Figure 7: Analogue 11 is functionally similar to 5-OP-RU (3c).

(a) Upregulation of surface expression of MR1 on C1R.MR1 cells at indicated time points with 10 μM 5-OP-RU 3c, compound 11 or Ac-6-FP (30, structure shown at right). Mean±s.e.m. from three independent experiments (for analogues 9 and 10, and concentration-response curves of 5-OP-RU 3c and 11 at defined time points see Supplementary Fig. 9). (b) Co-staining of human PBMCs with antibodies to CD3, CD161 and TCR TRAV1–2. Gated CD3⁺ lymphocytes are shown from one representative donor from four. Subsequently gated MAIT (TRAV1-2⁺CD161^hiCD3⁺) cells and conventional T cells expressing TRAV1–2 (TRAV1-2⁺CD161^hiCD3⁺) were co-stained with MR1 tetramers from 11 and 5-OP-RU 3c. See Supplementary Fig. 10 for gating strategy. (c) Cytokine profiles after activation of human PBMCs with 5-OP-RU (3c, 1.28 nM) and 11 (100 μM). See Supplementary Fig. 11 for gating strategy. (d) MAIT cells (TCRβ⁺, MR1-3c Tet⁺) as a percentage of αβ-T cells (TCRβ⁺) isolated from the lungs of mice intranasally inoculated with CpG plus 5-OP-RU 3c or 11 at indicated doses. Day 7 data are shown (Mean±s.e.m.). 4 mice per group. See Supplementary Fig. 12 for gating strategy. (e) Cytokine production by MAIT cells harvested from lungs (day 7) of mice inoculated with CpG (day 0) plus 5-OP-RU 3c (1 μM) or 11 (100 μM) four times on day 0, 1, 2 and 4, detected by intracellular cytokine staining either with or without further stimulation with PMA+ionomycin. One representative mouse (from 4) per group is shown. Experiments were performed twice. See Supplementary Fig. 12 for gating strategy.