RF catabolites modulate MR1 expression on the surface of APCs. (A–C) Bar graphs depict the expression of surface MR1*01 on C1R.MR1 cells incubated for 3 h (A) or 16 h (B and C) with titrated quantities of ligands followed by staining with 8F2F9 anti-MR1 antibody. Shown in B are overlay histograms for the expression of surface MR1*01 on C1R.MR1 cells after 16-h treatment with 200 µM of RF catabolites. (D–F) MR1 was then quantified after adding the indicated concentrations of the RF catabolites on THP-1.MR1 cells incubated for 3 h (D) or 16 h (E and F). Shown in E are overlay histograms for the expression of surface MR1*01 on THP-1.MR1 cells after 16-h treatment with 200 µM of RF catabolites. (G) Bars indicate the expression of surface MR1 on C1R.MR1 cells after treatment with lumichrome overnight and then staining with 26.5 vs. 8F2F9 MAb for comparison. (H) MR1 expression on the surface of PBMC-derived B lymphocytes was measured after treatment with RF catabolites for 16 h in the presence or absence of 5-OP-RU. Data in A–G represent the gMFI fold change from three independent experiments performed in duplicates, with standard error (SEM) represented by the error bars. Data in H are the average of three independent experiments done on PBMCs from three different donors performed in duplicate, with standard error (SEM) represented by the error bars. One-way ANOVA statistical analysis was performed for all samples with Dunnett’s multiple comparisons performed using NaOH as a control (ns: not significant, *P < 0.05, **P < 0.01, ****P < 0.0001). Statistical analysis for the 5-OP-RU competition experiment (H) used 5-OP-RU as a control. MR1, MHC-I–related protein. gMFI, geometric mean fluorescence intensity.