Evidence for MR1 Antigen Presentation to Mucosal-associatedInvariant T Cells*

Received for publication, January 31, 2005, and in revised form, March 9, 2005Published, JBC Papers in Press, March 31, 2005, DOI 10.1074/jbc.M501087200

Shouxiong Huang‡, Susan Gilfillan‡, Marina Cella‡, Michael J. Miley‡, Olivier Lantz§,Lonnie Lybarger‡¶, Daved H. Fremont‡, and Ted H. Hansen‡�

From the ‡Department of Pathology and Immunology, Washington University, St. Louis, Missouri 63110, §Laboratoired’Immunologie and INSERM U520, XCInstitut Curie, 26 rue d’Ulm, Paris, France, and ¶Department of Cell Biology andAnatomy, University of Arizona Health Sciences Center, Tucson, Arizona 85724-5044

The novel class Ib molecule MR1 is highly conservedin mammals, particularly in its �1/�2 domains. Recentstudies demonstrated that MR1 expression is requiredfor development and expansion of a small population ofT cells expressing an invariant T cell receptor (TCR) �chain called mucosal-associated invariant T (MAIT)cells. Despite these intriguing properties it has beendifficult to determine whether MR1 expression andMAIT cell recognition is ligand-dependent. To addressthese outstanding questions, monoclonal antibodieswere produced in MR1 knock-out mice immunized withrecombinant MR1 protein, and a series of MR1 muta-tions were generated at sites previously shown to dis-rupt the ability of class Ia molecules to bind peptide orTCR. Here we show that 1) MR1 molecules are detectedby monoclonal antibodies in either an open or foldedconformation that correlates precisely with peptide-in-duced conformational changes in class Ia molecules, 2)only the folded MR1 conformer activated 2/2 MAIT hybri-doma cells tested, 3) the pattern of MAIT cell activation bythe MR1 mutants implies the MR1/TCR orientation isstrikingly similar to published major histocompatibilitycomplex/��TCR engagements, 4) all the MR1 mutationstested and found to severely reduce surface expressionof folded molecules were located in the putative ligandbinding groove, and 5) certain groove mutants of MR1that are highly expressed on the cell surface disruptMAIT cell activation. These combined data strongly sup-port the conclusion that MR1 has an antigen presenta-tion function.

Classical MHC1 class I (or class Ia) proteins are highly poly-morphic, expressed on all nucleated cells, and have well de-fined peptide presentation functions (1). By comparison, non-

classical class I (or class Ib) typically have more limitedpolymorphism, more restricted tissue expression, and morediverse functions (2, 3). Interestingly, however, certain class Ibproteins have specialized antigen presentation functions suchas human HLA-E and mouse Qa-1 that present signal peptidesof other class I molecules to T cells or NK cells (2). Alterna-tively, mouse and human CD1 present glycolipid ligands to T orNK-T cells (4). However, other class Ib molecules such as HFEand ZAG have non-immunological functions, and still otherslike MR1 have unknown functions. Despite these functionaldifferences, sequence comparisons and recent crystallographicstudies indicate that class Ib proteins are structurally verysimilar to class Ia proteins (5–7).

Lymphocytes with restricted repertoires such as B1 B cells,some ��T cells, and NK-T cells that express autoreactive, in-variant antigen receptors have been called innate lymphocytes(8). The restricted repertoires of these cells may allow them torapidly respond to phylogenetically conserved antigens (9). Al-ternatively, lymphocytes with restricted repertoires may haveregulatory roles dependent upon the recognition of self-ligands.Until recently, NK-T cells were the only known T cell subsetwith an invariant TCRs conserved between mouse and man,perhaps reflecting an ancient and important physiologicalfunction (9, 10). More specifically, most mouse NK-T cells ex-press an invariant TCR� V-J junction (V�14-J�18) with aCDR3 of constant length paired with limited V� segments (11,12). Unlike conventional T cells, their development is not al-tered in TAP�/� mice. Consistent with this, NK-T cells recog-nize glycolipids such as �-galactosyl ceramide presented by theMHC class Ib molecule CD1d. It is noteworthy, however, thatthe endogenous ligand presented physiologically by CD1d wasdifficult to identify (13, 14). NK-T cells are thought to bridgeinnate and adaptive immune responses by secreting largeamounts of cytokines, particularly IL-4, upon stimulation, andNK-T cells have been implicated in T cell polarization, tumorrejection, and autoimmunity (15–17).

A second type of T cell with an invariant TCR was recentlydefined and, because they preferentially home to the gut mu-cosa, were named MAIT (mucosal-associated invariant T) cells(18, 19). Like most NK-T cells, MAIT cells express a TCR�chain encoded by a specific V�-J� rearrangement with a CDR3segment of constant length and minor sequence diversity thatpreferentially pairs with a limited number of V� segments (9).More specifically, the canonical sequence of the MAIT cell TCR(iV�7.2/19) is encoded by hV�7.2-J�33 in humans and thehighly homologous mV�19-J�33 in mouse and cattle. MAITcells reside within the CD4�CD8� T population in human,mice, and cattle as well as the CD8��� subset in humans (18).Like NK-T cells, MAIT cells are selected on hematopoietic cellsin a TAP-independent, �2m-dependent manner. Also similar to

* This work was supported by National Institutes of Health GrantsAI46553 and AI19687. The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

� To whom correspondence should be addressed: Dept. of Pathologyand Immunology, WA University School of Medicine, 4566 Scott Ave.,St. Louis, MO 63110. Tel.: 314-362-2716; Fax: 314-362-4137; E-mail:hansen@pathology.wustl.edu.

