The molecular basis for Mucosal-Associated InvariantT cell recognition of MR1 proteinsJacinto López-Sagasetaa, Charles L. Dulbergera, James E. Crooksa,b, Chelsea D. Parksa, Adrienne M. Luomac,Amanda McFedriesd, Ildiko Van Rhijne,f, Alan Saghateliand, and Erin J. Adamsa,b,c,1

aDepartment of Biochemistry and Molecular Biology, bBiophysics Program, and cCommittee on Immunology, University of Chicago, Chicago, IL 60637;dDepartment of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; eDepartment of Infectious Diseases and Immunology, UniversityUtrecht, 3584 CL, Utrecht, The Netherlands; and fDivision of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital/Harvard MedicalSchool, Boston, MA 02138

Edited by Philippa Marrack, Howard Hughes Medical Institute, National Jewish Health, Denver, CO, and approved April 5, 2013 (received for reviewDecember 27, 2012)

Mucosal-associated invariant T (MAIT) cells are an evolutionarilyconserved αβ T-cell lineage that express a semi-invariant T-cell re-ceptor (TCR) restricted to the MHC related-1 (MR1) protein. MAITcells are dependent uponMR1 expression and exposure to microbesfor their development and stimulation, yet these cells can exhibitmicrobial-independent stimulation when responding to MR1 fromdifferent species. We have used this microbial-independent, cross-species reactivity of MAIT cells to define the molecular basis ofMAIT-TCR/MR1 engagement and present here a 2.85 Å complexstructure of a human MAIT-TCR bound to bovine MR1. The MR1binding groove is similar in backbone structure to classical pep-tide-presenting MHC class I molecules (MHCp), yet is partially oc-cluded by large aromatic residues that form cavities suitable forsmall ligand presentation. The docking of the MAIT-TCR on MR1 isperpendicular to the MR1 surface and straddles the MR1 α1 and α2helices, similar to classical αβ TCR engagement of MHCp. However,the MAIT-TCR contacts are dominated by the α-chain, focused onthe MR1 α2 helix. TCR β-chain contacts are mostly through the vari-able CDR3β loop that is positioned proximal to the CDR3α loop di-rectly over the MR1 open groove. The elucidation of the MAIT TCR/MR1 complex structure explains how the semi-invariant MAIT-TCRengages the nonpolymorphic MR1 protein, and sheds light ontoligand discrimination by this cell type. Importantly, this structurealso provides a critical link in our understanding of the evolutionof αβ T-cell recognition of MHC and MHC-like ligands.

metabolite | molecular recognition | unconventional T cells |antigen-presentation

Mucosal-associated invariant T (MAIT) cells are a highlyconserved T-cell subset found in most mammalian species

(1–4). In humans, they can constitute up to 10% of circulatingdouble-negative T cells, although they are much less frequent inmice (1, 5, 6). Most MAIT cells lack expression of the CD4 orCD8 coreceptors, although many MAIT cells express the ααform of the CD8 coreceptor (1). In humans, these cells are foundat moderate frequency in the intestine and represent up to ∼50%of T cells in the liver (7). The cells exhibit an effector-memoryphenotype and express the CD161 receptor (6). Their presenceas mature effector cells in the periphery is dependent on B cellsand the gut commensal flora (6, 8). Stimulated human MAITcells can express both proinflammatory cytokines (IFN-γ, TNF-α,and IL-17) and cytolytic effectors (granzyme B) (7, 9, 10). MAITcells are known best for their reactivity against various micro-organisms from both bacterial and fungal origin (9, 10). Thesemicroorganisms include several important human pathogens,such as Mycobacterium tuberculosis, Salmonella typhimurium, andStaphylococcus aureus. Indeed, a significant proportion of thenonclassically restricted responding T cells in M. tuberculosis-infected individuals were determined to be of the MAIT lineage(9). MAIT cells have also demonstrated autoreactivity and havebeen associated with various autoimmune disorders (11, 12);they have also been found in both kidney and brain tumors (13).

MAIT cells are characterized by expression of a semi-invariantT-cell receptor that, in humans, is composed of Vα7.2 (TRAV1-2)/Jα33 α-chain paired with either Vβ13 (TRBV6) or Vβ2 (TRBV20)β-chains. MAIT T-cell receptors (TCRs), particularly the Vαdomain, are highly evolutionarily conserved, exhibiting highamino acid sequence identity between human and cow (75%)and human and mouse (72%) (1). This sequence identity reaches85% and 90%, respectively, when only the CDR loop-regionsequences are compared. The CDR3α loop, encoded by the re-gion of Vα-Jα rearrangement, is strictly conserved in length andhighly conserved in amino acid sequence in MAIT TCRs, bothwithin and between species. Junctional variability has been notedat only two of the CDR3α loop positions (underlined XXs):CAXXDSNYQL. In contrast, the CDR3β loop (encoded bythe Vβ-Dβ-Jβ rearrangement) is highly diverse in MAIT TCRs.This evolutionary and sequence conservation is reminiscent ofthe semi-invariant TCRs of type I invariant natural killer T(iNKT) cells that recognize lipids presented by the MHC-likeprotein CD1d.MAIT cells are restricted to the MHC class I-like molecule

MR1, which is similarly conserved throughout mammalian evo-lution. The MR1 gene is encoded outside of the MHC locus,found ∼23 Mb away from the CD1 genes on Chromosome 1 inhumans. Like CD1 molecules, MR1 exhibits low intraspeciesvariation and high-sequence conservation between mammalianspecies (3, 14). MR1 is ubiquitously expressed (14); however,cell-surface expression is low or undetectable in endogenous

Significance

Mucosal-associated invariant T (MAIT) cells are a highly con-served lineage of αβ T cells found in most mammals. These cellsexpress a T-cell receptor of low diversity that recognizes vita-min metabolites presented by the MHC-related protein, MR1.Despite the evolutionary divergence of MR1 from other MHCproteins, we have found that MAIT T-cell receptors recognizeMR1 using similar molecular strategies as that of the highlydiverse, conventional αβ T cells, which recognize classical MHCmolecules presenting peptide fragments. Our results also shedlight onto how MR1-presented antigens can modulate theMAIT–T-cell receptor affinity and MAIT cell stimulation.

