Volume 44, Issue 1, Pages 32-45 (January 2016) Diversity of T Cells Restricted by the MHC Class I-Related Molecule MR1 Facilitates Differential Antigen Recognition  Nicholas A. Gherardin, Andrew N. Keller, Rachel E. Woolley, Jérôme Le Nours, David S. Ritchie, Paul J. Neeson, Richard W. Birkinshaw, Sidonia B.G. Eckle, John N. Waddington, Ligong Liu, David P. Fairlie, Adam P. Uldrich, Daniel G. Pellicci, James McCluskey, Dale I. Godfrey, Jamie Rossjohn  Immunity  Volume 44, Issue 1, Pages 32-45 (January 2016) DOI: 10.1016/j.immuni.2015.12.005 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Identification and Characterization of Atypical MR1-Reactive T Cells (A) MR1 tetramer staining of αβ T cells in two example healthy donors. (B) Summary of 12 human samples examined across three separate experiments, showing (i) the percentage of MR1-Ag tetramer+ cells of total αβ T cells, (ii) the percentage of MR1-Ag tetramer+ cells of total TRAV1-2+ T cells, and (iii) the percentage of MR1-Ag tetramer+ cells of total TRAV1-2− T cells. (C) (i) Flow cytometry plots showing MR1-Ag tetramer staining on TRAV1-2− and TRAV1-2+ αβ T cells before and after magnetic bead enrichment from two representative healthy donors. (ii) Histogram overlays showing cell surface marker expression on T cell subsets as defined by gates in (i). (D) Histograms showing collective cell surface Ag expression data from five magnetically enriched healthy donor PBMC samples examined across three separate experiments and gated as per (C). (E) Representative transcription factor staining of MR1-reactive T cell subsets after magnetic bead enrichment. (i) shows flow cytometry plots and gates from a representative donor and (ii) shows transcription factor histogram overlays from gates derived in (i). Experiment is representative of six healthy donors analyzed across three individual experiments. (F) Flow cytometry plots showing MR1-Ac-6-FP and MR1-5-OP-RU dual tetramer staining on total αβ T cells pre-enrichment and TRAV1-2− or TRAV1-2+ αβ T post magnetic bead enrichment from two healthy donor PBMC samples, representative of six donors examined across two separate experiments. All box plots extend from 25th and 75th percentiles and whiskers extend to minimum and maximum values. See also Figures S1–S3. Immunity 2016 44, 32-45DOI: (10.1016/j.immuni.2015.12.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 TCR Gene Usage and Sequences of Atypical MR1-Reactive T Cells (A) Gene usage and CDR3α and CDR3β sequences derived from single T cells sorted based on MR1 tetramer and TRAV1-2 antibody staining. N.D., not determined; Red text represents residues encoded by non-templated nucleotides; ∗TRBV8-2 or TRBV8-3; ∗∗ TRBV4-2 or TRBV4-3; ∗∗∗ TRBV5-4 or TRBV5-8; ∗∗∗∗Open reading frame (Matulis et al., 2010; Tilloy et al., 1999; Eckle et al., 2014; Reantragoon et al., 2013). (B) Pie-charts showing distribution of (i) TRAV, (ii) TRAJ, and (iii) TRBV genes by single-cell sorted TRAV1-2− MR1-reactive T cells. Immunity 2016 44, 32-45DOI: (10.1016/j.immuni.2015.12.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Diverse Ag Reactivity by MR1-Reactive TCRs Histograms depicting staining intensity of MR1-Ag tetramer labeled 293T cells, transiently transfected to express surface TCRs as indicated (left column), stained with a panel of MR1 and CD1d tetramers (top row). White histograms represent TCRNEG cells and gray histograms represent TCR+ cells from the same file. Numbers in top right refer to MFI of gray profile. Samples were gated to equivalent expression of surface TCR. This experiment was performed twice with similar results. Immunity 2016 44, 32-45DOI: (10.1016/j.immuni.2015.12.