Fig. 4. Binding mode of CA7S to MR1 and MAIT TCR.

(A and B) Inhibition assay of 5-OP-RU (0.5 μM) and CA7S (500 μM) by anti-MR1 Ab (26.5) (A) or Ac-6-FP (B). Percentages of GFP⁺ cells were shown. (C) Effect of hMR1 mutations (Y7A, R9A, K43A, Y62A, L66A, W69A, M72A, R79A, R94A, W156A, and W164A) on the recognition of ligands. Cells expressing hMR1 mutants and a MAIT TCR were stimulated with vehicle control, 5-OP-RU, or CA7S and analyzed by flow cytometry after 6 hours. MR1 expression was shown as MFI of anti-MR1 staining subtracted by isotype control. (D) MR1-restricted ligand affinities (IC₅₀) determined by FP assay (31). Left: Titration curves of strong MR1 binders (5-OP-RU and Ac-6-FP), moderate MR1 binder (RL-6-Me-7-OH), weak MR1 binders (CA7S, CA3S, and DCF), and MR1 nonbinding substances [epigallocatechin gallate (EGCG) and NLV peptide] are displayed. Tetrahydroxy bile acid (THBA) was used as a bile acid control with similar hydrophilicity to CA7S and CA3S. The table represents a summary of IC₅₀ for all investigated compounds. (E) Effect of MAIT TCRα mutation on ligand recognition. Reporter cells transfected with vector alone (Mock), MAIT TCRβ together with WT MAIT TCRα (WT), or mutant TCRα (Y95F) were stimulated with vehicle control, 5-OP-RU, or CA7S and analyzed after 20 hours. (C and D) Data are presented as the means ± SD of triplicate assays. (A, B, and E) Data are presented as individual values of duplicate assays. All data are representative of at least two independent experiments. NB, no binding.