1 The abbreviations used are: MHC, major histocompatibility com-plex; class Ia, classical MHC class I proteins such as mouse Ld, Kd, andKb; class Ib, non-classical class I MHC proteins such as mouse CD1d,T22, M3, Qa1, or human CD1d and HLA-E; MR1, MHC-related protein1; hMR1, human MR1; mMR1, mouse MR1; NK-T, natural killer T cell,mAb, monoclonal antibody; TCR, T cell receptor; CDR3, complementa-rity determining region 3; �� versus ��, chains of alternative TCR; MFI,mean fluorescent intensity; TAP, transporter associated with antigenprocessing; IL, interleukin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 22, Issue of June 3, pp. 21183–21193, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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NK-T cells, MAIT cells are selected/activated by a class Ibmolecule. As recently shown by Treiner et al. (19), the devel-opment and activation of MAIT cells is dependent on the mono-morphic class I-related molecule MR1, which is remarkablyconserved among mammals (indeed more conserved thanCD1d) (20–22). In addition to MR1, MAIT cell development isdependent upon B cells and commensal flora, properties notshared with NK-T cells (19, 23). In regard to tissue distribu-tion, MAIT cells accumulate in the mucosal system, whereasNK-T cells are abundant in internal organs like the spleen andliver (9).

Properties of MAIT cells unique or shared with NK-T cellshave led to speculation about MAIT cell function (9). For ex-ample, based on their preferential accumulation in the gutmucosa, it was proposed that MAIT cells could contribute to thediscrimination between pathogens and commensals or be in-volved in a negative feedback loop to regulate IgA secretion,which plays a critical role in controlling the gut microbial flora.MAIT cells might also interact with dendritic cells, which areabundant in the mucosal lamina propria and are critical forantigen presentation during bacterial infections (24). Thus,resolving MAIT cell function could provide key insights intohow the immune system maintains the balance between toler-ance and immune responses in mucosal tissues.

Formidable obstacles must be overcome to test the validity ofany hypotheses regarding MAIT cell function. MAIT cells arerare and represent around 2% of CD4�CD8� lymph node Tcells but are somewhat more abundant in the intestinal mucosaof B6 mice. So far attempts have failed to generate MAIT cellclones from primary cells,2 but fortunately, MAIT T-T hybrido-mas were obtained for functional studies (9, 18). A number ofMAIT hybridomas were selected based on their expression ofthe TCR� canonical sequence, and some of these were shown tobe specifically activated by cells expressing MR1 by transfec-tion (9, 18). In addition to difficulties in obtaining MAIT cells,MR1 poses its own challenges for investigation. Most notably,endogenous expression of MR1 has not been defined, and thusfar there is no direct evidence that MR1 binds a ligand and, ifso, what its chemical nature might be.

Cells transfected or transduced with mMR1 cDNA wereshown to express low levels of MR1 on the surface; however,most MR1 protein remained intracellular (3). These findingsare consistent with, but not evidential of ligand limiting MR1expression. Furthermore, the low level of MR1 protein detectedon the cell surface after transfection/transduction was aug-mented by swapping the �3 domain of mMR1 with that of theclassical class I molecule H-2Ld (3). Biochemical characteriza-tion of insect cell expressed recombinant MR1 protein demon-strated stoichiometric association with �2m and provided evi-dence for N-linked glycosylation analogous to that found in allclass Ia molecules (3). These MR1 expression studies usedeither (i) anti-epitope tag mAb 64-3-7 that is specific for “open”(not associated with ligand) forms as shown with class Ia mol-ecules and H2-M3 (25–28) or (ii) mAb 4E3 generated in �2m-deficient mice immunized with a peptide derived from the �2domain of mMR1. Given this, we questioned whether ligandassociated or folded MR1 was missed in our detection system.Indeed, a soluble ectodomain of recombinant MR1 was secretedby insect cells, suggesting that it was folded. And this recom-binant MR1 was detected by mAb 64-3-7 only after denatur-ation, consistent with MR1 folding into a 64-3-7 negative con-former, analogous to class Ia molecules when they undergopeptide-induced folding (3). However, we have thus far been

unable to define a conventional peptide ligand bound to recom-binant MR1.2 Related to this uncertainty regarding an MR1ligand is the question of whether MAIT cell activation isligand-dependent.

To better characterize MR1 function in inducing MAIT acti-vation and the possible requirement for a potential ligand, wegenerated new mAbs against MR1 by immunizing MR1 knock-out mice with recombinant MR1 protein. Importantly thesenew mAbs to MR1 detected a folded conformer of MR1 missedin previous investigations. These new mAbs were used to showthat only folded MR1 activated both MAIT cells tested. Fur-thermore, a panel of MR1 mutants were used to (i) indicate thatthe TCR of MAIT cells engages MR1 in a very similar orienta-tion as that previously reported for ��TCR/MHC engagementsand (ii) make predictions about how MR1 interacts with aputative ligand that controls its expression and MAIT cellactivation.

EXPERIMENTAL PROCEDURES

Cell Lines—The B6 (H-2b) embryonic fibroblast WT3 (29) and TAP1-deficient line (25, 30) were used for retroviral transduction. MouseMR1-transfected WT3 (WT3.mMR1) was previously described (3) aswere the isolation and characterization of MAIT T-T hybridoma cells6C2 and 8D12 (18). The IL-2-dependent cell line CTLL-2 (31) was usedto test IL-2 secreted by the MAIT hybridomas. All cells were maintainedin RPMI 1640 or DMEM (Invitrogen) media supplemented with 10%fetal calf serum (HyClone, Logan, UT), 2 mM glutamine, 1 mM sodiumpyruvate, 0.1 mM nonessential amino acids, 1.25 mM HEPES, and 100units/ml penicillin/streptomycin.

