Mouse mucosal-associated invariant T cell receptor recognition of MR1 presenting the vitamin B metabolite, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil

Mucosal-associated invariant T (MAIT) cells can elicit immune responses against riboflavin-based antigens presented by the evolutionary conserved MHC class I related protein, MR1. While we have an understanding of the structural basis of human MAIT cell receptor (TCR) recognition of human MR1 presenting a variety of ligands, how the semi-invariant mouse MAIT TCR binds mouse MR1-ligand remains unknown. Here, we determine the crystal structures of 2 mouse TRAV1-TRBV13-2+ MAIT TCR-MR1-5-OP-RU ternary complexes, whose TCRs differ only in the composition of their CDR3β loops. These mouse MAIT TCRs mediate high affinity interactions with mouse MR1-5-OP-RU and cross-recognize human MR1-5-OP-RU. Similarly, a human MAIT TCR could bind mouse MR1-5-OP-RU with high affinity. This cross-species recognition indicates the evolutionary conserved nature of this MAIT TCR–MR1 axis. Comparing crystal structures of the mouse versus human MAIT TCR-MR1-5-OP-RU complexes provides structural insight into the conserved nature of this MAIT TCR–MR1 interaction and conserved specificity for the microbial antigens, whereby key germline-encoded interactions required for MAIT activation are maintained. This is an important consideration for the development of MAIT cell-based therapeutics that will rely on preclinical mouse models of disease.

Mucosal-associated invariant T (MAIT) cells can elicit immune responses against riboflavin-based antigens presented by the evolutionary conserved MHC class I related protein, MR1.While we have an understanding of the structural basis of human MAIT cell receptor (TCR) recognition of human MR1 presenting a variety of ligands, how the semi-invariant mouse MAIT TCR binds mouse MR1-ligand remains unknown.Here, we determine the crystal structures of 2 mouse TRAV1-TRBV13-2 + MAIT TCR-MR1-5-OP-RU ternary complexes, whose TCRs differ only in the composition of their CDR3β loops.These mouse MAIT TCRs mediate high affinity interactions with mouse MR1-5-OP-RU and cross-recognize human MR1-5-OP-RU.Similarly, a human MAIT TCR could bind mouse MR1-5-OP-RU with high affinity.This crossspecies recognition indicates the evolutionary conserved nature of this MAIT TCR-MR1 axis.Comparing crystal structures of the mouse versus human MAIT TCR-MR1-5-OP-RU complexes provides structural insight into the conserved nature of this MAIT TCR-MR1 interaction and conserved specificity for the microbial antigens, whereby key germlineencoded interactions required for MAIT activation are maintained.This is an important consideration for the development of MAIT cell-based therapeutics that will rely on preclinical mouse models of disease.
The MR1-MAIT cell axis is highly conserved across 150 million years of mammalian evolution, with 90% sequence identity between the α1 and α2 domains in mouse and human MR1 (15).This implies that a strong selection pressure exists to maintain a conserved population of MR1-responsive MAIT cells across mammalian species (16,17).The MAIT cell population is characterized by the expression of a semiinvariant αβ T cell receptor (TCR) where the α-chain is composed of TRAV1-2 + TRAJ33/12/20 + in humans and of orthologous TRAV1 + TRAJ33 + in mice (18)(19)(20)(21).While the TCR β-chain can be diverse, there is a preference for TRBV6/ 20 in humans and TRBV13/19 in mice (18,(22)(23)(24).Structural studies reveal that the capacity of the 5-OP-RU agonist to activate human MAIT cells is due to the formation of a structural motif termed an 'interaction triad' between the hydroxyl group of the conserved MAIT TCR TRAV1-2 CDR3α tyrosine 95 (Y95) residue, the ribityl moiety of 5-OP-RU, and tyrosine 152 (Y152) of MR1 (1,6,14).
Studies have been performed on TRAV1 + (Vα19) transgenic mice in an effort to functionally characterize mouse MAIT cells in vivo (18,(25)(26)(27)(28). Previously, mouse MR1 tetramers were used to isolate MR1-5-OP-RU tetramer + TRBV13-2 + stomach MAIT cells from C57BL/6 mice primed with the liveattenuated vaccine strain, Salmonella Typhimurium BRD509 and chronically infected with Helicobacter pylori (18).However, the structural basis underpinning mouse MAIT TCR recognition of MR1-5-OP-RU is unknown, yet important to determine in the context of development of MAIT cell centric therapeutics that will likely depend on preclinical mouse models of disease.
To gain insight into the structural requirements for mouse MAIT TCR-MR1-Ag recognition, here we determine the crystal structures of two mouse MAIT TRBV13-2 + TCRs (M2A and M2B) (10,18) which was identified from Vα19 transgenic mice (Vα19, paired with endogenous TCRβ chain) (10,29) in complex with mouse MR1-5-OP-RU.These mouse MAIT TCRs adopt a similar molecular footprint atop mouse MR1-5-OP-RU, as in human MAIT TCR-MR1-5-OP-RU complexes (1,14) and exhibited species cross-reactivity towards human MR1-5-OP-RU, albeit binding with weaker affinity for human MR1 indicating a species-specific preference for mouse MR1.Overall, this study has advanced our understanding of MAIT-MR1 co-evolution and MAIT TCR crossreactivity between human and mouse MR1 in the context of recognition of antigens derived during microbial riboflavin biosynthesis.

