Stabilizing short-lived Schiff base derivatives of 5-aminouracils that activate mucosal-associated invariant T cells

Abstract

Mucosal-associated invariant T (MAIT) cells are activated by unstable antigens formed by reactions of 5-amino-6-D-ribitylaminouracil (a vitamin B2 biosynthetic intermediate) with glycolysis metabolites such as methylglyoxal. Here we show superior preparations of antigens in dimethylsulfoxide, avoiding their rapid decomposition in water (t_1/2 1.5 h, 37 °C). Antigen solution structures, MAIT cell activation potencies (EC₅₀ 3-500 pM), and chemical stabilities are described. Computer analyses of antigen structures reveal stereochemical and energetic influences on MAIT cell activation, enabling design of a water stable synthetic antigen (EC₅₀ 2 nM). Like native antigens, this antigen preparation induces MR1 refolding and upregulates surface expression of human MR1, forms MR1 tetramers that detect MAIT cells in human PBMCs, and stimulates cytokine expression (IFNγ, TNF) by human MAIT cells. These antigens also induce MAIT cell accumulation in mouse lungs after administration with a co-stimulant. These chemical and immunological findings provide new insights into antigen properties and MAIT cell activation.

Figure 1. Glycolysis metabolites react with a vitamin B2 biosynthetic intermediate to create MAIT cell antigens.

(a) Enzymatic biosynthesis of vitamin B2 (top), and formation of antigens from biosynthetic intermediate 1 by condensation with glycolysis metabolites 5a-c (bottom). (b) Complex of antigen (blue) sandwiched between MR1 (purple) on the surface of an antigen-presenting cell APC and T-cell receptor protein TCR (orange) on the surface of a MAIT cell. (c) Interactions observed in a ternary crystal structure of a human MAIT TCR-3c-MR1 complex, showing antigen (blue)···MR1 (purple) and antigen (green)···TCR (orange) contacts, with an extended conjugated system involved in π-interactions (dashed circle).

Figure 2. Proposed mechanism for reaction between 1 and 5 to produce uracil and lumazine derivatives.

These reactions are reversible in water, but dehydrative cyclization drives formation of thermodynamically more stable aromatic lumazine derivatives 4. Polar aprotic solvents may facilitate kinetic control of these equilibria and enable trans-3 to persist in solution.

Figure 3. Formation and solution structures of uracil-based antigens that activate MAIT cells.

(a) Exclusive formation of 5-OP-RU 3c from 1 and 5c in DMSO-d₆, as monitored by ¹H NMR spectroscopy, with the characteristic signals of 1 and 5-OP-RU 3c as indicated. (b) Formation of 5-OP-RU 3c in DMSO (black) versus PBS buffer (red). In PBS buffer (red, 2.62 mM of 1, 7.86 mM of 5c, pH 7.4, 37 °C), 3c reached a maximum concentration corresponding to only 1.1% conversion of 1 at 5 min. In DMSO (black, 31.8 mM 1 and 34.5 mM 5c, room temperature), 5-OP-RU 3c reached 100% conversion after 2 days and then plateaued. (c) Stability of 5-OP-RU 3c in DMSO (black), PBS buffer (red) and after the DMSO solution was diluted into PBS buffer (blue). Data for PBS buffer were extracted from b (red) at the time the maximum 5-OP-RU 3c concentration was reached (that is, 5 min after mixing). (d–f) HMBC NMR spectra of 3a-c (DMSO-d₆), respectively. Square boxes indicate correlations for the aldehyde form of 3b, and circles for its hydrate form 3b′. Arrows and boxes indicate key ¹H–¹³C long-range correlations that unambiguously characterize the compounds. J refers to heteronuclear coupling through 1, 2, 3 or 4 bonds. (g) MAIT cell activation. Data represent mean±s.e.m. (n=3). The α-dicarbonyls 5a-c, lumazines 4a-b and the solvent DMSO-d₆ were inactive (up to 15 μM), while 4c and 4d had only very weak activity at the highest concentrations tested (EC₅₀>100 nM).

Figure 4. DFT optimized conformations of 3a-c and analogues 9–11.

(a) Optimized conformers of 3a–c and (b) optimized conformers of 9–11, which are analogues of 3c. Dihedral angles φ (red) and ψ (blue) define the angles formed by 5,6-substituents centred at uracil C5-C6 and 5-substituent to uracil C5-C4, respectively. In order to visualize the twisting of these substituents, the LUMOs (with orbital phases in cyan and blue) are shown for clarity. The incorporation of a methyl group to 3c (that is, 3a, 9 or 10) resulted in a twisting of the C-5 substituent relative to the ring and an inversion of the LUMO phase at the carbonyl carbon (3a and 10). These changes likely impact the ability of the ligands to form non-covalent interactions with MR1 and MAIT TCR (Fig. 1), as well as their reactivity towards the formation of Schiff base with K43 of MR1. (c,d) Plots illustrating the structural similarity of the compounds based on the dihedral angles φ (red) and ψ (blue), and the distances d₁ (purple) and d₂ (green).

Figure 5. Chemical syntheses of uracil-based analogues of 3c as possible antigens.

(a) Three step synthesis of 9 from 5-chloro-6-nitrouracil (13) and N-methylribitylamine (12). (b) Three step synthesis of analogue 10 from aldehyde 16 via successive Wittig, nucleophilic demethylation and Michael-type substitution reactions. (c) Seven step synthesis of 11 from the protected ribose-derived aldehyde 20. Successive Wittig and hydrogenation reactions on 20, followed by elaboration with Mander's reagent gave β-ketoester 25. Subsequent dehydrative cyclization with thiourea, base mediated desulfurization, ring alkylation and acid mediated global deprotection gave analogue 11.

Figure 6. Stability and MAIT cell activation of 9, 10 and 11.

(a) The aqueous stability of 3c and 9–11 measured by LCMS as a percentage of initial concentration (0.065 mM) over time (37 °C, PBS buffer pH 7.4). (b) MAIT cell activation. Data represent mean±s.e.m. (n=3). (c) Plot of antigen potency (pEC₅₀) versus calculated energy difference between the energy minimized conformers and postulated bioactive conformers of 3c and 9–11. The biologically active conformers of 9–11 were created by making the necessary atom connectivity changes to the antigen conformer present in the MR1-3c-TCR crystal structure.

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.