Gene Cloning and Retroviral Transduction—A biscistronic retroviralvector (pMSCV.IRES.Neomycin, pMIN) encodes the gene of interestfrom the upstream cistron and antibiotic resistance gene from thedownstream cistron (32). Generation of the epitope-tagged mMR1 andthe chimeric mMR1/Ld molecule (mMR1 (�1-�2 domain)/Ld (�3-cyto-plasmic tail) was previously reported (3). Genes encoding mMR1 andmMR1/Ld in the vector pIRES.neo (3) were cut with restriction enzymesand inserted into the multicloning sites of retroviral vector pMIN. Theinserted gene was confirmed with the BigDye terminator cycle sequenc-ing kit (ABI, Foster City, CA). Retrovirus-containing supernatants weregenerated using the Vpack vector system (Stratagene, La Jolla, CA) fortransient transfection of 293T cells (33) to produce ecotropic virus forthe infection of WT3 cells. Packaging cells were transfected using Fu-GENE 6 (Roche Applied Science). Virus-containing supernatants werecollected 48–72 h post transfection, and 2–3 ml was added to targetcells (�106) per infection. Cells were selected and maintained under 0.6mg/ml Geneticin (Invitrogen) or 0.3 mg/ml hygromycin (Sigma) post-transduction. These transduced cells were maintained as a single pop-ulation with stable expression of the targeted protein for more than 6months in culture.

mAb—mAb 4E3 was produced in �2m-deficient mice immunizedagainst a peptide corresponding to MR1 residues 130–153 (3). mAb64-3-7 is an epitope tag specific for open forms of class I molecules (27).mAb B8–24-3 (ATCC, Manassas, VA) detects folded H-2Kb, and mAb30–5-7 (26) detects folded H-2Ld. To produce new mAbs to MR1, MR1-deficient mice (19) were immunized with the soluble ectodomain ofinsect-expressed human MR1/�2m complexes (3). Hybridomas weregenerated by fusing splenocytes with Sp2/0 cells, and supernatantswere initially screened by enzyme-linked immunosorbent assay onplates coated with either hMR1/h�2m or h�2m alone. Those clones thatrecognized hMR1/�2m but not h�2m were then screened by flow cytom-etry using transfectants expressing hMR1 or mMR1 (3).

Sequential Immunoprecipitations and Western Blots—For sequen-tial immunoprecipitation, mMR1-transfected WT3 cells (107) werelysed in phosphate-buffered saline, pH 7.4, with 1.0% digitonin(Wako, Richmond, VA), 20 mM iodoacetamide (Sigma), 0.2 mM phen-ylmethylsulfonyl fluoride (Roche Applied Science) for 1 h on ice.Non-transfected WT3 cells were used as a control. Saturatingamounts of anti-MR1 mAbs 12.2, 26.5, 4E3, or epitope tag mAb 64-3-7were incubated with protein G-Sepharose 4 fast flow (AmershamBiosciences) for 2 h at 4 °C with rocking. The anti-Kb mAb B8–24-3was used as a negative control for pre-clearance. After washing withphosphate-buffered saline twice, antibody-bound protein G was incu-bated with post-nuclear lysates for 4 h. To assure complete removal ofreactive MR1 molecules the supernatants were subjected to a secondpre-clearance with protein G bound by the same antibodies. For the

2 S. Gilfillan, O. Lantz, D. H. Fremont, and T. Hansen, unpublishedobservations.

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third incubation, the same or an alternative antibody was used to testfor the remaining mMR1 proteins in each supernatant. mAb-coatedbeads were washed four times in phosphate-buffered saline contain-ing 0.1% digitonin, and proteins were eluted by boiling in lithiumdodecyl sulfate sample buffer (Invitrogen), and 2-mercaptoethanolwas added to a final concentration of 1%. Samples were analyzed onSDS-PAGE as described (34). For Western blots, biotinylated 64-3-7was used for MR1 detection. The staining signal was visualized by thechemiluminescence substrate using the ECL system (AmershamBiosciences).

Site-directed Mutagenesis—The mMR1/Ld site-mutation constructsin retroviral vector pMIN were generated using the QuikChange II XLsite mutagenesis kit (Stratagene) according to the manufacturer’s in-structions. For each mutation eight clones of carbenicillin (100 �g/ml)-resistant bacteria were screened by DNA sequence analyses for theexistence of the desired mutations only. Confirmed constructs wereused for virus production and transduction of WT3 cells.

Flow Cytometry—For surface staining, 106 cells per sample wereincubated on ice in microtiter plates with a saturating concentration ofmAb. After washing, phycoerythrin-conjugated goat anti-mouse IgG(BD Pharmingen) was used to visualize the primary antibody staining.Intracellular MR1 molecules were stained with fluorescein isothiocya-nate 64-3-7 as described (3). Flow cytometric analyses were performedusing a FACSCalibur (BD Biosciences). Data were analyzed usingCellQuest software (BD Biosciences).

MAIT Cell Activation and mAb Blocking—Biological activity of IL-2secreted by MAIT T-T hybridoma lines 8D12 and 6C2 was used toestimate the degree of MAIT cell activation. 105/ml mMR1/Ld-trans-duced WT3 cells were cultured with 106/ml hybridoma cells in completeRPMI 1640 medium. Purified anti-mMR1 mAbs were added at differentconcentrations. Supernatants were harvested and frozen at �80 °C forat least 1 h to lyse trace cells that may have carried-over. CTLL-2 cellswere washed and added to supernatants at a final cell density of 5 �103/200 �l/well in a 96-well plate. Triplicate wells were run for eachsample. CTLL-2 cells in the absence of culture supernatant were usedas the negative control, and the addition of serial IL-2 dilutions wasused as positive control. After 18–24 h of incubation, Alamar blue(BioSource international, Carmarillo, CA) was added at 20 �l/well, andrelative amounts of IL-2 in each supernatant were determined byfluorescence on a multi-detection microplate reader (Bio-Tek instru-ments, Winooski, VT).