Author contributions: J.L.-S., C.L.D., J.E.C., A.M.L., A.M., A.S., and E.J.A. designed research;J.L.-S., C.L.D., J.E.C., C.D.P., A.M.L., A.M., and E.J.A. performed research; I.V.R. contributednew reagents/analytic tools; J.L.-S., C.L.D., J.E.C., A.M.L., A.M., A.S., and E.J.A. analyzeddata; and J.L.-S., C.L.D., J.E.C., and E.J.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID code 4IIQ).1To whom correspondence should be addressed. E-mail: ejadams@uchicago.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1222678110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1222678110 PNAS | Published online April 23, 2013 | E1771–E1778

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cells but is robust in MR1-transduced or -transfected cells (2).Recently, the precursor to riboflavin, 6,7-dimethyl-8-(1-D-ribityl)lumazine (DMRL), and chemical derivatives of it were shown tobe presented by MR1 and could stimulate MAIT cells (15),shedding light on the mechanism of MAIT cell immunosur-veillance of microbial species.In addition to microbial reactivity, microbial-independent auto-

reactivity of human MAIT cells toward MR1-transfected cells hasbeen noted (3, 16, 17), suggesting MAIT reactivity is not com-pletely dependent on the presence of an exogenous, microbialantigen. Furthermore, microbial-independent cross-species re-activity between mouse and human MAIT cells and their MR1orthologs (3) has been previously observed. Position 151 on theMR1 α2 helix was implicated in cross-species reactivity (16), andwas further shown to enhance human MAIT cell reactivity whenmutated to an alanine (18). Taken together, these observationssuggest that MAIT cell reactivity to MR1 appears to be a balancebetween MR1 availability on the cell-surface, which may be mod-ulated by endogenous, small-molecule ligands that can supportMR1 expression and stability, and other features that can enhanceTCR binding, such as species-specific differences in the α-helices,in particular at position 151, or the presence of stimulatory small-molecule ligands from exogenous sources.MAIT cells have similarities to iNKT cells in their effector-

phenotypes, their recognition of highly conserved MHC-like mole-cules (MR1 and CD1d, respectively) and their use of semi-invariantTCRs. Like MAIT cell TCRs, iNKT TCRs similarly have a con-served α-chain. The α-chain in iNKT TCRs is completely invariantwithin the iNKT cell population, but iNKT β-chains have preferredVβ domains and diverse CDR3β loops. Structures of mouse andhuman iNKT TCR complexes with CD1d presenting various lipidshave established that TCR recognition of CD1d is unlike classicalαβ TCR recognition of MHC-peptide and follows a mostly con-served docking orientation mediated predominantly via theCDR3α loop via residues contributed by the Jα18 gene segment(19). Similarly, extensive mutagenesis of MAIT TCRs has estab-lished a bias of α-chain contribution to MR1 binding and muta-genesis of MR1 has provided a tentative footprint on the MR1 α1and α2 helices (18). However, lacking definitive structural evidenceof this interaction, the docking footprint of the MAIT TCR onMR1 remains unclear, as does the role of MR1-presented antigenin MAIT TCR engagement. We have taken advantage of the mi-crobial-independent cross-species reactivity of MAIT cells to studythe molecular basis for MAIT cell binding to MR1. Here wepresent the 2.85 Å structure of a human MAIT TCR in complexwith bovine MR1. Our structure reveals a docking orientationreminiscent of diverse αβTCR recognition, such as that observed inclassical αβ TCR recognition ofMHC/peptide (20) or noninvariantNKT recognition of CD1d/sulfatide (21, 22), and is distinctly dif-ferent from that of the iNKT TCR bound to CD1d (19). There isa clear bias toward contacts contributed by the conserved MAITα-chain CDR loops; however, the CDR2β and diverse CDR3β alsocontribute to binding. Our structure establishes the molecular basisfor MAIT cell xeno-reactivity, providing an excellent model bywhich to understand the bona fide MAIT cell recognition of MR1and how stimulatory ligand presentation from MR1 can enhanceMAIT TCR binding and lead to MAIT cell activation.

ResultsMR1 Has a Small Ligand-Binding Cavity Lined with Aromatic and BasicResidues. Gene sequences for human MAIT TCRs F7, G2, andAE6 were derived from the published sequences of Tilloy et al.(1) and were synthesized via overlapping PCR. These MAITTCRs were expressed in Escherichia coli and refolded to producefull-length heterodimeric TCRs, whereas a single-chain versionof bovine β2microglobulin (β2m)-MR1 was expressed in insectcells for our biophysical and structural studies. Crystals of theMAIT TCR F7/MR1 complex were optimized and used to col-

lect a complete dataset, which was finally refined to 2.85 Å res-olution (Table 1). There was one complex of MAIT TCR/MR1in the asymmetric unit.In the complex, bovine MR1 adopted an overall binary struc-

ture of a class I MHC fold associated with the β2m subunit (Fig.1A), highly similar to that of HLA-A2 and to the recent publishedhuman MR1 structure (Fig. 1B) (15). Of note was a slight shift inthe α2 helix of our structure in relation to unliganded humanMR1(Fig. 1B); the flexibility of this region was further reflected in thetwo molecules of human MR1 in the asymmetric unit (shown aslight and dark magenta), where a dramatic shift (up to 11 Å) wasevident in this region. The structural differences between thesemolecules suggest the α2 helix is highly flexible and can adaptstructurally in a context-dependent manner. MR1 has a cavitysmaller (∼760 Å) than that of classical class I MHC molecules,similar to that noted previously (15).With the exception of a smallopening or “portal,” this cavity is mostly closed on the left-mostside, where it is lined with aromatic (tryptophan, tyrosine, andphenylalanine) and basic (arginine and lysine) residues, giving thisregion an overall basic charge (Fig. 1C), suggesting it can ac-commodate small molecule species that are polar and potentiallynegatively charged. Due to its similarity in location to the Apocket of classical class I MHC and A′ cavity of CD1 molecules,we refer to this region as the A′ cavity, similar to the designationused in ref. 15.Clear electron density exists for a ligand in our MR1 structure

