Diversity of T Cells Restricted by the MHC Class I-Related Molecule MR1

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1 Diversity of T Cells Restricted by the MHC Class I-Related Molecule MR1 Facilitates Differential Antigen Recognition. Nicholas A. Gherardin 1,2*, Andrew N. Keller 3,4*, Rachel E. Woolley 3,4, Jérôme Le Nours 3,4, David S. Ritchie 2, Paul J. Neeson 2, Richard W. Birkinshaw 3,4, Sidonia B.G. Eckle 1, Ligong Liu 5,6, Adam P. Uldrich 1,7, Daniel G. Pellicci 1,7, David P. Fairlie 5,6, James McCluskey 1, Dale I. Godfrey 1,7# & Jamie Rossjohn 3,4,8# 1 Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010, Australia 2 Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia 3 Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia 4 ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, Australia 5 Division of Chemistry & Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia. 6 ARC Centre of Excellence in Advanced Molecular Imaging, University of Queensland, Queensland 4072, Australia. 7 ARC Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Clayton, Victoria 3800, Australia 8 Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK * joint first authors # joint senior and corresponding authors. Jamie.rossjohn@monash.edu, godfrey@unimelb.edu.au Running title: Autoreactive and atypical MAIT TCR recognition 1

2 Abstract Mucosal-associated invariant T (MAIT) cells predominantly express TRAV1-2 + T cell receptors (TCRs) activated by antigens produced during riboflavin biosynthesis and presented by MR1. However, the extent of TCR diversity and associated antigen (Ag) responsiveness of the MR1-restricted T cell repertoire remains unclear. We describe diverse populations encompassing human TRAV1-2 + and TRAV1-2 - MR1-restricted T cells, some exhibiting MR1 autoreactivity or reactivity towards folate derivatives. Autoreactivity and reactivity of folate-derivatives with TRAV1-2 + TCRs was attributable to CDR3b loopmediated effects. A TRAV1-2 - TCR docked more centrally on MR1 in comparison to the TRAV1-2 + TCRs, thereby adopting a markedly different molecular footprint. Accordingly, diversity within the MR1-restricted T-cell repertoire leads to differing Ag recognition modes and functional responsiveness to MR1 and associated small molecule antigens. Introduction αβtcrs can recognise a range of peptide, lipid and riboflavin metabolite-based Ags presented by the Major Histocompatibility Complex molecules, the CD1 family and the MHC-related protein 1 (MR1), respectively 1. MAIT cells, an evolutionarily conserved population of innate-like T cells, orchestrate the MR1-mediated T cell response 2, 3. MAIT cells in humans are highly abundant, accounting for up to 10% of the circulating T cell pool, and are enriched in human liver 4, 5. MAIT cells are activated by a wide array of bacteria and yeast 6, 7, 8, and thus are emerging as key effectors in antimicrobial immunity. Upon activation, MAIT cells rapidly produce pro-inflammatory cytokines including IFN-γ, TNF and IL-17a 4, 5, 9. They can also directly lyse bacterially infected cells in a perforin and granzyme dependent manner 10, 11. MAIT cells have also been implicated in diseases of non-microbial aetiology, including autoimmunity 12, 13, 14, 15, 16, 17, 18, 19, 20, cancer 21, 22, 23, 24, 25, 26, 27, and other inflammatory diseases 28, 29, 30. While MAIT cells can be activated in the absence of TCR ligation 31, the role of the MAIT TCR in non-microbial disease is unclear. Recognition of MR1-Ag complexes is driven by the MAIT TCR 2, 7, 32, 33, 34, which is characterised by highly biased TCR a-chain usage 35, 36. Specifically, the TCR a-chain consists of an invariant TRAV1-2 gene segment frequently recombined with TRAJ33 36, although TRAJ12 or TRAJ20 usage has also been observed in the human MAIT TCR repertoire 7, 36, 37. These TCR a-chains pair with an enriched set of TRBV genes, predominantly from the TRBV6 and TRBV20 families, but with diverse TRBJ, TRBD gene usage and non-germline encoded nucleotide additions 36. Accordingly, much of the diversity in the MAIT cell repertoire resides within the Complementarity 2

3 Determining Region (CDR) 3b loop, although a precise functional role of CDR3b hypervariability has not been clearly demonstrated 36, 37. Presently, two classes of MR1-bound ligands, folate- and riboflavin-derived Ags, have been identified. The folate (vitamin B9)-derivatives, which include the naturally occurring 6-formyl pterin (6-FP) and analogues, acetyl-6-fp (Ac-6-FP), 2-acetylamino-4-hydroxy-6-formylpteridinedimethyl-acetal and 2-acetylamino-4-hydroxy-6-formylpteridine, do not activate MAIT cells 32, 34, 38, 39. In contrast, microbially derived riboflavin (vitamin B2) derivatives can activate MAIT cells, the potency of which can vary. Namely, the bicyclic lumazines 7-hydroxy-6-methyl-8-Dribityllumazine (RL-6-Me-7-OH) and 6,7-dimethyl-8-D-ribityllumazine (RL-6,7-diMe) are weak agonists, while the chemically unstable transitory pyrimidine antigens 5-(2-oxoethylideneamino)- 6-D-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP- RU) represent very potent MAIT cell agonists 32, 34, 38. Consistent with the non-stimulatory and stimulatory properties of the folate- and riboflavin- associated ligands, respectively, MR1 tetramers loaded with the stimulatory Ags specifically bind MAIT cells 7. Indeed, MR1-5-OP-RU tetramers label essentially all human TRAV1-2 + MAIT cells and mouse TRAV1-2 + MAIT cells 7. The mechanism by which the TRAV1-2 + MAIT TCR discriminates between folate and riboflavinbased Ags has been resolved through a series of structural and mutational studies on the MAIT TCR-MR1-Ag axis 32, 33, 34, 38, 40. These studies showed that the MAIT TCR adopted a consensusdocking mode on MR1-Ag regardless of TRAJ or TRBV gene usage, CDR3β sequence, or the nature of the Ag, highlighting the innate-like pattern-recognition-like features of this interaction 41. Namely, the TCR a-chain extensively contacted the central region of the MR1-Ag binding cleft, thereby providing a basis for the nearly invariant TCR a-chain usage. Moreover, the TRAJ33- encoded Tyr95a residue directly contacted the riboflavin-based Ags, mutation of which markedly reduced MAIT cell activation in response to these Ags 33. The Tyr95a residue was also conserved in the TRAJ12 and TRAJ20 gene segments, thereby indicating the key role this residue plays in enabling MAIT cell activation 7. The neighbouring CDR3b loop of the MAIT TCR could subtly modulate this recognition in an Ag-dependent fashion 32. While these studies suggest that MR1-Ag recognition by MAIT TCRs is highly conserved, the scope of the MR1-restricted TCR repertoire, and the extent to which TCR heterogeneity may impact on MR1-restricted recognition of riboflavin and folate-based Ags, remains unclear. Here, using MR1 tetramers 38 loaded with riboflavin- and folate-based ligands, we identified populations of human MR1-restricted cells that were autoreactive towards MR1 or could be 3

