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. Author manuscript; available in PMC: 2024 Jun 3.
Published in final edited form as: Sci Immunol. 2024 Jan 26;9(91):eade6924. doi: 10.1126/sciimmunol.ade6924

Fig. 1. Purification and structural determination of a ligand for MAIT cells.

Fig. 1.

(A and B) Screening of MAIT cell agonists from mouse intestine. NFAT-GFP reporter cells expressing MAIT TCR and MR1 were stimulated with HPLC-separated fractions from SPF mouse intestine (A) and freshly prepared 5-OP-RU as a control (B) for 16 to 20 hours and analyzed by flow cytometry. (C and D) HRMS (C) and 1H NMR spectra (600 MHz, D2O) (D) of fraction #84–45 from SPF mice intestine. In (D), impurities are denoted by asterisks. (E) Chemical structure of CA7S. (F and G) HRMS/MS spectra (F) and extracted ion chromatogram (G) of fraction #84–45. (H and I) NFAT-GFP reporter cells were stimulated with 5-OP-RU, RL-7-Me, and CA7S. Percentages of GFP+ cells (H) and MR1 expression (I) were analyzed at 20 and 6 hours after stimulation, respectively. MR1 surface expression is presented as mean fluorescence intensity (MFI) values of stimulated cells subtracted by those of vehicle-treated unstimulated cells (ΔMFI). (J) MR1 surface expression at 0, 2, 4, 8, and 16 hours after stimulation with vehicle control (None), 5-OP-RU, or CA7S. (H to J) Data are presented as the means ± SD of triplicate assays and are representative of more than two independent experiments.