(Fig. 2), enclosed by the aromatic (Y7, Y62, W156), basic (R9,K43, R94), and polar (S24 and T34) residues lining the A′ cavity.The electron density is similar in nature to the 6-FP previouslyidentified as bound by human MR1 refolded in the presence ofmammalian cell-culture media (15). Because MR1 used for thisstudy was expressed in insect cells, there may be alternativecompounds that can be presented that are either synthesized byinsect cells or present in the insect cell media. The continuouselectron density of this ligand with lysine 43 suggested that some,

Table 1. Data collection and refinement statistics (molecularreplacement)

Data collection and refinement MAIT TCR F7 – MR1

Data collectionSpace group P 21 21 21Cell dimensions

a, b, c (Å) 82.045, 87.41, 156.372α, β, γ (°) 90.00, 90.00, 90.00

Resolution (Å) 50–2.85 (2.9–2.85)Rsym 0.079 (0.681)I/σI 14.63 (2.72)Completeness (%) 99.55 (97.57)Redundancy 4.1 (4.1)

RefinementResolution (Å) 2.86Total No. reflections 108,886No. unique reflections 26,556Rwork/Rfree 0.2129/0.2695No. atoms

Protein 6,285Ligand/ion 28Water 5

B-factorsProtein 65.20Ligand/waters 73.50

R.m.s. deviationsBond lengths (Å) 0.004Bond angles (°) 0.88

Values in parentheses are for highest-resolution shell.

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if not all, of this ligand was covalently attached via a Schiff base,as noted previously (15).

MAIT TCR Straddles MR1 α1 and α2 Domains in a Classical DockingOrientation. The F7 MAIT TCR docks on MR1 in a perpendic-ular fashion, reminiscent of conventional αβ TCR recognition ofMHC-peptide, with an orientation similar to that of the type II,noninvariant NKT TCR binding to CD1d presenting sulfatide(Fig. 3) (21, 22). The MAIT TCR/MR1 complex interface buries∼1180 Å2 of surface area, within the range of buried surfaceareas observed in αβ TCR/MHCp (23) and type II NKT TCR/CD1d structures (21, 22). The α-chain buries the majority of thesurface area (∼55%) with its contacts biased toward the α2 helixof MR1. The β-chain buries ∼45%, with contacts biased towardthe MR1 α1 helix (Fig. 4 and Table S1). This binding orientationis unlike that of the invariant NKT TCR recognition of CD1d,where the docking orientation is rotated more than 90° withmost of the CD1d contacts established with the α chain, theCDR3α loop in particular (Fig. 3B, Lower Right). The MAITTCR is oriented such that the CDR3α loop is situated over theA′ portal, perfectly positioned for contact with a suitable ligandshould it be presented in this cavity.Comparison of the human MAIT TCR structure in our com-

plex with bovine MR1 with an unliganded human MAIT TCRreported by Reantragoon et al. (18) reveals only minor structuraldifferences in the Vα domain conformation (Fig. S1), with anrmsd of 0.6 as determined by the DALI server (http://ekhidna.biocenter.helsinki.fi/dali_lite/start_). The CDR loop conforma-tions of the Vα domains are highly similar between the ligandedand unliganded TCRs (Fig. S1), suggesting that little confor-

mational adjustments of the Vα CDR loops are required forrecognition of MR1. Of note is the striking structural similarityof the CDR3α loop side-chains between these TCRs; eventhough these loops vary at two amino acid positions (90 and 91),the remaining side-chain conformations are remarkably conserved,including Y95. However, the positioning of the Vβ CDR loops aredifferent, mainly because of the use of different Vβ domains inthese two MAIT TCRs. This finding suggests that the Vβ CDRloops may be more adaptable during engagement of MR1.All three CDR loops of the MAIT TCR α-chain contribute to

MR1 recognition (Fig. 4 and Table S1). The germ-line–encodedCDR1α and CDR2α loops exclusively establish contacts withMR1 residues of the α2 helix. The majority of contacts are vander Waals (VDW); however, several key hydrogen-bonding con-tacts are established with these loops, in particular the main-chainatoms of F29 from the CDR1α loop hydrogen-bond with side-chains of N155 and E160 of MR1, and E55 of the CDR2α loopforms a salt-bridge and hydrogen-bond with R147 of MR1 (Ta-ble S1). One α-chain contact lies outside of the canonical CDRloops, R66 from β-strand-D of the Vα domain establishes VDWand weak electrostatic interactions with E159 of the α2 helix.The CDR3α loop establishes both VDW and hydrogen-bondcontacts with both the α1 and α2 helices, with the majority ofcontacts coming from Y95. This residue hydrogen-bonds withR61 of the α1 helix and W156 of the α2 helix and forms VDWcontacts with R61, L65, W69 of the α1 helix, and Y152 and W156of the α2 helix. Y95 is also positioned directly over the portalcontaining the ligand density, suggesting this residue may es-tablish direct contact with a stimulatory ligand. The main-chainnitrogen of Q96 of the CDR3α loop also hydrogen-bonds withthe R61 side-chain of MR1. Of note, all of the residues of theCDR3α loop that contact MR1 derive from the Jα33 (TRAJ33)segment used in MAIT TCRs. The MAIT TCR α-chain has beencharacterized in several different species and is highly conserved.Examination of the residues of the TCR α-chain observed tocontact MR1 in this complex structure reveals that only 2 of the12 α-chain residues used in contacting MR1 are variable acrosshuman, mouse, and cow (Fig. S2). Human E55 is a lysine in themouse Vα homolog, whereas human S93 is a glycine in the cowMAIT Vα domain.

