Fig. 5

The lipidome of CLL T cells is characterized by low cholesterol and phospholipids, and accumulation of triglycerides, compared to healthy T cells. CD4+ and CD8+ T cells of HD and CLL patients were FACS-sorted and subjected to liquid chromatography-mass spectrometry (LC-MS)-based lipidomics (A) at baseline or (B) after 2-day stimulation with αCD3/αCD28 antibodies. Abundance of each lipid molecule was normalized by internal standards and protein amount (A) or total lipidome pool (B). Results are visualized as principal component analysis plots, volcano plots, and heatmaps of differentially abundant lipid species. In the volcano plots, the three horizontal dotted lines indicate p values of 0.05, 0.01, and 0.001, respectively. The two vertical dotted lines indicate log2 (fold change) of −2 and 2, respectively. Lipid molecules with significantly different abundance in CLL T cells compared to HD T cells (p ≤ 0.01) are colored according to the lipid class they belong to. For heatmap representation of lipidomics data, a cutoff for variable importance in projection (VIP) score >1 from all lipid molecules analyzed was defined. Abundance of the different lipid classes relative to the total lipids sum is shown in Supplementary Fig. 5A, B. C Relative abundance of triacylglycerols and cholesterol esters after a 2-day stimulation with αCD3/αCD28 antibodies is plotted separately. D PBMC from HD and CLL patients were stimulated for 2 days with αCD3/αCD28 antibodies and protein levels of ATGL were measured. E FAO was assessed by flow cytometry on CD4+ T cells by FAOBlue in the same experimental conditions as in (D). F Abundance of acylcarnitines after a 2-day stimulation with αCD3/αCD28 antibodies is plotted separately. Data are presented as mean ± SEM (C–F) and differences were analyzed with t-test (C) or two-way repeated measures ANOVA with Tukey’s/Šidák’s multiple comparison test (D–F). **** = p < 0.0001; *** = p < 0.001; ** = p < 0.01; * = p < 0.05. PC phosphatidylcholine, PC[O] alkylphosphatidylcholine, PC[P] alkenylphosphatidylcholine, PC[O + P] alkyl/alkenylphosphatidylcholine, LPC lysophosphatidylcholine, LPC[O] alkyllysophosphatidylcholine, LPC[P] alkenyllysophosphatidylcholine, LPC[O + P] alkyl/alkenyllysophosphatidylcholine, PE phosphatidylethanolamine, PE[P] alkenylphosphatidylethanolamine, PE[O] alkylphosphatidylethanolamine, PE[O + P] alkyl/alkenylphosphatidylethanolamine, LPE lysophosphatidylethanolamine, LPE[P] alkyllysophosphatidylethanolamine, LPE[O + P] alkyl/alkenyllysophosphatidylethanolamine, PI phosphatidylinostiol, BMP bis(monoacylglycero)phosphate, PG phosphatidyglycerol, PS phosphatidylserine, CL cardiolipin, SM[d] sphingomyeline, SM[t] hydroxysphingomyeline, SPH[d] sphingosine, GM3 monosialdihexosylganglioside, Cer[d] ceramide, HexCer[d] hexosylceramide, Hex2Cer[d] dihexosylceramide AC acylcarnitine, TAG triacylglycerol, TAG[O] alkyltriacylglycerol, DAG diacylglycerol, CE cholesterol ester, FA fatty acid