4 activated by folate-based Ags in addition to, or in preference to, the riboflavin-based Ags. These cells included TRAV1-2 + and TRAV1-2 - populations, the latter of which showed a high degree of abtcr diversity. Autoreactivity and folate-based antigen reactivity was attributable to the CDR3b loop of the TRAV1-2 + MAIT TCRs, while structural analysis of a TRAV1-2 - TCR-MR1-Ag ternary complex revealed a markedly different docking mode to that of the TRAV1-2 + TCRs. Thus, the MR1-reactive T cell repertoire is much more diverse than previously recognised, and this diversity leads to differential responsiveness to MR1-restricted Ags, and in some instances MR1 autoreactivity. Our findings provide a mechanistic basis for MR1-mediated immunity in diseases of non-microbial aetiology. Results Identification of atypical MR1-restricted T cells A defining feature of human and mouse MAIT cells is the expression of an invariant TCR α-chain (TRAV1-2 TRAJ33). However in humans, TRAJ20 and TRAJ12 gene segments can also be used in the generation of TRAV1-2 + MAIT TCR a-chains 7, 32, 37. The recent advent of MR1-Ag tetramers has allowed the unequivocal identification of MR1-Ag-reactive T cells regardless of abtcr chain usage or any other cell surface markers 7. Given the central role of the MAIT TCR α-chain in mediating MR1-Ag recognition, we investigated whether any MR1-Ag reactive T cells exist that lack expression of the prototypical TRAV1-2 + TCR α-chain. Staining a cohort of blood samples derived from multiple myeloma patients with MR1-5-OP-RU tetramers together with a TRAV1-2-specific mab, we identified one patient who had a higher proportion of MAIT cells than the rest of the cohort (Supplementary Figure 1a). Further, these included three different subsets based on TRAV1-2 usage and MR1-5-OP-RU tetramer staining intensity, including TRAV1-2 + MR1-5-OP-RU HI, TRAV1-2 + MR1-5-OP-RU LOW and TRAV1-2 - MR1-5-OP-RU HI. Moreover, each population was present in both peripheral blood and bone marrow (Figure 1a), and displayed a surface phenotype (CD8α +, CD161 ++, IL-18Rα ++ ) akin to MAIT cells 4, 42 (Supplementary Figure 1b). Single cell TCR sequencing of these cells revealed that the TRAV1-2 + populations segregated into HI and LOW intensity staining based on TRBV gene usage. Interestingly, the TRAV1-2 - population exhibited clonal expression of a TRAV36- TRAJ34 TCR α-chain paired to TRBV28 (Supplementary Figure 1c), thereby representing atypical MR1-restricted TCR gene usage 7, 9, 36, 37. Notably, the sequences encoding the CDR1a, CDR2a and CDR3a loops of the TRAV36-TRAJ34 TCR differed markedly from those of the TRAV1-2-TRAJ33 TCR (Supplementary Figure 1c). We refer to this TCR as an atypical MR1- restricted T cell. 4

5 Given that this atypical MR1-restricted T cell population was derived from a multiple myeloma patient, we established whether similar cells were present in healthy control PBMC samples. Indeed, using MR1-5-OP-RU tetramers, we detected atypical MR1-restricted T cells in 4 healthy donor samples, albeit at variable frequency (Figure 1a ii). Interestingly, the MR1 tetramer staining intensity of these atypical TRAV1-2 - cells was more varied than for the TRAV1-2 + cells, suggesting a range of different TCR affinities within this population. In order to investigate whether the MR1 tetramer staining of atypical cells was dependent on the 5- OP-RU Ag, we stained a larger cohort of 12 healthy donors with MR1 tetramers loaded with agonist 5-OP-RU as well as two non-agonist folate-derivatives, 6-FP and its analogue Ac-6-FP. In addition to detecting the atypical MR1-5-OP-RU tetramer + TRAV1-2 - T cells in all donors, we also observed MR1-6-FP and MR1-Ac-6-FP tetramer reactive populations in both the TRAV1-2 + and TRAV1-2 - fractions. In order to determine whether this MR1-6-FP or MR1-Ac-6-FP tetramer staining was specific, we performed a dual tetramer labelling experiment where MR1-6-FP tetramer was used in one colour and CD1d-a-GalCer tetramer (specific for type I NKT cells) was used in another colour (Supplementary figure 2a). This showed that these tetramers stained mutually exclusive T cell populations, as expected if these tetramers are binding different T cells based upon their TCR specificity. Furthermore, we used flow cytometric cell sorting to enrich them from 7 donors, separating them into TRAV1-2 + and TRAV1-2 - fractions, and expanded them in vitro for 10 days. A broad sorting gate was applied to capture low intensity cells that stained with the MR1-6-FP or MR1-5-OP-RU tetramer (data not shown). Upon re-staining with MR1 tetramers two weeks later, it was clear that we had enriched MR1 restricted T cells that recapitulated the staining patterns of the cells prior to sorting (Supplementary figure 2b). MR1-5-OP-RU + cells were much more abundant than MR1-6-FP + and MR1-Ac-6-FP + as a percentage of total αβ cells with median values of 3.56%, 0.09% and 0.04%, and ranges of %, % and % for each, respectively (Figure 1c i). In the TRAV1-2 + T cell fraction, the frequency of MR1-6-FP/Ac-6-FP + T cells was lower (less than 5%) than the MR1-5- OP-RU + cells, with medians of 61.25%, 1.90 and 0.84% for MR1-5-OP-RU, MR1-6-FP and MR1- Ac-6-FP tetramers, respectively (Figure 1c ii). However, within the TRAV1-2 - T cell fraction, the frequency of MR1-6-FP/Ac-6-FP + T cells was similar to, or higher than the MR1-5-OP-RU + cells with medians of 0.12%, 0.18% and 0.16% of total TRAV1-2 - cells for each tetramer respectively (Figure 1c iii). This suggested that a subpopulation of MR1-restricted T cells were capable of 5