A B

C

α1

α2

α1

α2

α3β2m

K43K43H58H58

R94R94

R9R9

bMR1bMR1HLA-A2HLA-A2

760 A3

α1

α2

bMR1bMR1hMR1ahMR1ahMR1bhMR1b11Å

Fig. 1. MR1 has a small, basic, aromatic-lined cavity optimal for bindingsmall ligands. (A) Side view of a ribbon diagram of bovine MR1. The heavychain, composed of the α1, α2, and α3 domains, is shown in cyan, β2m in teal.(B) Comparison of bovine MR1 (bMR1) α1 and α2 platform domain (shown incyan) with that of HLA-A2 (PDB ID code: 1DUZ, shown in purple); rmsd of theCα backbones of the two platform domains is 0.581. At the bottom isa comparison of bovine MR1 with that of the two human MR1 structuresidentified in ref. 15 (PDB ID code: 4GUP). Bovine MR1 is shown in cyan andthe two MR1 human structures (molecule “a” and molecule “b”) are shownin lavender and magenta, respectively. The 11 Å shift between the α2 helicesof human MR1 molecules “a” and “b” is indicated by a red line. (C) Elec-trostatic surface representation of the MR1 cavity shown on a ribbon dia-gram of the MR1 CA backbone. The basic residues contributing to the overallbasic charge of the cavity are shown (Inset), highlighted in yellow.

6-FP6-FP

R94R94R9R9

Y7Y7

T34T34K43K43

Y62Y62

W156W156

Y95αY95α

S24S24

Fig. 2. Ligand presentation by MR1. An omit electron density map of theresidues and ligand found in the MR1 cavity. Side-chain electron density(1.5σ) is shown in green. 6-FP, identified in ref. 15, has been modeled intothe ligand electron density (1σ) shown in yellow.

López-Sagaseta et al. PNAS | Published online April 23, 2013 | E1773

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Contacts of the MAIT TCR β-chain to MR1 are restricted tothe germ-line–encoded CDR2 loop and the diverse CDR3 loop.With the exception of T54 and T55, all contacts from the CDR2β(Y48, A50, S51, and D56) are VDW; T54 and T55 establishhydrogen-bonds with the side-chain of Q64 (Fig. 4 and TableS1). Six CDR3β loop residues (W96, T97, G98, E99, G100, andS101) establish contacts with the α1 and α2 helices, the majorityof them from VDW interactions. S101 from the CDR3β loopmakes hydrogen-bond contacts with main-chain and side-chainatoms of E149 of the MR1 α2 helix. In contrast to the conservedα-chain contacts, those of the β-chain are less conserved bothwithin and between species. Only T55, which establishes VDWand hydrogen-bond contacts with Q64 of MR1, is conserved inthe human Vβ domains used in MAIT TCRs; this position isvariable in its mouse counterparts (Fig. S2).Mutagenesis analyses of autoreactive MAIT TCR/MR1 inter-

actions (17) and those in the presence of microbial infection (17,18) are both consistent with the overall footprint of the humanMAIT TCR on bovine MR1 described here. Both the mouse and

human studies describe an α-chain bias in MR1 recognition (18),consistent with the major contribution to binding coming fromthe MAIT TCR α-chain observed in this complex structure. Mostof the residues in either human or mouse MAIT TCR α-chainsthat were determined to be important for MR1 recognition (17,18) establish contacts with MR1 in our structure, specifically:G28α, F29α, N30α, Y48α, V50α, L51α, S93α, N94α, and Y95α. Itis important to note that these residues are strictly conservedbetween mouse and human MAIT TCRs. Of particular impor-tance is residue Y95α, which establishes eight contacts with MR1and is perfectly positioned to establish contacts with a ligandpresented in the A′ cavity. Mutagenesis of this residue abrogatedMAIT cell autoreactivity and microbial-dependent MR1 stimu-lation. As discussed later, modeling of stimulatory compoundsidentified by Kjer-Nielsen et al. (15) into the A′ cavity of MR1 inour complex structure supports the importance of this residue inligand recognition. The presence of a stimulatory ligand likelysupplies additional contacts that overcome the binding thresholdrequired for MAIT cell activation.

Positions 72, 147, and 151 Mediate the Xeno-Reactive InteractionsBetween Human MAIT TCRs and Bovine MR1. To identify positionscontributing to MAIT cell cross-reactivity, the amino acid dif-ferences between human and bovine MR1 were mapped ontoour MR1 structure (Fig. 5). Three differences are at contact siteswith the human MAIT TCR: A72 of the α1 helix and R147 andQ151 of the α2 helix, which are M72, Q147 and L151 in humanMR1, respectively (Fig. S3). Although position 72 is contactedonly by one residue of the CDR3β loop (W96), Q151 and R147

MAIT/MR1 αβTCR/MHC Type II-NKT/CD1d Type I-NKT/CD1d

αα

β

MR1MR1

αβ

α

β

MAITMAIT

α

β

MR1 MHC

CD1d/sulfatide

CD1d/αGalCer

α1

α2

α1

α2

β

α

β

α

β

α

β

A

B

3

3

3

1 1

3

2

2

1

3

3

1

3

3

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2

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2

2

2

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1

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Fig. 3. The MAIT TCR docking orientation on MR1. (A) Ribbon diagrams ofthe complex structures of the human MAIT TCR with bovine MR1; conven-tional αβ TCR with MHC-peptide; type II NKT TCR with CD1d-sulfatide; andtype I iNKT TCR with CD1d-α-Galactosylceramide (PDB ID codes: 2CKB, 4EI5,and 3HUJ, respectively). The TCR α- and β-chains are shown, respectively, inpink and marine (MAIT), yellow-orange and orange (αβ TCR), light green andgreen (type II NKT), and skyblue and darkblue (type I NKT). MR1 is shown incyan, classical MHC in yellow, CD1d in green and slate for the type II and typeI complexes, respectively. (B) Positioning of the CDR loops onto the respectiveMHC or MHC-like surfaces. MR1, classical MHC, and CD1d are all shown inwhite in a semitransparent surface representation. The CDR1, CDR2 and CDR3loops from the respective TCRs are shown as they are positioned in each ofthe complexes. The dashed black lines represent the axis of binding, derivedfrom a line extending between the conserved intrachain disulfides of the Vαand Vβ Ig domains. The loop coloring is the same as that defined in A.