RESULTS

mAbs Detect Two Distinct MR1 Conformers, Analogous to theLigand Empty (Open) and Ligand-associated (Folded) Class IaMolecules—We previously reported low levels of surface ex-pression of MR1 by transfection or transduction using theepitope tag mAb 64-3-7 that is specific for non-ligand associ-ated class heavy chains, H2-M3 (25–28). Furthermore, we pro-duced anti-mouse MR1 mAb 4E3 by immunizing �2m-deficientmice with a peptide derived from mouse MR1 residues 130–153(3). As expected, both of these mAbs detect denatured mouseMR1 as determined by Western blotting. Thus, we questionedwhether we might be missing a population of folded MR1 mol-ecules not detected by either mAb 64-3-7 or 4E3. To purpose-fully generate mAb to folded MR1, MR1 knock-out mice wereimmunized with the secreted recombinant ectodomain of hu-man MR1/�2m (3). Hybridoma clones reactive with MR1 werescreened and subcloned. Of the eight new anti-MR1 mAbsisolated, five were specific for human MR1 (hMR1), and three(mAbs 12.2, 26.5, and 4) cross-reacted with mouse MR1(mMR1) as determined by their staining profile on MR1 trans-fectants (Fig. 1A). The mAbs reactive with mMR1 were selectedfor characterization in this report. To test whether detection ofmMR1 by mAbs 12.2, 26.5, and 4 was conformation-dependent,each mAb was used to precipitate MR1 protein from theWT3.mMR1 cells. These precipitates were then blotted withbiotinylated mAb 64-3-7, demonstrating that each mAb precip-itated mMR1 protein. However, it should be noted that thesignal with 12.2 and 26.5 was consistently stronger than with4, suggesting mAb 4 is of lower affinity (data not shown).Furthermore, none of these new mAbs to MR1 reacted withdenatured MR1 protein by Western blotting. Thus mAb 12.2,

26.5, and 4 all appear to be conformation-dependent in theirdetection of MR1.

Our laboratory previously demonstrated that class Ia pro-teins and the class Ib protein H2-M3 are detected as alterna-tive conformers distinguished by their association with ligand(26–28,35). More specifically, nascent class I heavy chainstransition from an open conformer to a folded conformer whenthey bind a high affinity peptide in the endoplasmic reticulum,and reciprocally, surface class I heavy chains transition from afolded conformer to an open conformer after peptide dissocia-tion (36). Thus, it was of interest to determine whether thisparadigm also applied to MR1. Open conformers in these afore-mentioned studies were detected by mAb 64-3-7, and foldedconformers were detected by conformation-dependent, allele-specific mAbs. To determine whether MR1 also exists as alter-native open versus folded conformers, sequential immunopre-cipitation experiments were performed using mAb 64-3-7 andthe new conformational-dependent mAbs to MR1. A lysate ofWT3.mMR1 cells was precleared with a negative control anti-body (B8–24-3), the epitope tag mAb 64-3-7, or mMR1-reactivemAbs 12.2 and 26.5. Supernatants from each of these pre-cleared antigen preparations were then tested with the same oralternative mAb. As shown in Fig. 1B the pattern of reactivityin the sequential immunoprecipitation experiment was strik-ing and defined two MR1 conformers, 64-3-7� (12.2, 26.5)� and64-3-7� (12.2, 26.5)�. Evidence that both mAbs 12.2 and 26.5detect the same conformer was demonstrated by the fact thatthey cleared for each other. Although the apparent lower affin-ity of mAb 4 precluded its use as a clearance reagent, mAb4-reactive MR1 proteins were removed by both 12.2 and 26.5but not 64-3-7 (data not shown). Therefore, all three new anti-MR1 mAbs 12.2, 26.5, and 4 are conformation-dependent anddetect an MR1 conformer distinct from mAb 64-3-7. Given thatthese new mAbs were raised against folded MR1 protein andthe clear parallel findings with class Ia molecules and H2-M3(26–28, 35), it was concluded that mAbs 12.2, 26.5, and 4 alldetect the folded MR1 conformer.

Both Open and Folded Conformers of MR1 Are Expressed onthe Cell Surface but Only Folded MR1 Activates MAIT Cells—Given that the new mAbs 12.2, 26.5, and 4 detect a conformerof MR1 previously not detected by mAbs 4E3 and 64-3-7, it wasof interest to reevaluate MR1 surface expression. As shown inFig. 2A, mMR1-transduced cells express low levels of open(64-3-7�) MR1 as previously reported. However, these samecells expressed substantially higher levels of folded (12.2�)MR1. Comparable staining was observed with mAb 26.5 (datanot shown). Interestingly, higher levels of surface expressionwere obtained by transduction with a chimeric gene consistingof the mMR1 �1/�2 domains connected to the �3 transmem-brane and cytoplasmic domains of Ld (3) (Fig. 2A). Like nativeMR1, the chimeric molecule was detected as a folded conformerat higher levels than as an open conformer. It is of interest tocompare these findings with conformers of Ld, a class Ia proteinthat is a relatively poor peptide binder (37). Intact MR1, chi-meric MR1/Ld, and intact Ld have a steady state surface ex-pression of 70–90% folded conformer. By contrast, other classIa molecules such as Kb and Kd have higher percentages offolded conformers indicative of better overall ligand binding(28). In any case, surface MR1 is detected as alternative conform-ers similar to class Ia molecules where it has been establishedthat the open conformer arises after ligand dissociation (26).

The existence of alternative conformers of MR1 on the cellsurface raised the interesting question of which conformeractivates MAIT cells. To address this question, different anti-MR1-reactive mAbs were used to block MAIT cell activation.Two MAIT cell hybridomas, namely 8D12 and 6C2, were used

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in the current report (18, 19). WT3 cells expressing eitherintact mMR1 or the mMR1/Ld were found to activate both8D12 and 6C2 MAIT hybridomas (not shown). However, thecells expressing the mMR1/Ld chimeric molecule were consis-tently better stimulators of MAIT cells and, therefore, wereused for this antibody blocking study. As shown in Fig. 2B,mAbs 12.2 and 26.5 displayed a dose-dependent inhibition ofMAIT cell activation resulting in complete blockage at 10�g/ml. By contrast, neither mAb 4E3 or 64-3-7 inhibitedMAIT cell activation (Fig. 2B). Similar findings were seenwith both MAIT hybridomas in multiple assays. Thus, thefailure of mAb to the open MR1 to block MAIT cell activationand more importantly the complete blocking by mAb to thefolded MR1 indicate that only the folded MR1 conformeractivates these two MAIT cell hybridomas.

