T cell receptor interactions with human leukocyte antigen govern indirect peptide selectivity for the cancer testis antigen MAGE-A4

T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies. T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies. T cell–mediated immunity is essential during pathogen and cancer surveillance and plays a key role in autoimmunity and transplant rejection (1Coppieters K.T. 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Gostick E. Price D.A. Gao G.F. Jakobsen B.K. Sewell A.K. Germ line-governed recognition of a cancer epitope by an immunodominant human T-cell receptor.J. Biol. Chem. 2009; 284 (19605354): 27281-2728910.1074/jbc.M109.022509Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Additionally, recent evidence has demonstrated that it is not only the TCR-accessible residues in the peptide that can determine TCR selectivity (31Tynan F.E. Reid H.H. Kjer-Nielsen L. Miles J.J. Wilce M.C.J. Kostenko L. Borg N.A. Williamson N.A. Beddoe T. Purcell A.W. Burrows S.R. McCluskey J. Rossjohn J. A T cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class I molecule.Nat. Immunol. 2007; 8 (17259989): 268-27610.1038/ni1432Crossref PubMed Scopus (174) Google Scholar, 32Bianchi V. Bulek A. Fuller A. Lloyd A. Attaf M. Rizkallah P.J.P.J. Dolton G. Sewell A.K.A.K. Cole D.K.D.K. A molecular switch abrogates glycoprotein 100 (gp100) T-cell receptor (TCR) targeting of a human melanoma antigen.J. Biol. Chem. 2016; 291 (26917722): 8951-895910.1074/jbc.M115.707414Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 33Cole D.K. Edwards E.S.J. Wynn K.K. Clement M. Miles J.J. Ladell K. Ekeruche J. Gostick E. Adams K.J. Skowera A. Peakman M. Wooldridge L. Price D.A. Sewell A.K. Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.J. Immunol. 2010; 185 (20639478): 2600-261010.4049/jimmunol.1000629Crossref PubMed Scopus (93) Google Scholar). In this study, we solved the structure of a newly identified TCR in complex with an HLA-A*02:01 restricted melanoma antigen-A4 (MAGE-A4) peptide (GVYDGREHTV), hereafter referred to as A2-GVY. A2-GVY is a key oncology target because it is expressed by many tumors, including esophageal, head and neck, lung, ovarian, bladder, breast, lymphomas, and melanoma (34Marchand M. van Baren N. Weynants P. Brichard V. Dréno B. Tessier M.-H. Rankin E. Parmiani G. Arienti F. Humblet Y. Bourlond A. Vanwijck R. Liénard D. Beauduin M. Dietrich P.-Y. et al.Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1.Int. J. Cancer. 1999; 80 (9935203): 219-23010.1002/(SICI)1097-0215(19990118)80:2<219::AID-IJC10>3.0.CO;2-SCrossref PubMed Scopus (756) Google Scholar, 35Hillig R.C. Coulie P.G. Stroobant V. Saenger W. Ziegler A. Hülsmeyer M. High-resolution structure of HLA-A*0201 in complex with a tumour-specific antigenic peptide encoded by the MAGE-A4 gene.J. Mol. Biol. 2001; 310 (11502003): 1167-117610.1006/jmbi.2001.4816Crossref PubMed Scopus (45) Google Scholar). Our data demonstrated that the TCR bound in a position shifted toward the peptide N terminus. This enabled the TCR to achieve peptide selectivity for A2-GVY via a novel indirect mechanism whereby the TCR sensed the peptide features through an HLA residue, which acted as a tunable gateway or bridge. The altered side-chain conformation of this HLA residue was an antigenic feature for the TCR, correlating with the shifted binding footprint, to provide exquisite selectivity for the MAGE-A4 peptide N-terminal residue. Overall, our molecular characterization of TCR binding to a clinically relevant MAGE-A4 epitope extends our understanding of the general rules that underpin TCR antigen recognition. Following stimulation with peptide, we isolated an A2-GVY–specific TCR (GVY01) from CD8+ T cells from healthy HLA-A*02:01-positive donor peripheral blood mononuclear cells. The GVY01 TCR was identified from a panel of TCRs sequenced from A2-GVY tetramer-positive CD8+ T cells, and selected based on its ability to be expressed and refolded as a soluble molecule in bacteria and its ability to bind to A2-GVY in biophysical experiments. A structure of the GVY01-A2-GVY TCR-pHLA trimolecular complex was solved to 3 Å resolution (Table 1). The structure was characterized by well-defined observed electron density around the TCR-pHLA interface (Fig. S1). The GVY01 TCR bound to A2-GVY in a position shifted toward the peptide N terminus. Other TCR-pHLA interaction parameters were more typical (24Rossjohn J. Gras S. Miles J.J. Turner S.J. Godfrey D.I. McCluskey J. T cell antigen receptor recognition of antigen-presenting molecules.Annu. Rev. Immunol. 