−

+

G28G28

F29F29

N30N30

CDR1α

R66R66E55E55

L51L51

V50V50

Y48Y48

Q96Q96

Y95Y95

S93S93N94N94

Y48Y48A50A50

S51S51T54T54

D56D56

W96W96

T97T97E99E99

G98G98 G100G100

S101S101

α1

α2

α1

α2

Hydrogen Bond/Electrostatic

van der Waals

α chainα chain β chainβ chain bothboth

Fig. 4. Contacts mediated by the MAIT CDR loops on the MR1 surface.Shown in each panel are the side-chains of the CDR1α, CDR2α, CDR3α,CDR2β, and CDR3β loops and the residues they contact on the MR1 surface.No contacts were noted for the CDR1β loop. (Right, Upper) The distributionof VDW contacts (Upper) versus hydrogen-bond and electrostatic inter-actions (Lower). Contacts from the α-chain are shown in pink; β-chain inmarine and residues contacted by both α- and β-chains are colored purple.Hydrogen-bond contact distances are ≤ 3.3 Å and are shown as dashedyellow lines.

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are surrounded by residues V50, L51, and E55 of the CDR2αloop (Figs. 4 and 5). Position 151 has been noted to be a positionthat modulates MAIT cell reactivity during investigation of xeno-reactivity of MAIT cells to MR1 (3). In addition, mutagenesisof this position in human MR1 to alanine stimulated microbial-independent human MAIT cell reactivity (18), suggesting it playsan important role in MAIT TCR binding. This effect of position151 could be a direct result of a steric interaction of the 151 side-chain (a leucine in humans) interfering with the CDR2α loopbinding, or this residue may affect the positioning of the neigh-boring residue at 147. R147 forms a salt-bridge with E55 of theCDR2α loop, although the equivalent position in human, gluta-mine, would also be capable of forming hydrogen-bond inter-actions with E55.To evaluate the effect of these positions on MAIT TCR

binding to MR1, we first performed surface plasmon resonance(SPR) on the F7, G2, and AE6 TCRs (Fig. 6, Upper) with wild-type bovine MR1 to assess the affinity of this xeno-reactivity. F7and G2 MAIT TCRs bound wild-type bovine MR1 with low butsimilar affinity, 31 μM and 39 μM, respectively. The AE6 TCR,which uses a different Vβ domain (TRVB6_2 in lieu of TRVB6_1in F7 and G2), and thus differs from F7 and G2 in the CDR2βand CDR3β loops (Fig. S4), bound wild-type bovine MR1 withnearly a twofold weaker affinity, 74 μM.We then measured, usingbio-layer interferometry (Blitz), the affinity of the F7 MAIT TCRfor single bovine MR1 mutants expressing the human residuesat positions 72 (A to M), 147 (R to Q), and 151 (Q to L) anda “humanized” version containing all three mutations. The A72Mmutation enhanced binding ∼190% (Fig. 6, Lower) (∼19.5 μM)(Fig. S5), whereas the R147Q mutation decreased binding to∼40% of wild-type levels (∼91 μM). Mutation of Q151 to leucinedecreased binding to an undetectable level. However, the F7 TCRbound the triple “humanized” mutant with ∼55% the affinity ofwild-type (∼65 μM). Although position 151 played the most sig-nificant role in MAIT TCR recognition as revealed through ourmutagenesis, both positions 72 and 147 modulated binding,consistent with our observed footprint of the MAIT TCR on theMR1 surface.Finally, to probe the influence of the CDR3β loop on MAIT

TCR recognition of MR1, we have included a mutation of po-sition E149 of MR1 to alanine in our binding studies. E149 ofMR1 is the only residue to which hydrogen-bonds are formed bythe CDR3β (S101); however, it also establishes VDW contacts

with E99, G100, and S101 (Fig. 3 and Table S1). Mutation of thisposition to alanine had little effect upon MAIT TCR binding,suggesting that specific, side-chain–mediated contacts of theCDR3β may not play a central role in MR1 recognition.These results establish that: (i) MAIT TCRs recognize MR1

with a range of affinities with contributions from the Vβ domain(TRVB6_1 TCRs in this study bind more strongly than TRVB6_2);(ii) positions 72, 147, and 151 of MR1 act in concert to modulateMAIT cell xeno-reactivity by enhancing the MAIT TCR bindingaffinity of MR1 approximately twofold; and (iii) the variableCDR3β loop of this MAIT TCR (F7), although establishingnumerous VDW contacts with MR1, may not play a critical rolein MR1 recognition. This finding does not, however, rule out thepossibility that the CDR3β loop may play an important role inmodulating MAIT TCR binding and that variability at this loopbetween MAIT TCRs may result in different thresholds of MR1-dependent stimulation, similar to what was observed in iNKTcell modulation by CDR3β loop variability (24).

Optimized Ligand Docking in MR1 Provides Additional Contacts to theMAIT TCR Through Y95 in the CDR3α. To explore the effect of MR1presented ligand on MAIT TCR activation, we used AutodockVina to model how the stimulatory compounds identified in ref.15 could be presented by MR1 and enhance binding of the MAITTCR. The riboflavin precursor, DMRL and its chemical derivative,reduced 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH2OH),

α1

α2

2β2β

3β3β

W96W96

E55E55

L51L51

V50V50

3α

1α

A72

R147

Q1512α

Fig. 5. Sites of variability between human and bovine MR1. Variable sitesare indicated in pink on a semitransparent surface representation of bovineMR1. The contacting CDR loops of the MAIT TCR are shown, with the side-chains where contacts with variable residues are established. One residue inthe α1 helix differs between human and cow, position 72, which is an alaninein cow and a methionine in human. Two positions vary in the α2 helix, 147and 151, which are arginine and glutamine in cow, respectively, and gluta-mine and leucine in human, respectively. Of note is position 151, notedpreviously to mediate species cross-reactivity (3) and, upon mutation to al-anine, induce potent autoreactivity by MAIT cells (18).