6 detecting folate-derivate Ags, and furthermore, that these were enriched within the atypical (TRAV1-2 - ) T cell fraction (Figure 1c iv). Similar to MR1-5-OP-RU tetramer + MAIT cells, the MR1-6-FP or Ac-6-FP tetramer + cells were almost all CD161 + within the TRAV1-2 + fraction, while the MR1-6-FP or Ac-6-FP tetramer + TRAV1-2 - cells were heterogeneous for CD161 expression, the extent varying between donors (Figure 1d and data not shown). Thus, there is evidently diversity within the MR1-restricted T cell repertoire. MR1-restricted TCR heterogeneity and variable antigenic reactivity We next established the abtcr gene usage within the MR1-reactive TRAV1-2 - and the MR1-6- FP + or MR1-Ac-6-FP + TRAV1-2 + T cell populations. Multiplex nested PCR was used to determine the matched a and b-chain sequences of single cells sorted from in vitro-expanded MR1-reactive T cells. With the exception of one TCR that utilised TRAJ12, the remainder of the TRAV1-2 + folatereactive TCRs paired with TRAJ33 7 (Figure 2a). The invariant TCR a-chains paired with b-chains that utilized TRBV6, TRBV20 and TRBV4 gene families. Furthermore, there was no obvious conservation in CDR3β sequence motifs, indicating no discernable TCR bias that was associated with MR1-6-FP or MR1-Ac-6-FP tetramer + cells within the TRAV1-2 + fraction (Figure 2a). The TRAV1-2 - TCRs were markedly different from the TRAV1-2 + MAIT TCRs in that they utilized a diverse repertoire of TRAV and TRBV genes with extensive non-template-encoded nucleotides at the CDR3α junction (Figure 2a and b). Amongst the TRAV1-2 - TCRs, only one TCR utilized a TRAJ gene associated with the TRAV1-2 + invariant chain (TRAJ33; Clone AT11), and only one b chain used a TRBV gene normally associated with the MAIT repertoire (TRBV6; Clone AT12). Further, the TRAV1-2 - TCRs expressed a wide range of TRAV gene elements, including TRAV36, TRAV12-2, TRAV14, TRAV21, TRAV19, TRAV3, TRAV8 and TRAV4 (Figure 2a and b). These data reveal a far greater level of diversity within the MR1-restricted T cell repertoire than previously appreciated. To show that these TCR sequences conferred MR1 reactivity and investigate their respective antigen specificities, we transiently transfected seven TRAV1-2 + and three TRAV1-2 - TCRs into HEK293T cells and screened them with a panel of MR1-Ag tetramers, including human and mouse MR1-5-OP-RU, MR1-Ac-6-FP and MR1-6-FP and unloaded MR1 tetramer (Figure 3). As controls, we included four invariant MAIT TCRs previously characterized based on reactivity with 5-OP-RU 7, 32 as well as a type I NKT TCR (Figure 3a). As expected, the type I NKT TCR failed to bind any 6

7 of the MR1 tetramers but did bind to human CD1d-a-GalCer tetramers. Conversely, all of the MR1-reactive TCRs failed to bind CD1d-a-GalCer yet did bind to distinct MR1 tetramers, establishing that we had identified MR1-reactive TCRs. All TRAV1-2 + TCRs tested, regardless of their reactivity to MR1-folate-derivatives, bound strongly to MR1-5-OP-RU tetramers (Figure 3a). Furthermore, the TCRs derived from T cells reactive to MR1-6-FP also recapitulated this antigen reactivity and were labeled by the respective tetramers. More variable staining was observed with MR1-Ac-6-FP tetramers, suggesting that the additional acetyl group on this derivative can be distinguished by different TRAV1-2 + MAIT TCRs. For example, the AM2 TCR bound more strongly to MR1-Ac-6-FP, whereas the AM3 TCR bound more strongly to MR1-6-FP (Figure 3a). Intriguingly, both the M33.64 and AM1 TCRs bound to MR1-Lys43Ala mutant tetramers that refold in the absence of Ag (unloaded MR1 tetramer) 7, indicating that these TCRs are autoreactive towards MR1. Furthermore, the M33.20, AM1, AM2 and AM3 TCRs all utilised TRBV20, while TCR5 and M33.64 both utilized TRBV6-4, underscoring that the differences in antigen reactivity/autoreactivity between these respective groups is attributable to CDR3β sequence variation. None of the TRAV1-2 - TCRs investigated (MAV14, MAV21 and MAV36) bound to the unloaded MR1-Lys43Ala mutant tetramers, suggesting a requirement for the presence of Ag to enable TCR binding. Indeed, similar to the TRAV1-2 + TCRs, variation in Ag recognition was observed between the three TRAV1-2 - TCRs investigated. For example, MAV36 was clearly dependent on the presence of 5-OP-RU, whereas MAV14 recognised 5-OP-RU, 6-FP and Ac-6-FP, albeit to varying degrees (Figure 3a). Interestingly, MAV21 barely reacted with 5-OP-RU bound to human MR1, and failed to bind mouse MR1-5-OP-RU. Nevertheless, MAV21 bound MR1 tetramers loaded with 6-FP and Ac-6-FP with high intensity, suggesting that some TRAV1-2 - TCRs have the propensity to preferentially react to folate-derived ligands. To confirm the ability of MR1-Ag complexes to induce TCR-mediated signaling and activation via these atypical MR1-reactive TCRs, stable Jurkat cell lines expressing an autoreactive MAIT TCR (clone M33.64) and a TRAV1-2 -, MR1-5-OP-RU-restricted TCR (clone MAV36) were generated. As expected, the MAV36 TCR was activated in an MR1- and dose-dependent manner when cocultured with C1R.MR1 cells in the presence of both 5-OP-RU and E. coli (Figure 3b (i)). While the M33.64 cell line showed a similar pattern of activation towards 5-OP-RU and E. coli, it also exhibited robust, MR1-dependent, activation in the absence of additional Ag (Figure 3b (i) & (ii)). 7

