C24 1

All solvents and additives were LCMS grade and purchased from Sigma-Aldrich. Internal standard of sphingosine-1-phosphate, ceramides C16, C18, and C24:1, and deuterated ceramide mix were purchased from Avanti Polar Lipids (Alabaster). For the extraction of sphingolipids, human plasma (20 μl) was diluted to 400 μL with Milli-Q water (Sigma-Aldrich), and 3 μl of the internal standard was added, after which samples were extracted by the addition of 400 μL of isopropanol/ethyl acetate 18/85 (v/v); the solution was vortexed for 20 seconds and centrifuged at 16,110 x g for 5 minutes. The upper organic phase was collected, while the aqueous phase was acidified with 20 μL of formic acid and reextracted with 400 μL of fresh isopropanol/ethyl acetate 18/85 (v/v%). The obtained solution was vortexed and centrifuged. The supernatants were pooled and dried under a gentle stream of nitrogen. For sphingolipid extraction, cell media (500 μL) was diluted with 1 mL isopropanol/ethyl acetate 18/85 (v/v%) and processed as described above. Samples were resolubilized with 150 μL of methanol and 1 mM HCOONH₄ plus 0.2% HCOOH (v/v). UHPLC-MS/MS analysis was carried out with a Shimadzu Nexera coupled online to a triple-quadrupole LCMS 8050 (Shimadzu) by an ESI source.

Synthetic lipids (KRN7000, C24:0, C24:1, and βGalCer C24:1) were purchased from Avanti Polar Lipids (Alabaster). C24:2, pC24:0, pC24:1, pC24:2, and βGalCer C24:2 were synthesized in our laboratory at the University of Connecticut, as detailed in Supplemental Methods. All lipids were dissolved in the vehicle (0.5% polysorbate-20) and diluted in PBS or complete RPMI 1640 medium with 10% FBS. For the hybridoma assay, sulfatide analogs and βGalCer analogs were dissolved in DMSO (Life Technologies, Thermo Fisher Scientific). The purified anti-CD1D1 antibody (clone 20H2) was purchased from Harlan. Rat IgG was purchased from MilliporeSigma. The anti–mouse CD3 antibody was purchased from BioLegend (clone 145-2C11). Mouse CD1D1 monomers were provided by the NIH Tetramer Core Facility (Emory University, Atlanta, Georgia, USA). The fluorescent protein–labeled mAbs used for flow cytometry are detailed in the Supplemental Methods. Bafilomycin A1 (MilliporeSigma) was dissolved in DMSO and added to cell cultures at a final concentration of 50 nM, 5 minutes before adding lipid antigens. Sodium sulfite (MilliporeSigma) was dissolved in PBS and used at the concentrations shown.