Mutagenesis Strategy—The high degree of sequence similar-ity of MR1 with classical class Ia proteins (�40% identity in�1/�2) readily enabled us to design a structure-based mutagen-esis strategy to investigate the antigen presentation function ofMR1. To identify sequences interacting with TCR or putativeligand, we mutated 23 residues in the �1/�2 domains of MR1.These residues are at positions corresponding with ones previ-ously implicated in class Ia interaction with peptide or TCR bymutagenesis or crystallographic studies (38–49) (Fig. 3A).Most residues (17 of 23) were replaced with amino acids locatedin the corresponding positions in class Ia molecules so as not todisrupt overall structures. Four MR1 mutations (Y7L, H59A,W159A, F171A) were made at positions where all class Iaheavy chains have invariant tyrosines involved in A pocketanchoring of the peptide N terminus (50). And three MR1

FIG. 1. mAbs to mMR1 detect twoalternative mMR1 conformers. A, newmAbs were generated to recombinanthMR1 in MR1 knock-out mice, and theirspecies specificity was determined bytesting on cells expressing either hMR1(upper panels) or mMR1 (lower panels). Ofthe eight mAbs that displayed strongstaining with MR1, five were hMR1 spe-cific, whereas three mAbs (designated 4,12.2, and 26.5) cross-reacted stronglywith mMR1. Profiles representative ofeach type of new mAb (shaded) are shownwith isotype control antibodies (un-shaded). B, sequential precipitation wasperformed by preclearing a cell lysate ofWT3.mMR1 with mAbs listed along theleft side of the figure. Each preclearedlysate was then tested with mAbs listedalong the top. Eluted samples were run onSDS-PAGE and blotted with biotinylatedmAb 64-3-7 under denaturing conditionsto detect MR1. mAb B8–24-3 (anti-foldedKb) was used as a negative control mAb(Ctl) for preclearance. Similar sequentialprecipitations were performed multipletimes with consistent findings.

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mutations (H80T, H84A, A146K) were made at positions whereclass Ia molecules have conserved threonine, tyrosine, or lysineresidues, respectively, involved in F pocket anchoring of thepeptide C terminus (50). Each of the 23 MR1 mutants wasexpressed by transduction as a MR1/Ld chimeric molecule inWT3 cells to achieve the highest level of functional MR1 ex-pression. Cells expressing each mutant were then tested forsurface MR1 expression and its ability to activate MAITcell hybridomas.

MR1 Mutants Predict a TCR Engagement Similar to That ofMHC/Peptide—To identify residues of MR1 that are requiredfor MAIT cell activation, MR1 mutants in helical residues withhigh levels of surface expression of folded molecules were con-sidered relevant based on our above findings. Fortunately wehad three mAbs for these analyses, mitigating problems ofmutations affecting antibody detection. Indeed, as noted in Fig.3, B and C, mutant Y155R ablated detection of MR1 by mAbs12.2 and 26.5 but not by mAb 4. Thus, to assess expression of

FIG. 2. Surface expression of and MAIT cells activation by open and folded MR1 conformers. A, comparison of folded versus openconformers of mMR1/Ld, mMR1, and Ld. B6/WT3 cells transduced with mMR1/Ld, mMR1, and Ld were stained with mAb 64-3-7 (epitope tag specificfor open forms) as shown in the upper panels. Alternatively mAb 12.2 (folded MR1) or mAb 30–5-7 (folded Ld) was used for staining cells shownin the lower panels. % folded is a relative comparison based on the calculation folded/folded � open � 100. Negative control (thin line) representssecondary reagent only (comparable findings were also obtained using an isotype-matched negative control mAb). Mean fluorescent intensity (MFI)minus the negative control is shown above each peak. Surface expression was tested numerous times and remained constant. B, the mMR1/Ld-transduced WT3 cells were incubated with MAIT T-T hybridoma line 6C2 for 24 h with or without different concentration of listed mAbs. Theamount of IL-2 in samples was determined using IL-2-dependent CTLL-2 cells, and fluorescence of alamar blue product was used to quantify theamount of proliferation. Percent activity was determined after subtracting the background of CTLL-2 cells alone and then dividing by readings ofsamples without antibody. Background levels caused by non-transduced WT3 cells without additional antibody are indicated as a thin line. Similarfindings were obtained using MAIT cell line 8D12.

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FIG. 3. Effects of mutations on mMR1 surface expression and MAIT activation. A, the predicted MR1 structure is imposed upon atemplate of the Kb �1/�2 domains (81) with the accommodations of minor deletions (red) and additions (blue). MR1 mutations characterized hereare indicated by purple spheres (helical residues), green spheres (�-sheet residues), and yellow spheres (terminal ligand anchor residues). B,analyses of MFI of mMR1 mutants. Residues are shown as the same color scheme in Fig. A. MFI of mMR1 surface expression is normalized onnegative controls, and the ratio to wild type (MFImutant/MFIwildtype) is shown. Shaded areas highlight folded mMR1 with the ratio to wild type �0.5.C, surface expression of seven of the MR1 mutants (red line) compared with wild type (green line). The black line is the negative control andrepresents the secondary reagent only. Consistent findings were seen in several different assays. Note all MR1 mutants have similar levels ofexpression of MR1 as detected by conformation-independent mAb 4E3. This finding supports the fact that retrovirus expression results incomparable levels of expression. By contrast, all mutants shown except R9Vand A149Q displayed significant differences as detected by theconformation-dependent mAbs 12.2, 26.5, and 4. As discussed, these differences are important for mapping epitopes and defining the role of a