-50 0

50 100 150 200 250

0 20 40 60 Time, s

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200

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50

0wildtype M/Q/L E149AR147QA72M Q151L

% B

indi

ng o

f wild

type

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N.D.

Fig. 6. Affinity of human MAIT TCRs for bMR1 and humanized bMR1.(Upper) SPR curves are shown between three human MAIT TCRs [F7, G2, andAE6 (1)] and bovine MR1. Dissociation constants (KD) were derived fromequilibrium analysis, the fits are shown for their respective SPR curves.(Lower) Residues that differ between human and bovine MR1 (72, 147, and151) were converted to the human sequence and measured for changes inbinding by the human F7 MAIT TCR by bio-layer interferometry (Blitz).Values shown are percent binding of wild-type (KDmut/KDwt).

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each underwent 10 independent Vina docking runs. Side-chainswithin the MR1 binding pocket, as well as Y95α of the MAITTCR, were allowed to be flexible. Each round generated nineligand conformations, ranked by their calculated binding ener-gies, yielding the same top-scoring structure for each ligand in 8of the 10 runs. The best poses reported energies of −9.2 and−8.4 kcal/mole for DMRL and rRL-6-CH2OH, respectively.Shown in Fig. 7 are the orientations of these two ligands in theMR1 binding cavity. In each case, the lumazine group is stackedbetween the aromatic residues Y7 and Y62 and enclosed by thepolar residues R9, S24, K43, and R94. W156 also contributes toVDW contacts with both ligands. The orientation of the luma-zine group, although in the same plane, is flipped between thetwo ligands. DMRL has its two methyl groups at the 6 and 7position oriented toward the more nonpolar side of the bindingcavity, whereas the two carbonyl groups are oriented towardthe polar residues. rRL-6-CH2OH, in contrast, has its hydrox-ymethyl group oriented toward the polar side-chains and thecarbonyl groups oriented toward the hydroxyls of Y7 and Y62.Despite the flipped orientations of the lumazine group, theribityl chain is essentially positioned in the same spot. In bothcases, the first hydroxyl of the ribityl chain is positioned withinhydrogen-bonding distance to Y95 of the CDR3α loop, providingan important ligand-mediated contact that could enhance thebinding affinity of the MAIT TCR and initiate T-cell activation.We also performed an identical docking analysis of the non-stimulatory, noncovalently bound compound, 6-FP (Fig. S6), andfound similar conformations of this ligand in the MR1 groove,

suggesting these are the preferred orientations of the aromaticrings in the binding pocket.

DiscussionMAIT cells constitute a considerable percentage of circulatingCD8−/CD4− and CD8αα+ T cells and have been shown to respondto important human microbial pathogens such as M. tuberculosis,S. typhimurium, and S. aureus through their recognition of MR1.The broad conservation of MAIT cells in mammals, restriction toan equally highly conserved MHCmolecule, and their prevalencein barrier, mucosal tissues suggests that MAIT cell microbialdetection has been an adaptation widely used in mammalian hostdefense. In addition to microbial detection, MAIT cells havebeen shown to demonstrate microbial-independent autoreactivitywhen MR1 is expressed at high levels on antigen-presenting cells,which may provide a link to MAIT cell involvement in autoim-mune disorders and tumor immunosurveillance (11, 12, 25). Thismicrobial-independent reactivity of MAIT cells is further en-hanced by modification of a single amino acid residue, position151 on the α2 helix. This residue was found to be central to thecross-species reactivity used in our study. We have capitalized onthe close sequence identity between bovine and human MR1 andthe microbial-independent reactivity between bovine MR1 andhuman MAIT cells to provide insight into how MAIT cells,through their semi-invariant TCR, recognize MR1. We also ex-tend a model for the role of the riboflavin precurosor, DMRL andits derivative, rRL-6-CH2OH (15), in MAIT TCR engagementduring microbial detection.Despite the similarity of the semi-invariant MAIT TCR to that

of iNKT cells, the MAIT TCR engages MR1 with a footprint andorientation unlike that seen in iNKT/CD1d complex structures.Instead, the orientation of the MAIT TCR on MR1 is highlysimilar to that of diverse type II NKT TCRs bound to CD1d/sulfatide (21, 22), and classical αβ TCR recognition of MHC-peptide (20). Of particular note is the focus of the germ-line–encoded CDR loops (CDR1 and CDR2) onto the MR1 helices;this is highly reminiscent of classical αβTCR recognition, wherebythe CDR1 and CDR2 loops of the α-chain establish contacts withthe α2 helix and those of the β-chain contact the α1 helix (20). Inlieu of a peptide, the MAIT TCR CDR3α and -β loops establishcontacts with both helices and, in the case of the CDR3α loop, actsas a sensor for MR1-presented antigens. Several lines of evidencesupport the dominance of the Vα domain in MR1 recognition.This domain is essentially invariant in the MAIT cell population(only two residues in the CDR3α loop vary) and in our structurethere is a bias of the docking footprint on MR1 toward the VαCDR loops. Previous mutagenesis of MAIT TCR CDR loops (17,18) are consistent with the Vα dominance in our structure, andfinally, there is no significant conformational change of theCDRα loops, or their side-chains, between liganded and unli-ganded MAIT TCRs. Finally, mutation of an MR1 contact resi-due with the CDR3β loop, E149, showed little effect on MAITTCR binding. Taken together, these data suggest that MAITTCRs engage MR1 predominantly through the Vα CDR loops,whereas the contacts mediated by the Vβ CDR loops provideancillary contacts that can vary across the different Vβ domainsand the diverse CDR3β loops present in MAIT TCRs.The MAIT α-chain has only two variable positions located

within the CDR3 loop, where the V-J junction occurs duringrearrangement. All of the CDR3α loop contacts noted in ourstructure, however, derive from the amino acids encoded by theJα33 gene segment used during MAIT Vα rearrangement, a keysimilarity to that of invariant NKT cells, where the contributionof the Jα18 gene segment is pivotal for iNKT TCR recognition.Y95 is a particularly important residue in the MAIT CDR3αloop as it not only establishes contacts with many MR1 residues,but is also positioned directly over the opening to the MR1binding cavity where 6-FP and related compounds are bound and