8 Thus, a diverse array of TCRs allows responses to discrete patterns of MR1 autoreactivity and Ag recognition. Binding to atypical MR1-reactive TCRs To further investigate the M33.64 MAIT TCR autoreactivity towards MR1, the M33.64 TCR + cell line was co-cultured with C1R cells transduced with MR1 and sorted using flow cytometry based on expression of graded MR1 expression levels (Figure 4a i). When the M cells were cocultured with C1R.MR1 cells in the absence of added antigen, the M cells upregulated CD69 in an MR1-dose dependent manner (Figure 4a ii). Interestingly, the addition of 5-OP-RU antigen to these co-cultures only moderately enhanced this activation (Figure 4b). Thus, in contrast to other MAIT TCRs, 32 the M33.64 TCR is clearly capable of interacting with MR1 leading to TCR mediated activation in the absence of 5-OP-RU antigen. Given the previously established role for Tyr95α in mediating recognition of ribityl-containing Ags 33, we next sought to establish whether the M33-64 TCR was also dependent on this amino acid for recognition of MR1-5-OP-RU complexes. We thus transiently transfected HEK293T cells to express the M33-64 TCR, as well as 4 previously characterised MAIT TCRs 7, 43 (Figure 4c). For each TCR, we expressed a Tyr95αPhe mutant counterpart and for 3 of these TCRs we also expressed a Tyr95αAla mutant. While MR1-5-OP-RU tetramers bound the Tyr95αPhe mutant of the M33.64 TCR with equal affinity to the wildtype TCR, the other 4 Tyr95αPhe mutants resulted in a reduction in staining intensity, albeit to varying degrees (Figure 4c). Furthermore, while the M33.64.Tyr95αAla TCR bound MR1-5-OP-RU tetramers, with approximately 70% of the intensity observed for the wildtype TCR, staining of the M12.64 and M20.64 Tyr95αAla mutant TCRs was almost entirely ablated (Figure 4c). These data suggest that the M33.64 TCR is far less dependent on Tyr95α than other TCRs. Further, since the M33.64 and TCR#6 TCRs predominantly differ in their CDR3β amino acid sequence, CDR3β mediated interactions likely alleviate the role of Tyr95α in the M33.64 TCR. To gain insight into the relative affinity of an autoreactive TRAV1-2 + TCR and a TRAV1-2 - TCR for MR1-5-OP-RU complex, we recombinantly expressed and purified the M33.64 and MAV36 TCRs and performed tetramer inhibition assays, where increasing concentrations of soluble TCRs can specifically inhibit tetramer binding to TCR + cells, the efficiency of which is related to TCR affinity Here, the Jurkat.M33.64 cell line was stained with MR1-5-OP-RU tetramers, and titrating amounts of soluble MR1-restricted TCRs were added. The positive control was a previously characterized MAIT TCR (clone AF-7 32 ) and the negative control was a CD1a- 8

9 restricted TCR (clone BK6 46 ). All of the MR1-restricted TCRs inhibited the tetramer labelling of the Jurkat.M33.64 cells in a dose-dependent manner, whereas the CD1a-restricted BK6 TCR did not interfere with this interaction (Figure 4d). The greatest inhibitory effect was observed for the M33.64 TCR, which was moderately stronger than the AF-7 TCR. The atypical MR1-restricted TRAV1-2 - TCR (MAV36) provided weaker inhibition than the TRAV1-2 + TCRs. These data suggest that these TCRs vary in affinity for MR1-5-OP-RU where M33.64 affinity is greater than AF-7, and that both of these are greater than MAV36 TCR. To establish formal binding affinity values for these TCRs, we conducted surface plasmon resonance (SPR) studies in which a series of MR1-Ag complexes was coupled to the sensor chip (Figure 4e). While the M33.64 TCR bound to MR1-5-OP-RU (K D 0.7µM) with a moderately higher affinity compared to the AF-7 MAIT TCR (K D 2.7µM), the M33.64 TCR exhibited higher affinity towards MR1-Ac-6-FP (K D 69.5 µm) and MR1-6-FP (K D 81.4 µm), whereas the AF-7 TCR showed no detectable affinity towards these folate-derivatives (Figure 4e). The MAV36 TCR displayed no detectable affinity for MR1-6-FP or MR1-Ac-6-FP but, consistent with the tetramer inhibition assay, did bind to MR1-5-OP-RU with >10 fold lower affinity (K D 10.4 µm) compared to the autoreactive M33.64 TCR (K D 0.7µM). Thus, the SPR data correlated well with the cellular data (Figure 3 and 4a-c). Interestingly, while the Tyr95aAla mutant of the M33.64 TCR ablated recognition, the corresponding Tyr95aPhe mutant did not impact on recognition of MR1 bound to 6-FP, Ac-6-FP or 5-OP-RU (Figure 4e). This indicated that Tyr95, despite its strong conservation between the TRAJ33/TRAJ20 and TRAJ12 genes, is not an obligate requirement in mediating a TRAV1-2 TRAJ33 + response towards MR1-Ag. Accordingly, the TRAV1-2 + and TRAV1-2 - TCRs possess variable binding properties towards MR1-Ag. Molecular basis underpinning TRAV1-2 + MAIT TCR autoreactivity To determine the molecular basis for M33.64 TCR autoreactivity, we determined the structure of the M33.64 TCR, and the Tyr95aPhe mutant thereof, in complex with MR1-5-OP-RU (Table 1) and compared these complexes with that of a non-autoreactive MAIT TCR ternary complex in which the TCR utilised the same germline-encoded regions (TRAV1-2-TRAJ33-TRBV6-4; TCR#6) (Figure 5). The structures of the two M33.64 TCR ternary complexes were very similar to each other, and to that of previously reported TRAV1-2 + MAIT TCR ternary complexes, revealing that the inherent binding properties of the M33.64 TCR were not attributable to deviation from the consensus MAIT TCR-MR1 docking mode (Figure 5c). Accordingly, this implicated the CDR3b loop of the M33.64 TCR in conferring the ability to tolerate the impact of the Tyr95Phe mutation 9

10 and the MR1 autoreactivity. The CDR3b loop did not contact the bound Ag (Figure 5a), and thus the enhanced binding properties of the M33.64 TCR were related to MR1-mediated contacts and it s impact on the proximal environment. Namely, Thr100b and Asn99b from the CDR3b loop acted like a pincer around Gln149 of MR1, with Thr100b interdigitating between His148 and Glu149, the latter of which also hydrogen bonded to Thr100b (Figure 5a). Further, in addition to these CDR3b-MR1 contacts, the CDR3b loop played an important role on stabilising its own conformation as well as neighbouring CDR loops of the M33.64 TCR. For example, Asn99b wedged between the main chain backbone of the CDR3b loop; Thr100b packed against Tyr48a; and most noticeably, the double glycine motif, Gly97b-Gly98b enabled the main chain of the CDR3b loop to nestle closely against Met91a and Tyr95a of the MAIT TCR a-chain (Figure 5a). In contrast, the CDR3b loop of the MAIT TCR#6 neither contacted Tyr95a, Tyr48a or His148 of MR1 (Figure 5b). Collectively, the interactions mediated by the M33.64 TCR CDR3b loop were more extensive and possessed greater shape complementarity than the corresponding, shorter, MAIT TCR#6 CDR3b loop. Toleration of the Tyr95aPhe mutation of the M33.64 TCR was attributed to two factors. Firstly, the more extensive CDR3b loop mediated interactions reduced the dependency of the energetic contribution of the hydroxyl moiety of Tyr95 forming a hydrogen bond with the ribityl tail of 5-OP- RU and the Tyr152 of MR1. Secondly, the Tyr95aPhe mutant caused a compensatory change in that Tyr152 of MR1 swivelled upwards to form a direct hydrogen bond with the main chain of Gly98b (Figure 5d). Thus, subtle features inherent to the CDR3b loop can, for certain MAIT TCRs, engender a degree of autoreactivity towards MR1. Molecular basis of MAIT TCR recognition of folate-derivatives presented by MR1. Next, to understand the structural basis of how a MAIT TCR can recognise folate-derivatives presented by MR1, we determined the structure of the M33.64 TCR in complex with MR1-Ac-6-FP (Table 1). Unexpectedly, the M33.64 TCR-MR1-Ac-6-FP and the M33.64 TCR-MR1-5-OP-RU complexes were very similar, although subtle key differences were evident in the vicinity of the Ac- 6-FP ligand. Namely, Tyr95a was drawn 1.5 Å closer to the Ac-6-FP ligand, thereby enabling its hydroxyl group to form a direct hydrogen bond with the pterin ring (Figure 5e). The movement of Tyr95a meant that it no longer hydrogen bonded to Tyr152 from MR1, thereby differing from the TRAV1-2 + MAIT TCR ternary complex (Figure 5f) 32, 33, 38. To compensate for this loss of a polar interaction, the Tyr152 from MR1 swivelled form a direct hydrogen bond with the main chain of the CDR3b loop (Figure 5e). Notably, the conformation of Tyr152 in the M33.64 TCR-MR1-Ac- 10