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folded MR1, we considered mutations detected at �60% of thewild type level by at least one of the mAbs to folded MR1. Thislevel of expression was determined in functional assays to bewell above the level required for MAIT cell activation (data notshown). As expected from studies of class Ia molecules, mostMR1 mutations in alleged helical residues (13/16) were ex-pressed at high levels as folded MR1 proteins. The three ex-ceptions were MR1 mutations H59A, W159A, and F171A, alllocated at positions in class Ia molecules involved in anchoringof the N terminus of the peptide. Thus, to avoid ambiguitiesbetween peptide binding and TCR contacts, we excluded allMR1 positions where class Ia molecules have residues involvedin terminal peptide binding (these are considered as a group inthe last section of “Results”). Of the remaining 11 MR1 mu-tants in helical residues, 4 (L66K, G69R, A166E, and Y155R)ablated activation of both MAIT cell hybridomas, and anothermutant (K75R) sharply reduced activation of both MAIT cell

hybridomas (Fig. 4A). Furthermore, mutations A76V and Q82Ractivated the 6C2 hybridoma significantly better than the8D12 hybridoma, suggesting either a subtle difference betweenthese two hybridomas in affinity for, or orientation with, MR1.In any case, these MR1 mutations that ablate MAIT cell acti-vation are at positions previously shown in mutagenesis stud-ies of several different mouse and human class Ia molecules toaffect TCR interaction. Although co-crystallographic studieshave demonstrated some variation in the orientation that TCRengages MHC/peptide, the MR1 mutations that ablate MAITcell activation clearly reside within the collective TCR inter-faces (Fig. 3D). More specifically, the TCR mutagenesis foot-print on MR1 is concordant with the co-crystallographic foot-print of the N15 or 2C TCRs bound to Kb/cognate peptides (39,48). This finding strongly suggests that the �1/�2 antigen bind-ing platform of MR1 has an analogous interaction with TCR asMHC-peptide complexes (38–49).

putative MR1 ligand in the expression of a folded MR1 conformer. D, MR1 mutants predict TCR orientation and the role of ligand in MAIT cellactivation. Location of mMR1 mutants that affect MAIT activation are shown on the H-2Kb surface-accessible model (generated using softwareGRASP) (81). The pink indicates helical mutations with surface expression �60% but not activating MAIT cells (the mutant K75R may slightlyactivate 6C2), except that mutant A76V and Q82R display the different degree in activating two MAIT cell lines. The green area designates sitesof groove mutations expressed as folded molecules at the surface at levels that are sufficiently high enough to activate MAIT cells but that do not.Enclosed by the dotted line is an area that represents TCR binding area. The line is drawn around the C� atoms of the 4 TCR contacting the MHCresidues, which have been previously defined (38–43, 45).

FIG. 4. MAIT cell activation by MR1 mutants. A, MAIT hybridoma cell activation by mMR1 mutations of residues in the presumed helicalregions of the �1/�2 domains. B, MAIT interaction with mMR1 mutations corresponding to residues involved in the ligand binding by class Iamolecules. Only MR1 mutants expressed at �0.6 of wild type are shown to obviate concerns of low level expression precluding MAIT cell activation.Accordingly, mutants F22Y, Y95I, and L114Q are not shown. For both panels WT3 cells expressing the indicated mutant were incubated withMAIT T-T hybridoma line 8D12 (upper) or 6C2 (lower) for 24/48 h with or without mAbs 12.2, 26.5, or an isotype control mAb 34-2-12 (anti-H-2Dd).Relative IL-2 production was determined by proliferation of CTLL-2 cells as monitored by fluorescence of alamar blue product (y axis). The baseline indicates the background level of MAIT activity when co-culturing with non-transduced WT3 cells. The S.D. represents triplicates in an alamarblue assay. None signifies no antibody.

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MR1 Mutants Located in the Putative Ligand BindingGroove Affect MAIT Cell Activation and Expression of FoldedMR1 Conformers—We next assessed whether mutations in theputative MR1 ligand binding groove affected MAIT cell activa-tion. Again, only mutants expressed at �60% of the wild typelevel were evaluated. Of the seven mutations made in the MR1putative ligand binding groove (Y7L, R9V, F22Y, Y95I, R97E,F113Y, and L114Q) only four (Y7L, R9V, R97E, F113Y) wereexpressed at a sufficiently high levels for MAIT cell testing. Asshown in Fig. 4B, mutations Y7L and R97E ablated activationof both MAIT cell hybridomas, and F113Y was detected by bothhybridomas. Interestingly, R9V ablated activation of the 6C2hybridoma but strongly activated the 8D12 hybridoma. Thus,these substitutions at MR1 positions 7, 9, and 97 allowed foldedMR1 conformers to be expressed but dramatically affectedMAIT cell activation. As noted in Fig. 3D, residues 7, 9, and 97are predicted to be clustered in the center of the putative MR1ligand binding groove, well within the predicted interface withthe TCR. Drawing parallels with similar mutagenesis studiesof class Ia molecules (51–54), it is attractive to speculate thatresidues 7, 9, and 97 are involved in MR1 ligand positioning orligand discrimination.

Of the seven mutations discussed above in the putative MR1ligand binding groove, three (F22Y, Y95I, and L114Q) had lessthan half the levels of folded molecules at the cell surface asdetected by all three mAbs to folded MR1. However, all three ofthese mutations had levels of open MR1 comparable with wildtype as detected by mAbs 64-3-7 or 4E3. It is noteworthy thatclass Ia mutagenesis studies have implicated each of theseresidues as dramatically affecting peptide binding (55–60).Based on these parallels, the phenotype of these three mutantsis suggestive of overall poor ligand binding and a requirementfor ligand occupancy to retain a folded conformation.