Reporter cells expressing a mouse MAIT TCR respond to 5-OP-RU
We have previously used human Jurkat.MAIT reporter cell lines to test reactivity to 5-OP-RU and other MR1-ligands (1,3,14).Therefore, we employed a similar co-culture system here.BW58 reporter cells expressing mouse CD3 and the uncharacterized M2A TCR generated by retroviral transduction (previously named "BW58.CD3.MAIT Vβ8.2 cells") (10) were further modified to delete β2m (removing MR1dependent T-T presentation capacity).These cells were cocultured with the MR1-overexpressing M12.C3 cells and 5-OP-RU, and activation was measured by the upregulation of cell surface CD137 and downregulation of cell surface TCR expression.As expected, there was a dose-dependent activation in response to 5-OP-RU, but not Ac-6-FP, which could be blocked by anti-MR1 monoclonal 8F2.F9 (30), but not an isotype control mAb (Fig. 1).This assay further confirms the functionality of mouse MAIT TCRs and their reactivity towards 5-OP-RU presented by MR1.

Discussion
MAIT cells have been implicated in a range of immune settings, including bacterial and viral infections, autoimmune diseases, tissue repair, and cancer, which makes them attractive vaccine targets for modulating the immune responses (13,32,33).Central to MAIT-MR1-mediated immunity is the recognition of the riboflavin-biosynthesis metabolite, 5-OP-RU, displayed by MR1 to a TCR on MAIT cells.Activation of MAIT cells relies on engagement of the evolutionarily conserved MAIT CDR3α-Y95 residue with 5-OP-RU and the species-conserved Y152 residue of MR1 (1,14).Whereas human MAIT TCR-MR1-ligand interactions are now well defined, elucidating mouse MAIT TCR-MR1 binding has not advanced.Several studies have used transgenic mice expressing only the invariant orthologous TRAV1 + (Vα19) chain that defines mouse MAIT cells, to allow the functional characterization of MR1-restricted TRAV1 + T cells in the contexts of T cell development (34,35), microbial infection (24), and autoimmune diseases (36,37).
To examine how mouse TCRs can bind to MR1, we have analyzed two mouse MR1-5-OP-RU reactive MAIT TCRs; one from TRAV1 transgenic mice (paired with endogenous Vβ chain) and the other from WT mice, identified in the context of chronic H. pylori infection (18), named M2A and M2B, respectively.BW58 reporter cells expressing the M2A TCR were stimulated by 5-OP-RU presented by mouse MR1 expressing antigen presenting cells in an MR1-dependent manner and did not recognize MR1 Ac-6-FP.The affinity data revealed that these mouse MAIT TCRs exhibit high affinity for the cognate mouse MR1-5-OP-RU (M2A: K D 1.79 μM; M2B: K D 0.45 μM) yet have a markedly lower affinity for human MR1-5-OP-RU (by 13-fold for M2A: 24.2 μM; by 12-fold for M2B: 5.59 μM), and the dissociation of the complex is slower than for mouse MR1-5-OP-RU.Intriguingly, the human A-F7 MAIT TCR also exhibited a higher affinity for mouse MR1-5-OP-RU than its cognate human MR1-5-OP-RU.Moreover, this disparity in affinity values of the M2A, M2B, and A-F7 TCRs for mouse versus human MR1-5-OP-RU is likely due to the stronger engagement of TCRs with mouse-specific residues of MR1, such as the polar Q151 and T72 residues, in the antigen-binding cleft.