DMRLDMRL rRL-6-rRL-6-CHCH22OHOH

Y95αY95αY95αY95α

N N

NHN

H3C

H3C

HO

HO

HOO

O

OHHO

HO

HO

HO NH

O

O

HNN

N

OHDMRL rRL-6-

CH2OH

Fig. 7. MAIT TCR engagement of stimulatory antigens revealed throughligand modeling. Optimized positions of the DMRL (and its chemical de-rivative, reduced rRL-6-CH2OH) are shown in the MR1 ligand binding cavity.DMRL is shown in yellow, rRL-6-CH2OH in green. The side-chains lining thecavity, as well as residue 95 of the CDR3α loop, were allowed to move; thealternate conformations of these side-chains from that of our original MR1structure are shown in white. These positions represent the most energet-ically favorable of eight alternate conformations, with energies of −9.2and −8.4 kcal/mole for DMRL and rRL-6-CH2OH, respectively. The con-formations of both ligands are such that the first hydroxyl group of theribityl chain engages the MAIT TCR through residue Y95 of the CDR3α loop(shown in pink), via a hydrogen bond (≥3.3 Å, shown as yellow dashed line).The chemical structures of DMRL and rRL-6-CH2OH are shown beneath theirmodeled positions.

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where our modeling has favored binding of the stimulatory ri-boflavin derivatives DMRL and rRL-6-CH2OH. It is likely thatcontacts established by this residue “sense” presentation of stim-ulatory ligands by MR1, contributing to enhanced T-cell engage-ment and subsequent MAIT cell activation. The minor contactsthat the MAIT TCR establishes with our modeled stimulatorycompounds is in direct contrast to what is observed in conven-tional αβ TCR recognition of MHC-peptide, where TCR contactswith the peptide comprise 20–30% of the contact surface (23), andiNKT TCR contact with CD1d/aGalCer, where ∼36% of TCRcontacts are with the lipid headgroup (19). Indeed, differences inthe headgroup of the lipid antigen presented by CD1d that arerecognized by iNKT TCRs can have profound effects on iNKTeffector outcome (26). It is conceivable that other, yet to be iden-tified, stimulatory compounds might establish more contacts withthe TCR and could modulate MAIT cell activity; however, the datato date suggests a minor role for ligand in TCR engagement.The F7 and G2 MAIT TCRs examined in our study retain the

ability to bind our “humanized”MR1 (bovineMR1 with the threespecies-specific differences at positions 72, 147, and 151 mutatedto their respective human amino acids: M, Q, and L) with a Kd of∼60 μM, suggesting that human MAIT TCRs can bind humanMR1 in the absence of exogenous stimulatory ligands. Thisfinding is consistent with the previously observed microbial-in-dependent autoreactivity and may be the basis for MAIT cellreactivity in autoimmunity and tumor surveillance. Presentationof certain ligands that can provide additional contacts to theTCR, such as DMRL or rRL-6-CH2OH, would enhance activa-tion. However, these contacts with the proposed stimulatorycompounds that we have noted in our complex structure andthrough modeling are not substantial in relation to the othercontacts between the TCR and MR1, suggesting that T-cellstimulation through TCR engagement is a very finely tunedprocess. Although mutagenesis of Y95 of the CDR3α chain wasshown to abrogate microbial-dependent MAIT cell activation(18), this residue establishes numerous contacts with MR1 in-dependent of bound ligand and only participates in one hydrogenbond with our modeled stimulatory compounds. Thus, the triggerfor MAIT cell activation likely comes from a combination of twofactors, the first being enhanced stability of MR1 on the cellsurface; we suggest this can be through binding a range of ligandsthat are suitable for docking in the cavity of MR1, and second,through presentation of compounds that can provide additionalcontacts to the MAIT TCR, such as our modeled conformationsof DMRL and rRL-6-CH2OH.Although DMRL and rRL-6-CH2OH have been shown to in-

duce MAIT cell stimulation, we propose that there are additionalclasses of endogenous and exogenous antigens that may stabilizeMR1 expression on the cell surface and may also provide addi-tional contacts to the MAIT TCR through position Y95α. Thesecompounds may be acidic in nature (electrostatically compatiblewith the basic MR1 groove) and aromatic, such as what is ob-served with DMRL, rRL-6-CH2OH, and other derivatives alsoshown to stimulate MAIT cells.Self/nonself recognition by T cells is a key feature of immunity;

this includes self/foreign peptide recognition (via classical MHCmolecules) by classical αβ T cells and lipid recognition by CD1-restricted T cells. However, the closest analogy to MAIT cellrecognition of small molecule metabolites presented byMR1maybe detection of metabolite phosphoantigens by human Vγ9Vδ2 Tcells, whereby both foreign and self metabolites can provide po-tent stimulatory signals to this T-cell lineage. Although the mo-lecular mechanisms behind this activation process have onlybegun to be elucidated (27, 28), it is testament to the importanceof metabolic processes to immune assessment of the health of thecell. It may very well be thatMAIT surveillance is based on similarendogenous and exogenous metabolite profiling.

Materials and MethodsRecombinant MR1 Expression and Purification. The cDNAs corresponding tothe ectodomain region of the bovine β2m and bovine MR1, linked througha glycine-serine flexible linker, were cloned in-frame with a C-terminal 6- or12xHis tag into the pAcGP67A vector (BD Biosciences). This single-chainbovine β2m-Gly-Ser-MR1 construct (scMR1) was expressed in Hi5 cells viabaculovirus transduction. The recombinant protein was first captured fol-lowing addition of Nickel NTA Agarose (Qiagen) and then subjected to an-ion exchange chromatography in a MonoQ column (GE Healthcare). Forcrystallographic purposes, the protein was then treated with Carboxypep-tidase A (Sigma) as previously described (29), followed by a final step of size-exclusion chromatography.