As shown in Fig. 3B there are some interesting inconsisten-cies between MR1 detection by mAbs 12.2 and 26.5 versus mAb4. As noted earlier, Y155R ablated detection of mAbs 12.2 and26.5 but not 4. This strongly suggests that residue 155 is partof the 12.2 and 26.5 epitope but not the 4 epitope. And muta-tions W159A and F171A sharply reduce detection by all threemAbs, suggesting they may also contribute to each epitope.However, it cannot be ruled out that these latter mutationsmore profoundly prevent MR1 from attaining a folded confor-mation. Perhaps most intriguing of the serologic differenceswas the observation that mAb 4 is clearly more sensitive toperturbation in the putative ligand binding groove than mAbs12.2 and 26.5. For example, Y95I, R97E, and L144Q all sharplyreduced mAb 4 staining compared with mAb 12.2 and 26.5staining. There are now several examples of mAbs madeagainst Ia or class II molecules loaded with endogenous ligandsthat display some ligand discrimination (61–64). Thus, a pos-sible explanation of this finding is that reactivity of mAb 4 withMR1 is also influenced by the conformation of a bound ligand.

MR1 Mutations at Sites Involved in Terminal Peptide Bind-ing in Class Ia Molecules—There are nine invariant class Iaresidues that are critical for peptide binding (Fig. 3A). ResiduesTyr-7, Tyr-59, Tyr-159, and Tyr-171 in the A pocket anchor theN terminus, and Thr-80, Tyr-84, Thr-143, Lys-146, and Trp-147 in the F pocket anchor the C terminus (50). Of these nineinvariant residues, mMR1 contains two significant (T80H,K146A) and four conservative (Y59H, Y84H Y159W, Y171F)substitutions. Mutations were made at all six of these posi-tions. In addition to these six mutants at class Ia invariantsites, we substituted the consensus Tyr at mMR1 position 7with Leu to potentially disrupt its pocket chemistry. Interest-ingly, the three MR1 mutations corresponding to the Phepocket of class Ia molecules had little effect on surface expres-

sion of folded MR1 and MAIT cell activation. By contrast, thefour mMR1 mutations corresponding to the class Ia A pockethad a profound effect on both surface expression of folded MR1and/or MAIT cell activation (Fig. 3B and Fig. 5). More specifi-cally, Y7L was expressed as a folded MR1 protein but did notactivate MAIT cells. H59A had reduced levels of folded MR1and activated only one of the MAIT cells, whereas both W159Aand F171A had severely impaired expression of folded MR1 andMAIT cell activation. Thus, MR1 expression and MAIT cellactivation are highly sensitive to perturbation of single resi-dues in the A rather than the F pocket. Interestingly class Iamolecules also have a greater susceptibility to A pocket versusF pocket perturbations which can be explained by their pocketarchitecture and the atomic basis of ligand anchoring. Forexample, the stability of MR1 itself is more likely to be per-turbed by changes in the buried A pocket compared with thesolvent-exposed F pocket based on classic studies of lysozyme(65). Furthermore, in specific regard to class Ia, the N terminusof the peptide is buried and needs to make specific hydrogenbonds with invariant A pocket residues and, thus, is suscepti-ble to unbalanced substitutions. By contrast, the C terminus ofthe peptide has more surface exposure when bound in the Fpocket, and certain rearrangements can be accommodated (50).Thus, the greater susceptibility of MR1 to substitutions in theA versus F pockets suggests it may have a similar mechanismof terminal ligand anchoring as class Ia molecules. In addition,susceptibility to A pocket perturbations may suggest that MR1binds ligands with a unique N terminus like the strong prefer-ence of H2-M3 for formylated peptides (66). Regardless of theexplanation, the phenotype of MR1 mutants at sites involved interminal peptide binding of class Ia molecules provides furthersupporting evidence that MR1 binds a ligand.

DISCUSSION

MR1 proteins have two properties that predict that theyhave a conserved function of physiological relevance. FirstmMR1 and hMR1 share 90% identity in their �1/�2 domainsthat far exceeds the 70% or less identity shared by theseregions of mouse and human class Ia and Ib proteins (20, 21).Second, MR1 is the activation and restriction element for apopulation of T cells preferentially expressed in the gut lam-ina propria with an invariant CDR3� called MAIT cells (19).Extending these findings, in this study we report multiplelines of evidence supporting the model that MR1 functions asa unique antigen presentation molecule with propertiesshared with both class Ia and class Ib molecules. Key to thisconclusion, several findings reported here provide compellingcircumstantial evidence that MR1 binds a ligand that deter-mines the expression of folded MR1 proteins as well as theactivation of MAIT cells. For example, 1) MR1 is detected inan open versus folded conformation analogous to class Iamolecules when they bind peptide, 2) certain MR1 groovemutations known to affect class Ia peptide binding impair theexpression of folded MR1 protein, 3) only the folded MR1conformer activates MAIT cells based on 2/2 hybridomastested, 4) MR1 engages the TCR in an orientation strikinglysimilar to ��TCR engagements of MHC/peptide complexes asimplied by MAIT cell activation by our panel of MR1 mu-tants, and 5) certain MR1 groove mutations with high surfaceexpression levels affect MAIT cell activation. Further sup-porting the role of a ligand in MR1 folding, we were unable torefold recombinant MR1 after bacterial expression using fold-ing conditions that yielded folded non-ligand binding class Ibproteins T10, T22, MICA, MICB, RAE-1 (3, 67–71). However,a folded recombinant ectodomain of MR1 was secreted byinsect cells in the presence of highly supplemented media.Thus, accumulating evidence supports the notion that MR1

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folding and MAIT cell activation are both ligand-dependent.Despite the above-listed similarities between MR1 and class

Ia proteins, there are significant differences. Whereas class Iaproteins are ubiquitously expressed, this is certainly not thecase for MR1. Indeed, endogenous expression of MR1 has yet tobe identified despite considerable effort on our part. Further-more, unlike the abundance of T cells that are activated andrestricted by class Ia molecules, MAIT cells are very rare,particularly in mice (9, 18). However, MAIT cells are abundantwhen compared with the number of antigen-specific, class Ia-restricted T cells. As noted earlier, several properties of MR1mirror those of CD1d proteins. Most notably, like MR1 detec-tion by MAIT cells, CD1d is detected by a unique population ofT cells with an invariant TCR�, namely NK-T cells. However,there are also important differences between MAIT cells andNK-T cells, including their tissue distribution and the uniquedependence of MAIT cells on gut flora and B cells (9, 19).