Previous functional studies have shown that the inability of human MR1 to activate a mouse MAIT hybridoma was due to   (17).Based on both the structural and affinity data obtained in this study, it can be speculated that the reason the mouse MAIT TCRs and human A-F7 TCR could bind mouse MR1-5-OP-RU at higher affinity than human MR1-5-OP-RU is due to the presence of the essential activating Q151 residue in MR1, which is contacted by the shared CDR2α motif between mice and humans.This report also reveals key structural requirements for particular germline-encoded residues in mouse MAIT CDR1α (i.e., the TRAV1-2/TRAV1-unique "G-F-N" motif), CDR2α (i.e., the shared TRAV1-2/TRAV1-"V-L" motif), CDR3α (the TRAJ33exclusive "S-N-Y-Q" motif), and TRBV13-associated residues from CDR2-FWβ, which explains the preferred gene usage of mouse MR1-restricted TRAV1 + /TRBV13 + MAIT cells in the context of infection with riboflavin-producing bacteria.This study highlights the evolutionary conserved nature of the MAIT-MR1 interaction across mice and humans.Further, the MR1 molecules of non-human primates (NHPs), including macaques, is more closely related to that of human MR1 (95% sequence similarity), where MAIT cell levels are maintained at frequencies similar to that of humans (38).Comparison of MR1 α1 and α2 domains from a range of NHP species with that of human MR1 revealed all 12 previously described human MR1 residues that bind 5-OP-RU are conserved (39).Despite this conservation, cross-reactivity of human MR1 tetramers was diminished when used to isolate macaque MAIT cells (38,39).This is due to the presence of three amino acid substitutions at positions 72, 151, and 159, in all NHPs excluding chimpanzees, which were described as contact residues for human MAIT TCRs (39).Given these human MAIT TCR-contact residues are retained in chimpanzee MR1, the phenotype and functional properties of their MAIT cells are likely to be very similar to their human counterparts.
This cross-species reactivity is also observed in the context of type I natural killer T cell (NKT) recognition of lipid-based Ags presented by nonclassical MHC class-I molecule, CD1d (40).Crystal structures of human and mouse NKT TCRs in complex with CD1d-lipid Ags reveal the highly conserved docking mode dominantly mediated by the invariant NKT TCR α-chain, with the CDR1α loop binding CD1d, and CDR3α loop (as is the case in MAIT-MR1 complexes), playing a central role in contacting both the antigen-presenting molecule and the ligand (41,42).Nevertheless, finespecificity differences between mouse and human NKT TCRs towards CD1d-lipid can occur, which has made development of α-GalCer-related therapeutics more challenging (43).Whether there are fine specificity differences between human and mouse MAIT TCR responses towards MR1 ligands remains to be established.Such considerations are crucial for understanding the physiology of MAIT cells and will be critically important for rationally developing humanbased MAIT cell therapeutics that will likely depend on the use of pre-clinical mouse models.

Protein expression, refolding, and purification of soluble MR1-Ag and MAIT TCRs
Soluble human A-F7 MAIT TCR was refolded from inclusion bodies as described previously (6,8) exchange chromatography, followed by size-exclusion chromatography (Superdex 200, GE Healthcare) and lastly, anion exchange (HiTrap-Q HP) chromatography, as previously described (3).Protein purity was determined by SDS-PAGE, and concentrations were calculated from absorbance values at A 280nm using a NanoDrop spectrophotometer.