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Activation and Affinity of Autoreactive and Atypical MR1-Restricted TCRs (A) Bar graphs depicting activation of Jurkat.TCR cell lines co-cultured with C1R.MR1 cells in the presence of titrating quantities of (i) fixed E. coli bacteria or (ii) purified 5-OP-RU. Activation is expressed as fold-change from basal CD69 expression by Jurkat.TCR cells alone. Experiments were performed twice with similar results. Error bars refer to SEM of two replicate wells. (B) (i) Histogram overlay of MR1 surface expression on wild-type C1R (black; isotype = white) and C1R cell lines engineered to express low (mid gray) and high (light gray) amounts of MR1. (ii) Bar graph depicting CD69 expression on Jurkat.M33.64 cell line after overnight co-culture with graded MR1 expressing C1R cell lines as shown in (i). CD69 expression is expressed as fold-change from Jurkat.M33.64 cells cultured alone. Bars are average of duplicate wells from one of two independent experiments. (C) Bar graphs depicting MFI of MR1-5-OP-RU tetramer staining of HEK293T cells transiently transfected with wild-type MAIT TCRs and Tyr95α mutants. N.D., not done. Experiment was performed twice with similar results. Error bars depict SEM of duplicate wells. (D) Bar graphs depicting activation of Jurkat.TCR cell lines co-cultured with C1R.MR1 cells in the presence of titrating quantities of purified 6-FP (white bars), Ac-6-FP (gray bars), or 5-OP-RU (black bars). Activation is expressed as fold-change from basal CD69 expression by Jurkat.TCR cells alone. Experiment was performed three times with similar results. Error bars refer to SEM of two replicate wells. (E) The affinity of TCR-MR1-Ag binding was measured by surface plasmon resonance, testing M33.64, M33.64.Tyr95αPhe, A-F7, and MAV36 TCRs against MR1-5-OP-RU, MR1-6-FP, and MR1-Ac-6-FP. Relative binding affinities for each TCR is depicted. Error bars denote SE. Data are representative of two independent experiments. Immunity 2016 44, 32-45DOI: (10.1016/j.immuni.2015.12.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Folate-Metabolite Responsiveness and MR1 Autoreactivity The CDR3α and CDR3β loops of M33.64 TCR (A) and TRAV1-2+ TCR (TRBV6-4, PDBID: 4PJ7) (B), interacting with MR1. (C) Superimposition of the CDR loops of M33.64 (darker colored loops) and TRAV1-2+ (lighter colored loops) sitting atop MR1. (D) Position of Tyr152 when bound to M33.64 TCR (white) compared to the Tyr95αPhe mutant (gray) when ligating 5-OP-RU. (E) Position of Tyr152 when M33 TCR ligates Ac-6-FP (white) compared to 5-OP-RU (gray). (F) The position of Tyr152 in relation to the CDR3β, in the MAIT TCR (TRBV6-1 – A-F7; PDBID 4NQC) ternary structure. Image color coding: CDR1α, teal; CDR2α, pink; CDR3α, yellow; CDR1β, cyan; CDR2β, red; CDR3β, orange; Ac-6FP/5-OP-RU, green; MR1, gray; and dotted lines signify H-bonds (black) or salt-bridges (red). See also Table S1. Immunity 2016 44, 32-45DOI: (10.1016/j.immuni.2015.12.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 MAV36 TCR Docking Mode (A) The TRAV1-2+ ternary complex (TRBV6-1 – A-F7; PDBID: 4NQC) structure and associated footprint on the MR1 surface, and (B) the MAV36 ternary complex and associated footprint. Spheres represent the center of mass of the TCR Vα (slate) and Vβ (purple) domains. Color of CDR loop contacts, are consistent with the CDR loop colors in Figure 5, while TCR α chain and β-chains are shown in slate and purple, respectively. (C) Comparison of the MAV36 ternary complex relative to the TRAV1-2+ TCR docking position (yellow). Arrows detail TCR rotation around the center of mass of the MR1 α1α2-domains, as well as displacement along MR1 binding cleft. See also Tables S1 and S2. Immunity 2016 44, 32-45DOI: (10.1016/j.immuni.2015.12.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Molecular Contacts of the MAV36 TCR-MR1-Ag Complex Interactions between MR1 and (A) the CDR1α, CDR2α and Vα framework, (B) CDR3α and CDR3β, and (C) CDR1β, CDR2β and Vβ framework. (D) Superimposition of the CDR loops of MAV36 (darker colored loops) and TRAV1-2+ (TRBV6-1 – A-F7, lighter colored loops) in relation to the internal surface of the MR1 binding cleft. (E) Interaction of the CDR1α and CDR3α with 5-OP-RU, in the MAV36 ternary complex. (F) Interaction of the CDR3a with 5-OP-RU, in a TRAV1-2+ TCR ternary complex. Image color-coding consistent with Figures 5 and 6. See also Table S2. Immunity 2016 44, 32-45DOI: (10.1016/j.immuni.2015.12.005) Copyright © 2016 Elsevier Inc. Terms and Conditions