11 6-FP complex was very similar to that in the M33.64 TCR-Tyr95aPhe-MR1-5-OP-RU complex (Figure 5d). Accordingly, conformational flexibility in the intricate interaction network of the ligand and its environs enabled this MAIT TCR to be activated by folate-derivative based ligands. Differential docking modes for an atypical MR1-restricted TCR To establish how a TRAV1-2 - TCR engaged an MR1-Ag complex, we determined the structure of the MAV36 TCR (TRAV36-TRAJ34-TRBV28-TRBJ2-5) in its unliganded state and in complex with MR1-5-OP-RU (Table 1). The electron density for the TCR, and at the TCR-MR1-Ag interface, was clear (not shown), providing an opportunity to analyse the plasticity of the interaction, the intermolecular contacts, and compare it to the TRAV1-2 + MAIT TCR-MR1-Ag docking mode. While there was some relative juxta-positioning of the TCR a- and TCR b-chains upon binding (not shown), the CDR loops of the MAV36 TCR underwent limited conformational change upon binding, indicating essentially a lock and key interaction. The MAV36 TCR was located centrally, oriented at a 65 angle across the MR1 Ag-binding cleft, whereas the TRAV1-2 + TCR docked orthogonally across MR1-Ag. (Figure 6a,b). The buried surface area (BSA) at the interface was 1080Å 2, in which the TCR a-chain and b-chains contributed 52 and 48%, respectively, closely matching values for the TRAV1-2 + TCR-MR1-5-OP-RU ternary complex (BSA of 1200Å 2, equal contributions from the TCR a- and b-chains) (Figure 6b). Here, the MAV36 TCR b-chain and a-chain interacted with residues spanning and of the a1 and a2-helices of MR1, respectively (Supplementary Table 1). In comparison, the MAIT TCR interacted with residues ranging and of the a1 and a2 helices of MR1, respectively. Accordingly, the footprint of the TRAV1-2 - TCR and TRAV1-2 + TCR was more extensive over the a1 and a2 helix of MR1, respectively. Further, the centre of gravity of the MAIT TCR a- and TCR b-chains was displaced approximately 9Å towards the A -pocket in comparison to the corresponding chains of the MAV36 TCR (Figure 6c). Moreover, the atypical TCR b-chain was rotated 18 towards MR1, which enabled it to participate in more gemline-encoded interactions with MR1 in comparison to the MAIT TCR b-chain. Thus, the salient features of this atypical TRAV36 + TCR- MR1 interaction deviated from those of the TRAV1-2 + TCR-MR1 interaction. Atypical TCR-MR1-mediated contacts Given that the sequence of the MAV36 TCR a-chain was distinct from the invariant MAIT TCR a- chain (Supplementary Figure 1c), we compared how these two TCRs mediated an MR1-restricted response. Regarding the MAV36 TCR, the CDR1a loop participated extensively at the interface 11

12 (BSA 21.0%), contacting residues from the a1 and a2 helices of MR1. The interactions principally arose from Arg31a extending across the a2-helix, with its aliphatic moiety packing against Tyr152, and the guanidinium group forming van der Waals contacts with His148 and a salt-bridge with Glu149 (Figure 7a). In addition, Thr28a abutted Arg61, while Asn29a was wedged between the helical jaws of the Ag-binding cleft, contacting Tyr62 and Tyr152. Most unusual for TCR interactions 1, the N-terminal residues of the MAV36 TCR a-chain interacted with the Agpresenting molecule, namely, Asp2a, packed against and salt-bridged to Arg61 of MR1. The CDR2a loop (7.8 BSA%) played a lesser role in the interactions with MR1, running across the main axis of the a2-helix, between residues His148 and Tyr 152, and made a number of vdw contacts in this vicinity in addition to Thr50a hydrogen bonding to His148 (Figure 7a). The CDR3a loop (BSA 14.0%), while sitting centrally within the Ag-binding cleft (Figure 7b), played a moderate role in the interaction with MR1, with most of the interactions being hydrophobic in nature. Here Tyr92a, which is non-equivalent to the Tyr95a from the TRAJ33 region 33, packed against Glu100b with it s hydroxyl group hydrogen bonded to Gln99b. Further, other residues at the tip of the CDR3a loop, namely, Asn93a, Thr94a and Asp95a, packed against Leu65, Tyr152 and the main chain of Gly68 and Leu65, respectively (Figure 7b). Thus, whilst the CDR3a loop of the TRAV1-2 + MAIT TCRs played the prominent role in the interaction with MR1 33, the CDR1a loop of the TRAV36 + TCR was the key determinant. The role of the CDR1b loop (BSA 1.9%) was restricted to a Glu30b salt bridge to Arg79 (Figure 7c), while the CDR2b loop (BSA 16.9%) and neighbouring framework regions (BSA 13.8%) played much greater roles in mediating MR1 interactions. For example, Phe48b and Tyr50b converged onto a region spanning residues of the a1-helix, with this interaction network extended by Met54b packing against Val75 and Gln71, and Glu56b salt bridging to Arg41 of MR1 (Figure 7c). As such, the TRBV28 region of the MAV36 TCR appeared to participate in more favourable MR1 interactions in comparison to the germline encoded TRBV regions of the TRAV1-2 + MAIT TCR 33. The CDR3b loop was distal from the A -pocket of MR1 and was exclusively involved in MR1-mediated contacts (BSA 15.8%) (Supplementary Table 1, Figure 7d). Here, the CDR3b loop comprised a trio of bulky residues (Tyr98b-Gln99b-Glu100b) that contacted MR1, with Glu100b and Tyr98b sandwiching Asn146, and Glu100b salt bridging to Arg31 of MR1 (Figure 7b). Thus, while the CDR3b loops of TRAV1-2 + TCRs play predominant roles in MR1 interactions, and can directly fine-tune Ag-reactivity 32, the CDR3b loop of the MAV36 TCR 12