SPR measurements
All SPR experiments were conducted in duplicate (at least two independent experiments) at 25 C on a BIAcore T200 instrument using HBS buffer: 10 mM HEPES-HCl pH 7.5, 150 mM NaCl, and 0.005% surfactant P20 supplied by the manufacturer (GE Healthcare), as described previously (6).Biotinylated C-terminally cysteine-tagged human and mouse MR1-Ag complexes (generated as described previously in (8) were immobilized on streptavidin sensor chips with a surface density of 2000 response units (RU).Each streptavidin-chip comprises four flow cells; one was loaded with mouse MR1-5-OP-RU, the other was loaded with human MR1-5-OP-RU, and the last cell was left empty to subtract for nonspecific binding.Various concentrations of MAIT TCRs (serially diluted from 200 μM) were injected over the chip at a rate of 5 μl/min.Data were plotted using the 1:1 Langmuir-binding model in Prism version 10 (GraphPad) software (https:// www.graphpad.com/features).

Crystallization, structure determination, and refinement
For ternary complexation, purified mouse TCR was mixed with mouse MR1-β2m-5-OP-RU in a 1:1 M ratio at a concentration of 4 to 6 mg/ml and incubated on ice for 1 h.Crystals were obtained via hanging drop vapor diffusion method.Crystals of ternary mouse MAIT TCRs in complex with mouse MR1-5-OP-RU formed in 0.1 M Bis-Tris Propane (pH 7.5-8.5),16 to 22% PEG 3350, and 0.2 M sodium acetate.Before flash freezing in liquid nitrogen, crystals were soaked in reservoir solution supplemented with 10 to 15% glycerol for cryoprotection.X-ray diffraction data were collected at 100 K at the Australian Synchrotron at either MX1 or MX2 beamlines.Diffraction images were indexed, integrated, and scaled using XDS (45) and were further processed using Aimless in the CCP4 suite (46) and Phenix package (47).Phases were calculated by molecular replacement, and the initial search model for MR1 complex was human MR1 complex (PDB 4GUP), where the ligand was removed, to minimize model bias, and a A-F7 TCR (PDB ID: 4L4T) was used as a search model for the mouse MAIT TCRs.Model building was undertaken in COOT (48) accompanied by iterative rounds of refinement using Phenix.refinesoftware (https://phenix-online.org/documentation/ reference/refine_gui.html).The GradeWeb server and Phenix tools were used to build and generate ligand restraints.Validation of models was achieved using MolProbity (49) and all graphical representations were generated using the PyMOL Molecular Graphics System, version 2.5.Calculations of BSAs were achieved using CCP4 AreaIMol in the CCP4 suite (46).
declare that they have no conflicts of interests with the contents of this article.

Figure 2 .
Figure 2. Steady-state affinity measurements of mouse MR1-restricted TCRs.The affinity of TCR-MR1-Ag interactions were determined using SPR, by measuring the binding of soluble mouse MAIT TCRs, M2A (top panels), M2B (middle panels), and human MAIT, A-F7 (bottom panels), against mouse MR1 refolded with 5-OP-RU and Ac6-FP, and human MR1 refolded with 5-OP-RU.The K D values represent mean ± s.e.m values obtained from two independent experiments (n = 2) performed in technical duplicates using different batches of proteins.NB, no binding.RU, response units.The SPR sensorgrams,