All ��T cells described thus far detect MHC/peptide orCD1d-lipid complexes (12, 72, 73). Therefore, MR1 would be theexception to this rule if it does not bind a ligand involved in ��Tcell activation. However, it is certainly worthwhile consideringthis possibility. Relevant to this issue are studies of T22, a

non-ligand binding class Ib protein detected by ��T cells (68,74). Indeed, a mutagenesis study of the groove of class Ibmolecule T22 was interpreted as evidence that it binds a ligandin a similar manner as class Ia molecules (74). Subsequentcrystal structure analysis, however, showed T22 does not binda ligand and has a severe truncation of its �2 helix exposing its�-sheet floor for direct contact with the ��TCR (68). And the��TCR has extruded CDR3 loops (75). Thus, T22 groove resi-dues are potential direct contacts for ��TCR interaction. How-ever, this is clearly not the case for MR1 groove mutations.Unlike T22, MR1 is predicted to have intact � and � domainsthat appear to engage the TCR in a manner strikingly similarto MHC/peptide (Fig. 3D), and the CDR3s of MAIT TCR are ofnormal length (18). Furthermore if MR1 does not bind a ligandrequired for stable surface expression and MAIT cell function,then one might expect its groove would need to close in amanner analogous to other ligand-independent MHC mole-cules. However, if this were the case one would expect a differ-ent set of solvent-exposed residues to protrude from the MHChelices and be involved in MAIT cell activation. Because thiswas not found, our data suggest that MR1 makes direct contactonly with helical and not groove residues in a manner analo-

FIG. 5. MAIT activation by MR1 mu-tations of residues corresponding tothe A or F pockets of the Kb molecule.The overall approach is the same as thatin Fig. 4. However, some of these mutantshave significantly reduced surface expres-sion of folded MR1 (12.2 staining) thatcould clearly impact of MAIT cell activa-tion. To make this point clear surface ex-pression relative to wild type is indicatedby a circle for each mutant and indexed onthe right axis.

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gous with ��TCR engagements with MHC/peptide.Difficulty in defining endogenous expression of MR1 has

been interpreted as evidence that MR1 may be inducible. Thus,in light of findings reported here it is attractive to speculatethat a limiting ligand may control expression of MR1. If so,MR1 would be like H2-M3, HLA-E (76), and Qa-1 (77–79) thatstay in the endothelial reticulum due to limiting endogenousligands. If MR1 expression is ligand-dependent, it is of interestto consider the mechanisms of MR1 expression by transduc-tion/transfection. One striking feature of our expression of mu-tants reported here is that they all have similar levels of openMR1 conformer expressed at the cell surface (Fig. 3, B and C).Open conformers at the cell surface could be accounted for byhigh levels of MR1 expression bypassing the endoplasmic re-ticulum quality control or forcing MR1 to bind suboptimalligands, which dissociate after endoplasmic reticulum egress.In any case the open MR1 conformer is irrelevant for MAIT cellactivation, whereas the folded MR1 conformer activates MAITcells. And importantly, expression of the folded MR1 conformeris dramatically affected by certain mutations in the groove,consistent with ligand control of folded MR1 expression.

It is important to note that we have expressed both intactMR1 and chimeric MR1 in several different cell lines includingtwo different fibroblast cell lines, a DC cell line and a B cell line(not shown). All of these were found to express folded MR1molecules capable of MAIT cell activation. Thus, if there is anMR1 ligand, it is likely to be a self-protein expressible, at leastat low levels, in the absence of gut flora. However, one mustconsider that MR1 could be similar to H2-M3 that binds a lowlevel of self-ligands derived from mitochondrial proteins,whereas its physiological function is to present bacterial-de-rived peptides (80). Thus, our findings can be incorporated intoa model whereby the commensal flora induces the physiologicexpression of folded MR1 and thus MAIT cell activation.

In summary MR1 displays a unique combination of proper-ties shared with class Ia and class Ib proteins. Like class Iaproteins, MR1 is detected by the ��TCR in a manner verysimilar to all ��TCR engagements with MHC/peptide definedto date based on our mutagenesis studies. Furthermore, exten-sive mutagenesis of the MR1 groove defines several parallelswith class Ia, strongly supporting the notion that MR1 binds aligand in a manner similar to class Ia. However, like severalclass Ib proteins, MR1 appears to have a highly restrictivetissue expression. More specifically, our data suggest that ex-pression of folded MR1 is controlled by a limiting self-ligand,making it analogous to H2-M3, HLA-E, and Qa-1. And likeCD1d, MR1 is detected by relatively rare T cells with an in-variant TCR�. These combined finds strongly support the con-clusion that MR1 has a novel function in antigen presentation,thus underscoring the importance of defining endogenousand/or exogenous MR1 ligands.

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Structure-Function Analyses of MR1 21193

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Lybarger, Daved H. Fremont and Ted H. HansenShouxiong Huang, Susan Gilfillan, Marina Cella, Michael J. Miley, Olivier Lantz, Lonnie

Evidence for MR1 Antigen Presentation to Mucosal-associated Invariant T Cells

doi: 10.1074/jbc.M501087200 originally published online March 31, 20052005, 280:21183-21193.J. Biol. Chem. 

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