13 played a less prominent role and was distant from the bound Ag. Accordingly, TRAV1-2 + MAIT TCR-MR1-Ag and TRAV36 + TCR-MR-Ag interfaces were markedly distinct from one another. Atypical TCR-Antigen contacts The MAV36 TCR was dependent upon 5-OP-RU for activation, MR1 tetramer staining and measurable affinity using SPR, indicating that a direct interaction between the MAV36 TCR and 5- OP-RU underpinned these observations. Indeed, Asn29a from the CDR1a loop, which intercalated between Tyr62 and Tyr152 of MR1, hydrogen bonded to the ribityl tail of 5-OP-RU (Figure 7e). In comparison, Tyr95a from the CDR3a loop of the MAIT TCRs represented the contact point between the 5-OP-RU Ag and the invariant TRAV1-2TRAJ33 a-chain (Figure 7f). Interestingly, the positioning of Asn29a from the MAV36 TCR closely aligned with that of Tyr95a from the MAIT TCR (Figure 7e & f). Therefore, convergent recognition mechanisms amongst the diverse MR1-restricted T cell repertoire can permit the specific recognition of riboflavin-based metabolites. Discussion Highly conserved TCR a-chain usage, including by type I NKT cells (TRAV10-TRAJ18), Germline-Encoded Mycolyl-reactive (GEM) T cells (TRAV1-2-TRAJ9) and MAIT cells (TRAV1-2-TRAJ33), is a common feature in lipid and metabolite-mediated T-cell immunity 47, 35, 48. The TCR-b chains of these T cell populations are variable, contributing to TCR-specificity and finetuning Ag recognition 32, 45, 48, 49, 50. In addition, other CD1d and CD1b restricted T cells co-exist that express diverse TCR a-chains. For example, type II CD1d restricted NKT cells express a more diverse ab T-cell repertoire and can respond to a range of lipid Ags distinct from that of type I NKT cells 51. Like GEM T cells, other CD1b-restricted T cells known as LDN5-like T cells similarly utilise more diverse TCRs to recognise mycolate Ag presented by CD1b 52. Accordingly, we sought to investigate whether the MR1-restricted T cell repertoire displayed abtcr heterogeneity that could result in different functional responses. Ags derived from riboflavin biosynthesis are found in a wide range of bacteria and yeast that possess an intact riboflavin metabolic pathway and can activate MAIT cells in an MR1-restricted manner 38, 39, 41. The invariant TRAV1-2TRAJ33 a-chain of the MAIT TCR plays a critical role in mediating contacts with MR1 and enabling specific reactivity towards the microbially derived riboflavin-associated pyrimidine antigens. In particular, Tyr95a, a conserved residue encoded by the TRAJ33/TRAJ12/TRAJ20 gene elements, made a crucial hydrogen bonding contact with the ribityl tail of the ribityllumazines and transitory pyrimidines formed during riboflavin biosynthesis, 13

14 explaining how metabolites derived from riboflavin biosynthesis can act as MAIT cell agonists In contrast, antigens derived from folate metabolism do not possess a ribityl tail and fail to activate MAIT cells despite binding to MR1. These findings were supported by MR1 tetramer based studies, in which MR1-riboflavin-metabolite tetramers effectively stained all MAIT cells, in contrast to MR1-6-FP tetramers that failed to stain the majority of MAIT cells Recently it has been suggested that variation in the MAIT TCR b-chain repertoire, including CDR3b hypervariability, can modulate reactivity to riboflavin-associated metabolites 53. Here, we have shown that MR1-restricted TCR repertoire diversity can operate on two major and distinct levels. Firstly, within the TRAV1-2 + MAIT cell population, we describe MAIT cells with particular TCR b-chains that are activated by folate-derived Ags or display autoreactivity towards MR1. Moreover, some MAIT cells can discriminate between different types of folate-derivative antigens. Both MR1 autoreactivity and folate-metabolite responsiveness were attributable to subtle conformational shifts in an intricate interaction network that involved the hypervariable CDR3b loop, the ligand itself, Tyr95a, and neighbouring MR1 residues. The extent of contacts, and the shape complementarity of the CDR3b loop with its environment played a key role in mediating these effects. Moreover, this lessened the dependence of Tyr95a in mediating the MR1-restricted recognition, which is consistent with some TRAV1-2 + MR1-restricted T cells using TRAJ-encoded elements that do not possess this conserved Tyrosine within other TRAJ genes 53. Thus, within the TRAV1-2 + MAIT cell repertoire, populations exist that are responsive to folate-derived Ags and display MR1 autoreactivity. These findings share some similarities with autoreactive type I NKT cells 50, 54 and CD1a-restricted T cells 7, 55, 56, 57, and may support observations where MAIT cells have been implicated in autoimmune diseases and cancers where they presumably do not require presence of microbially-derived metabolites 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27. Further, we demonstrate that a diverse range of T cells utilise distinct TRAV-TRAJ genes that mediate MR1-restricted Ag recognition. Some of these atypical MR1-restricted T cells did not recognise 5-OP-RU, yet bound to folate-derivative Ags, indicating that these cells can exhibit diverse and non-overlapping MR1-restricted Ag specificities. While the TRAV1-2 + MAIT TCRs act as innate-like pattern recognition receptors 32, the atypical TRAV36 + TCR adopted a more central docking mode above MR1. The TRAV36 + TCR was positioned more towards the empty F pocket of MR1, thereby indicating that any ligands occupying this pocket could potentially be recognised by some atypical MR1-restricted TCRs. These findings suggest that MR1-restricted T cells can exhibit diverse docking strategies, in a manner that is analogous to the type I and type II NKT TCR recognition of CD1d 1. Interestingly, despite variations in TCR repertoire, the 14