Figure 3 .
Figure 3. Crystal structures of 2 mouse MAIT-MR1 complexes compared with a typical human MAIT-MR1 ternary complex.A-C, mouse M2A (TRAV1-TRBV13-2)-mMR1-5-OP-RU (D-F) M2B (TRAV1/TRBV13-2)-mMR1-5-OP-RU (G-I), A-F7 (TRAV1-2/TRBV6-1) TCR-MR1-5-OP-RU (PDB ID: 6PUC).A, D, and G, top panels are cartoon representations of the ternary complexes.For simplicity, the equivalent TCR α-chains of the mouse TCRs are colored dark blue; the shared mouse TCR β-chains (TRBV13-2/TRBJ2-3) are colored dark pink.The human TCRα A-F7 is shown in light blue and the human A-F7 TCRβ is shown in light orange.The mouse MR1 heavy chain and β2-microglobulin (β2m) molecules are colored light pink and pale yellow, respectively, whereas human MR1 heavy chain and β2m are colored white and pale cyan, respectively.The 5-OP-RU antigen is presented as green sticks.Pie charts represent the relative contribution of each segment of the TCRs, M2A (A), M2B (D), and A-F7 (G) to the buried surface area (BSA) directed against their cognate MR1-5-OP-RU complex.The corresponding complementarity determining region (CDR) loops, namely CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β are shown in teal, sky-blue, purple, red, orange, and yellow, respectively, and the mouse TCR framework (FW) residues of the αand β-chains are shown in dark blue and dark pink, respectively, while the human TCR FWα is colored light orange and FWβ in light blue.The middle panels show the CDR loops of the M2A (B), M2B (E) and A-F7 (H) TCRs docking onto MR1.Each docking angle is shown as a black dashed line connecting the center of mass of Vα with the centre of mass (COM) of Vβ which are represented as a sphere colored consistent with chain colors in the upper panels.The lower panels illustrate the respective mouse M2A (C) and M2B (F), and A-F7 (I) TCR footprints on the molecular surface of MR1-5-OP-RU.The atomic footprint is colored based on the TCR segment making contact.The surface of mouse MR1 is colored light pink whereas human MR1 is colored white.5-OP-RU, 5-(2oxopropylideneamino)-6-D-ribitylaminouracil; MAIT, mucosal-associated invariant T; TCR, T-cell receptor.

Figure 5 .
Figure 5. Interface comparison of TCR α-chain of mouse MAIT versus human MAIT bound to relevant MR1-5-OP-RU.A-C, interactions of the CDR3α of the MAIT TCR (A) mouse M2A, (B) mouse M2B and (C) human A-F7, as well as MR1 residues with 5-OP-RU (colored as in Fig. 1).The MAIT TCR α and β-chains are colored as in Figure 4, A, D, and G. D-F, interactions of the CDR1α and CDR2α loops of MAIT TCR (D) mouse M2A, (E) mouse M2B and (F) human A-F7, with mouse MR1 and human MR1, respectively.Mouse MR1 and human MR1 are colored as in Figure 1.Hydrogen bonds are represented as black dashes (and those involved in forming the interaction triad are colored light blue) and van der Waals contacts are represented as dotted teal lines.Residues that differ between mouse MR1 and human MR1 are labeled in bold lettering.5-OP-RU, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil; MAIT, mucosalassociated invariant T; TCR, T-cell receptor.

Figure 6 .
Figure 6.Interface comparison of TCR beta chain of mouse MAIT TCR versus human MAIT TCR bound to MR1-5-OP-RU.A-C, interactions of the CDR2β (in orange as in Fig. 4) and framework β-chain residues of the (A) mouse M2A, (B) mouse M2B and (C) human A-F7 MAIT TCRs, where the FWβ residues are colored as in Figure 4, A, D and G. D, interactions of the CDR3β loops of the superimposed mouse MAIT TCRs, M2A (colored in gold) and M2B (colored light yellow).E, interactions of the human A-F7 CDR3β (colored in yellow) with MR1.Hydrogen bonds and van der Waals contacts are colored as in Figure 6.Salt bridges are represented as red dashes.Residues that differ between mouse MR1 and human MR1 are labeled in bold lettering.5-OP-RU, 5-(2oxopropylideneamino)-6-D-ribitylaminouracil; MAIT, mucosal-associated invariant T; TCR, T-cell receptor.

Table 1
Data collection and refinement statistics Statistics for the highest-resolution shell are shown in parentheses.