Recombinant MAIT TCR Expression and Purification. The cDNA samples for theMR1-reactive MAIT TCR clones F7, G2, and AE6 were synthesized via over-lapping PCR from sequences reported by Tilloy et al. (1). These cDNAs weremodified to bear T48C and S57C mutations at the α- and β-constant chaindomains, respectively, and were separately cloned into different versions ofthe pAcGP67A. Each chain contains a C-terminal 3C protease site followed byeither acidic or basic zippers and a 6xHis tag. For each clone, both α- andβ-chains were coexpressed in Hi5 cells via baculovirus transduction. Theheterodimeric TCRs were captured with Nickel NTA Agarose and furtherpurified by anion exchange and size-exclusion chromatography. For SPRstudies, the purified TCRs were treated with 3C protease and the digestedsample loaded onto a Nickel NTA agarose column. The His-tag–free fractionwas collected from the flow-through and used as analyte for SPR. For crys-tallization purposes, the MAIT TCR chains were separately expressed inE. coli as inclusion bodies and then each clone refolded, treated with Car-boxypeptidase A and purified as previously described (29).

Generation of MR1 and MAIT TCR Mutants. The scMR1 mutants: A72M, R147Q,E149A, Q151L, and the triple mutant, A72M/R147Q/Q151L were generatedthrough overlapping PCR with specific primers containing the desiredmutations. This mutant was expressed in insect cells as described for the wild-type counterpart.

MR1 and MAIT TCR Biophysical Interaction Analysis. The 12xHis tag scMR1 orthe Q151L mutant were captured to a stable level of 1,500 RUs on the surfaceof a NiCl2-treated NTA sensor chip (GE Healthcare). Insect cell-derivedrecombinant FC was captured in the flow channel 1 to subtract nonspecificbinding events. Increasing concentrations (0.625, 1.25, 2.5, 5, 10, 20, and40 μM) of each TCR clone were injected at a flow rate of 30 μL/min using 10mM Hepes pH 7.4, 150 mM NaCl, and 0.005% Tween-20 as running buffer.The traces of the reference-subtracted binding signals were plotted andGraphPad Prism was used to determine the affinity constants for the inter-actions with the immobilized MR1.

All interaction analyses of the MR1 mutants were carried out in real time bybio-layer interferometry in a Blitz System Package (Fortebio). The 12xHis scMR1or mutants of it were captured to a stable level of 4 ηmunits on a Ni-NTA (NTA)Biosensor.A seriesofMAITTCR concentrations ranging from0.37 to90 μMwereran over the immobilized MR1 protein in 10 mM Hepes pH 7.4, 150 mM NaCl,and 0.005%Tween-20 and the association and dissociation binding traces wererecorded. FC was used as a “nonbinding” control partner. Responses frommeasurements with FC were subtracted from those with MR1 and in-teraction affinity Kds were calculated with GraphPad Prism by plotting thebinding values at equilibrium against the TCR concentrations.

Ternary Complex Formation and Crystallization. Equimolar amounts of refol-ded F7MAIT TCR and scMR1weremixed and concentrated to 8.5mg/m. Initialcrystals were found in 0.1 M [4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid] pH 7.0 and 1.5 M ammonium sulfate. These crystals were crushed andused for microseeding fresh drops. This procedure yielded single crystals thatwere used for data collection.

Crystallographic Data Collection, Structure Determination, and Refinement.Crystals were cryo-cooled in mother liquor supplemented with 20% (vol/vol)glycerol before data collection. All datasets were collected on a MAR300 CCDat beamline 23 ID-D at the Advanced Photon Source at the Argonne NationalLaboratory and processed with HKL2000 (30). The structure of the ternarycomplex was solved by molecular replacement with the program Phaser (31)and using as search model entities the coordinates of the ligandless HLA-B*4103 (PDB ID code 3LN4) for β2m-MR1 and the previously solved MAITTCR coordinates (PDB ID code 4DZB) with the CDR loops omitted for the F7MAIT TCR. Refinement was accomplished with Phenix software suite (32) and

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Refmac5 (33) by initially dividing the molecules into rigid bodies and sub-sequent jelly-body and restrained refinement. Next, extensive cycles of manualbuilding in Coot (34) and restrained refinement were carried out and missingregions were built in accordance to the electron density maps. PRODRG (35)was used for the generation of the ligand-modified lysine in MR1 and includedin subsequent refinement and manual building steps. All of the refinementprocedure was performed taking a random 5% of reflections and excludingthem for statistical validation purposes (Rfree).

Structure Analysis. Intermolecular contacts and distances were calculated usingthe program Contacts from the CCP4 software package (36), interface surfaceareas were calculated using the PISA server (www.ebi.ac.uk/msd-srv/prot_int/pistart.html), and all structural figures were generated using the programPymol (Schrödinger). Coordinates and structure factors for the MAIT TCR–scMR1complex have been deposited in the Protein Data Bank under the ID code 4IIQ.

Docking of Stimulatory Ligands. Autodock Vina v1.1.2 (37) was used toperform docking calculations on the ligands DMRL, rRL-6-CH2OH, and 6-FPto the complex structure. Because of the stochastic nature of AutodockVina, we ran each ligand calculation 10 times, yielding the same top scoringstructure for each ligand in 8 of the 10 runs. Vina was run using an x, y, z boxsize of 24, 18, 28 centered at x, y, z coordinates −17.1, 37.0, 38.6 with flexibleresidues. The flexible residues were TYR7, ARG9, SER24, LYS43, TYR62,ARG94, and TRP156 on MR1 (chain C) and Y95 on the MAIT TCR α-chain(chain A). All other Vina parameters were set to the default.

ACKNOWLEDGMENTS.We thank the staff of the Advanced Proton Source atGM/CA-CAT (23ID) for their use and assistance with X-ray beamlines; andRuslan Sanishvili, Steven Corcoran, and Michael Becker in particular for helpand advice during data collection. This study was supported by NationalInstitutes of Health Grant R01AI073922 (to E.J.A.).

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