15 convergence of key germline-encoded residues in mediating contacts with the ribityl moiety of the Ag was still apparent. The extent to which these atypical MR1-restricted T cells are functionally aligned to TRAV1-2 + MAIT cells is unclear, however the MAV36 TCR was isolated from a population of cells with a surface phenotype similar to that of MAIT cells, suggesting a similar developmental history. On the otherhand, other TRAV1-2 - populations lacked expression of these markers (data not shown), suggesting that TCR diversity can engender diversity in the developmental and functional programming of atypical MR1 restricted T cells. The population of the folate-derivative and autoreactive TRAV1-2 + and TRAV1-2 - MR1-restricted T cells were relatively small compared to the TRAV1-2 + riboflavin responsive MAIT cells. While MAIT cells are highly abundant in humans, these atypical MR1-restricted T-cells are in lower abundance but similar in number to type I NKT cells. Collectively, we have shown here that MR1- restricted immunity can be orchestrated by a diverse repertoire of T cells with fine-specificities towards chemically diverse small organic molecules, which suggests differing roles for MAIT cell subsets in health and disease. 15

16 Methods Human Samples Healthy human blood buffy coats were obtained from the Australian Red Cross Blood Service. Ethics approval was granted by both the Red Cross and the University of Melbourne Human Research and Ethics committee ( ). Peripheral blood mononuclear cells were isolated by standard density gradient (Ficoll-Paque Plus, GE Healthcare Life Science) and cryopreserved. Multiple myeloma patient blood and bone marrow samples were obtained from Peter Macallum Cancer Centre patients enrolled in the LITVACC clinical trial (Trial Number ; details available at after approval from its human ethics committee. Peripheral blood and bone marrow mononuclear cells were isolated by standard density gradient (Ficoll-Paque Plus, GE Healthcare Life Science) and cryopreserved. Cell Surface Staining Healthy human mononuclear cells were stained in FACS buffer (PBS + 2% FBS) with MR1-6-FP and MR1-Ac-6-FP tetramers for 45 min at room temperature, washed once with FACS buffer and either magnetically enriched by anti-phycoerythrin-mediated MACS purification (Miltenyi Biotec) or stained with surface antibodies in FACS buffer for a further 20 min on ice, prior to re-suspension in FACS buffer ready for flow cytometric acquisition. MR1-5-OP-RU tetramers were stained with the surface antibodies for 20 min on ice. Surface antibodies including those specific for CD3ε (UCHT1, BD Biosciences), CD4 (OKT4, Biolegend), CD8α (SK1 Biolegend), CD14 (M5E2, Biolegend), CD19 (HIB19, Biolegend), CD33 (IV M-505, Biolegend), CD57, CD69 (FN50 BD Biosciences), CD161 (HP-3G10, Biolegend), IL-18Rα (H44, Biolegend), pan-tcrγδ (11F2, BD Biosciences), TRAV1-2 (3C10, Biolegend), and viability dyes 7-aminoactinomycin (7-aad; Sigma) and Zombie Yellow (Biolegend). Flow Cytometry Data Analysis: All flow cytometry data was analysed using Flowjo Software (Treestar) and acquired using an LSRFortessa Analyser (BD Biosciences) equipped with a Yellow-Green laser. Unless stated otherwise, all FACS plots of primary human samples are gated on CD14 -, CD19 -, TCRγδ -, CD3 + lymphocytes after dead cell and doublet exclusion. CD33 + cells were also removed from bone marrow sample analysis. Jurkat cell lines are gated on viable, GFP + cells after dead cell and doublet removal. C1R cells were removed from analysis by CD19 exclusion where appropriate. Expansion of primary human T cells 16

17 Fresh, unenriched primary human T cells were sort purified and stimulated in vitro for 48 h with soluble anti-cd3 (OKT3 10 µg/ml), anti-cd28 (CD28.2, 2 µg/ml), phytohemagglutinin (1 µg/ml) plus rhuil-2 (200 U/ml, Peprotech) and rhuil-7 (50 ng/ml, ebioscience) and irradiated human PBMCs from 3 different allogeneic donors (mixed at 1:1:1; 50:1 irradiated PBMCs: sorted T cells). After 48 h, cells were removed from CD3, CD28 and Phytohemagglutinin and 2 weeks with irradiated PBMCs. After 2 weeks, T cells were stimulated again with plate-bound anti-cd3 (10 µg/ml), anti-cd28 (2 µg/ml) and phytohemagglutinin (1 µg/ml). Cells were allowed to expand for 2-3 weeks in rhuil-2 and rhuil-7 before cryopreservation. Cells were cultured in complete human T cell culture media consisting of a 1:1 (v/v) mix of RPMI-1640 and AIM-V (Invitrogen, Life Technologies) supplemented with 10% (v/v) FBS (JRH Biosciences), 2% (v/v) Human AB serum (Sigma) Penicillin (100 U/ml), Streptomycin (100 µg/ml), Glutamax (2 mm), sodium pyruvate (1 mm), nonessential amino acids (0.1 mm), HEPES buffer (15 mm), ph (all from Invitrogen, Life Technologies) and 2-mercaptoethanol (50µM, Sigma). Single Cell TCR Sequencing cdna from single cell sorted MR1 tetramer + αβt cells was produced by addition of 2 µl per well of buffer containing SuperScipt VILO (Invitrogen) and 0.1% Triton X-100 (Sigma) and incubated according to manufacturers instructions. Vα and Vβ transcripts were amplified by two rounds of semi-nested multiplexed PCR 58 using primers complementary to human TRAV, TRAC, TRBV and TRBC genes as previously described 59. PCR products were sequenced (Applied Genetics Diagnostics, Australia) and analysed using the IMGT database. Expression and purification of soluble proteins Human MR1-5-OP-RU, 6-FP 38 and Ac-6-FP 32, human MR1.K43A 7, mouse MR1-5-OP-RU, and 60, 61 human CD1d-αGalCer tetramers were generated in-house as previously described. TCR proteins were produced as previously described 62. In brief, genes encoding truncated extracellular ectodomains of human TCRα and TCRβ genes of either the M33.64 (and mutants thereof), MAV36 and AF-7 TCRs were synthesized (Life Technologies) and cloned into the pet30 (Novagen) expression vector. Genes were then expressed as inclusion bodies in Escherichia coli strain BL21 by recombinant methods. Purified inclusion bodies were solubilised in 8 M Urea, 20 mm Tris-HCl (ph8.0), 0.5 M Na-EDTA and 1mM DTT. Soluble TCRs were produced by oxidative refolding in 5M Urea, 10 mm Tris ph8.0, 2 mm Na-EDTA, 500 mm L-arginine-HCl, 0.5 mm oxidized glutathione, 5 mm reduced glutathione, PMSF and pepstatin A. Protein was dialysed in 10 mm Tris prior to purification by size-exclusion and ion-exchange chromatography (GE Healthcare). TCR proteins contained an engineered disulfide linkage between the TRAC and TRBC domains as 17

