123346 prevent the conversion of Cer to HexCer. Consequently, treatment of GENZ-123346 with Th17 and iTreg cells caused accumulation of Cer and depletion of glycosphingolipid, including HexCer, DiHexCer, GD1, and AcGD1 (Supplementary Fig. 6eh). These data indicated that glycosphingolipid metabolism is required to regulate Th17 and iTreg cells differentiation.

Fig. 5: Glycosphingolipid biosynthesis pathway is selectively upregulated in Th17 and iTreg cells compared to Th0, Th1 and Th2 cells.
figure 5

a, b PCA depicts profiles of lipid (a) or protein related to lipid metabolism (b) in Th0, Th1, Th2, Th17, and iTregs cells. c, Heatmap depicts the expression levels of each lipid species in Th1, Th2, Th17, and iTreg cells compared to Th0 cells. d, Relative levels of 3-ketosphinanine was shown in Th1, Th2, Th17, and iTreg cells compared to Th0 cells. e, Lipidomics analysis shows the relative contents of lipid species related to ceramide glycosphingolipid in Th1, Th2, Th17, and iTreg cells compared to Th0 cells. f-h, Heatmap depicts the expression levels of each ceramide species (f), each glycol-ceramide species (g), or each ganglioside species (h) was shown in Th1, Th2, Th17, and iTreg cells compared to Th0 cells. i, Cell number of Th17 or iTreg cells treated with DMSO or 2.5 μM GENZ-123346 was shown. j, Naive CD4+ T cells were labelled with e670 proliferation dye and stimulated with immobilized anti-TCRβ mAb and anti-CD28 mAb in the presence of GENZ-123346 under Th17 or iTreg polarization condition. k, BrdU incorporation by Th17 or iTreg cells treated with GENZ-123346 was examined by flow cytometry. l, Intracellular staining of IL-17A and IL-17F in Th17 cells treated with myriocin was shown. m, Surface staining of PD-1 in iTreg cells treated with GENZ-123346 was shown. Four biological replicate was prepared for metabolomics and proteomics analysis. Three independent experiments were performed and showed similar results (i-m). Error bar indicates SD. The source data for the figures is provided in Supplementary Data 1 and Supplementary Data 2.

It has also been reported there was differences in the metabolic profiles and requirement of pathogenic Th17 cells and non-pathogenic Th17 cells22. Therefore, we differentiated naïve CD4+ T cells into pathogenic (p) Th17 cells in the presence of IL-1β, IL-6, and IL-23, and evaluated whether the inhibition of sphingolipid or glycosphingolipid metabolism suppresses the generation of pTh17 cells. Although we found that the number of pathogenic Th17 cells decreased with the addition of myriocin or GENZ-123346, the production of cytokines was not substantially affected under these conditions (Supplementary Fig. 6i–k). Previous studies have indicated that glycosphingolipids and cholesterol may affect early TCR signaling, we evaluated the effects of myriocin and GENZ-123346 on phosphorylation of proteins downstream TCR signaling23. After 30 min TCR stimulation, inhibition of sphingolipid or glycosphingolipid metabolism by myriocin or GENZ-123346 in Th17 or iTreg cells did not substantially affect the phosphorylation of Src, Lck, and Lat (Supplementary Fig. 6I, m). We also investigated whether myriocin inhibited Th17 and iTreg cell differentiation after early TCR stimulation. Although treatment with myriocin from 48 h after T cell activation failed to suppress differentiation of Th17 or iTreg cells, myriocin treatment with 24 h after TCR stimulation suppressed IL-17A production or PD-1 expression (Supplementary Fig. 4e–h). Taken together, impaired Th17 and iTreg cell differentiation did not appear to be highly dependent on TCR stimulation.