18 previously described 63. Soluble MR1-5-OP-RU 38 and MR1-Ac-6-FP 32 proteins were produced as previously described. In brief, genes encoding human MR1A heavy chain and β2-microglobulin were expressed and solubilised as per TCR chains above. MR1 protein was produced by oxidative refolding in the presence of 5-ARU and pyruvaldehyde in 10 mm Tris ph8.0, 2 mm Na-EDTA, 500 mm L-arginine-HCl, 0.5 mm oxidized glutathione, 5 mm reduced glutathione, PMSF and pepstatin A. Protein was dialysed in 10 mm Tris prior to purification by size-exclusion and ionexchange chromatography (GE Healthcare). Production of transient and stable TCR and MR1-expressing cell lines. Full-length TCRα and β chain genes were cloned into a 2A-peptide-linker pmig expression vector. Vectors were used to transiently transduce HEK293t cells, together with a similar pmig expression vector encoding CD3ε, δ, γ and ζ, using FUGENE transfection reagent (Promega). Cells were harvested after 48 h and stained with a panel of tetramers. pmig vectors described above were also used to generate stable cell lines by retroviral transduction of αβtcr-deficient Jurkat-76 cells, using HEK293T cells as packaging cells as previously described 64. C1R cells were similarly transduced to expressing human MR1 and sort purified to produce stable cells lines with graded expression of human MR1, as previously described 40. MR1-Alanine mutant cell lines were made previously 40. Activation assays For Assays using Jurkat.TCR cells, 5x10 4 Jurkat.TCR cells were incubated at 1:1 ratio with C1R cells for 18 h in 200 µl RF10 complete media (consisting of RPMI-1640 (Invitrogen, Life Technologies) supplemented with 10% (v/v) FBS (JRH Biosciences), 2% (v/v) Penicillin (100U/ml), Streptomycin (100µg/ml), Glutamax (2 mm), sodium pyruvate (1 mm), nonessential amino acids (0.1 mm), HEPES buffer (15 mm), ph (all from Invitrogen, Life Technologies) and 2-mercaptoethanol (50µM, Sigma)) with 5-OP-RU or PFA-fixed Escherichia coli. Cells were harvested, washed once in PBS and stained with CD19 (HIB19, Biolegend), CD69 (FN50 BD Biosciences), and 7-aminoactinomycin (7-aad; Sigma). For assys using primary cells, T cells were sort purified and cultured with 1x10 4 wildtype C1R cells treated with 5-OP-RU with or without MR1 blocking antibody (26.5) or C1R.MR1 cells in complete human T cell culture media, as described above. Cells were harvested, washed once with PBS and stained with CD19 (HIB19, Biolegend), CD69 (FN50 BD Biosciences), and 7-aminoactinomycin (7-aad; Sigma). Surface Plasmon Resonance 18

19 Surface plasmon resonance (SPR) experiments were conducted as described previously 33 in duplicate at 25 C on a BIAcore 3000 instrument using HBS buffer consisting of 10 mm HEPES- HCl at ph 7.4, 150 mm NaCl, and 0.005% (v/v) surfactant P20 (GE Healthcare). Biotinylated C- terminal cysteine-tagged-mr1-ag was immobilized on a SA-Chip with a surface density of between response units (RU). Concentrations of MAIT TCRs between µm, were injected over the captured MR1-Ag at 10 µl/min. The final response was calculated by subtracting the response of a biotin-labelled flow cell alone from that of the TCR-MR1-Ag complex. The affinity constants were calculated from equilibrium data using GraphPad Prism. Crystallization, structure determination and refinement. Purified MR1-b 2 M-Ag was mixed with purified MAIT TCR in a 1:1 molar ratio to a final concentration of between 1-8 mg/ml, which was then crystallised using the vapour diffusion hanging drop method, by mixing protein solution with precipitant in a ratio of 1:1 or 1:2, and storing at 20 C in the dark. The precipitants used consisted of 100 mm bis-tris propane, which varied in ph from , 0.2 M sodium acetate and 8-20% (w/v) PEG3350. Crystals grew over 1-14 days. Samples were cryo-protected in a solution that consisted of precipitant solution, which also contained 10% (w/v) glycerol, prior to being drop frozen in liquid N 2. Diffraction data was collected at 100K on the Australian synchrotron MX1 and MX2 beamlines, where reflections were indexed and integrated using either XDS 65 or imosflm, and scaled using pointless 66. Phases were calculated by molecular replacement using PHASER 67, whereby the MR1 ternary complex, which had the CDR loops and ligand removed, was used as a search model for the M33 ternary complex structures. The TRBV6-1 TCR only was used as a search model for the unliganded MAV36 TCR (PDB ID: 4L4T; 33 ). For the MAV36 ternary, the unliganded MAV36 TCR was used in combination with the MR1-b 2 M (PDB ID: 4L4T; 33 ), without ligand. Refinement was performed using phenix.refine 68, with the initial refinement rounds including simulated annealing. Model building was performed with COOT 69, with MolProbity used in validation 70. The Grade Web Server was used to generate ligand restraints for Ac-6FP and 5-OP-RU. The buried surface area and the centre of mass of select domains, was calculated by the CCP4 71 implementation of Areaimol and molecular interactions were determined by the CCP4 implementation of CONTACT. All molecular graphics were constructed using PyMOL. Competing Financial Interests The authors declare no competing financial interests Acknowledgments 19

20 We thank John Waddington, Marcin Ciula and Bronwyn Meehan for technical assistance and advice. We thank the Monash Macromolecular Crystallization Facility for support and the Australian Synchrotron staff for assistance with data collection. We thank staff from the Flow Cytometry facility at The University of Melbourne and Melbourne Brain Centre. We are grateful to Dr Dario Vignali (St. Jude Children s research hospital) and Prof. Stephen Turner (The University of Melbourne) for providing pmig expression vector; Dr Paul Savage (Brigham Young University, UT, USA) for a-galcer analogue PBS-44. This work was supported by the National Health and Medical Research Council of Australia (NHMRC; ) and the Australian Research Council (ARC; CE and LE ). NAG is supported by a Leukaemia Foundation of Australia Postgraduate Scholarship; APU is supported by an ARC Future Fellowship; DGP is supported by an NHMRC ECF Fellowship ( ); JR is supported by an NHMRC Australia Fellowship (AF50); DIG and DPF are supported by NHMRC Senior Principal Research Fellowships ( , ). Author Contributions NG and AK are joint first authors; JLN, RWB,APU, DGP, JMc, DSR, PJN, DPF and LL either undertook/supervised experiments and or/ analysed data, and/or provided key reagents or patient samples; JR and DIG co-led the investigation and are co-senior authors. 20