2015; 33 (25493333): 169-20010.1146/annurev-immunol-032414-112334Crossref PubMed Scopus (382) Google Scholar): the TCR having a crossing angle of 101°, with the α-chain positioned over the HLAα2 helices and N terminus of the peptide, the TCR β-chain positioned over the HLAα1 helices and C terminus of the peptide, and the TCR CDR3α and CDR3β loops sitting directly above the peptide (Fig. 1, A and B).Table 1Data collection and refinement statisticsA2-AVYGVY01-A2-GVY TCR-pHLA complexPDB accession code6TRN6TROData collection Space groupP21P64 2 2 Cell dimensions a, b, c (Å)56.0, 80.7, 58.7220.0, 220.0, 96.8 α, β, γ (degrees)90.0, 115.1, 90.090.0, 90.0, 120.0 Resolution (Å)48.31–1.35 (1.37–1.35)67.91–3.00 (3.05–3.00) Rmerge (%)4.7 (92.1)9.4 (180) Rpim (%)2.8 (65.9)3.7 (68.7) CC½0.999 (0.552)0.998 (0.499) I/σI12.2 (1.0)13.5 (1.21) Completeness (%)99.5 (99.7)98.6 (99.6) Multiplicity3.6 (2.9)6.60 (6.80)Refinement Resolution (Å)48.31–1.35 (1.39–1.35)67.91–3.00 (3.08–3.00) No. of reflections97,575 (7596)26,360 (2023) Rwork/Rfree16.1/20.0 (29.2/32.5)21.3/27.0 (34.1/37.3) No. of atoms Protein31906572 Water43923 Ethylene glycol12 B-Factors Protein24.1111.6 Water36.873.8 Ethylene glycol25.9 Root mean square deviations Bond lengths (Å)0.0080.002 Bond angles (degrees)1.4671.203 Open table in a new tab Despite this N-terminal shift, the GVY01 TCR made conserved contacts (with respect to many other TCR-pHLA complex structures (24Rossjohn J. Gras S. Miles J.J. Turner S.J. Godfrey D.I. McCluskey J. T cell antigen receptor recognition of antigen-presenting molecules.Annu. Rev. Immunol. 2015; 33 (25493333): 169-20010.1146/annurev-immunol-032414-112334Crossref PubMed Scopus (382) Google Scholar)) with two of the HLA restriction triad residues, Arg-65 and Ala-69, in addition to the central solvent-exposed residues in the peptide (Asp-4, Gly-5, Arg-6, and Gln-7) (Table S1 and Fig. 1C). However, its N-terminal shift abrogated its ability to make direct contacts with the third HLA restriction triad residue, Gln-155. Aside from these residues, the GVY01 TCR made most contacts with HLA residues Glu-166 and Trp-167, and peptide contacts were focused toward Asp-4, with Arg-6 making the second highest number of contacts with the GVY01 TCR (Table S1 and Fig. 1C). These interactions resulted in a total TCR-pHLA buried surface area of 2018 Å2 and ∼30% of the total contacts with the GVY peptide, which are both within the normal range for TCR-pHLA complexes (24Rossjohn J. Gras S. Miles J.J. Turner S.J. Godfrey D.I. McCluskey J. T cell antigen receptor recognition of antigen-presenting molecules.Annu. Rev. Immunol. 2015; 33 (25493333): 169-20010.1146/annurev-immunol-032414-112334Crossref PubMed Scopus (382) Google Scholar). Comparison of the previously published unligated A2-GVY co-complex (35Hillig R.C. Coulie P.G. Stroobant V. Saenger W. Ziegler A. Hülsmeyer M. High-resolution structure of HLA-A*0201 in complex with a tumour-specific antigenic peptide encoded by the MAGE-A4 gene.J. Mol. Biol. 2001; 310 (11502003): 1167-117610.1006/jmbi.2001.4816Crossref PubMed Scopus (45) Google Scholar) and the GVY01-A2-GVY TCR-pHLA trimolecular complex suggested some stabilization of the peptide residues Arg-6 and His-8 upon TCR ligation (Fig. 2A); however, significant conformational changes to A2-GVY were not apparent from the structural data. Importantly, the TCR-pHLA contact interface at Asp-4 and Arg-6 did not require major structural remodeling of A2-GVY (Fig. 2B), as has been observed for other TCR-pHLA interactions (31Tynan F.E. Reid H.H. Kjer-Nielsen L. Miles J.J. Wilce M.C.J. Kostenko L. Borg N.A. Williamson N.A. Beddoe T. Purcell A.W. Burrows S.R. McCluskey J. Rossjohn J. A T cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class I molecule.Nat. Immunol. 2007; 8 (17259989): 268-27610.1038/ni1432Crossref PubMed Scopus (174) Google Scholar, 36Madura F. Rizkallah P.J. Holland C.J. Fuller A. Bulek A. Godkin A.J. Schauenburg A.J. Cole D.K. Sewell A.K. Structural basis for ineffective T-cell responses to MHC anchor residue-improved "heteroclitic" peptides.Eur. J. Immunol. 2015; 45 (25471691): 584-59110.1002/eji.201445114Crossref PubMed Scopus (44) Google Scholar, 37Madura F. Rizkallah P.J. Legut M. Holland C.J. Fuller A. Bulek A. Schauenburg A.J. Trimby A. Hopkins J.R. Wells S.A. Godkin A. Miles J.J. Sami M. Li Y. Liddy N. et al.TCR-induced alteration of primary MHC peptide anchor residue.Eur. J. Immunol. 2019; 49 (31091334): 1052-106610.1002/eji.201948085Crossref PubMed Scopus (11) Google Scholar). Further inspection of the GVY01-A2-GVY TCR-pHLA trimolecular complex revealed that TCR CDR1α residue Pro-29 stacked closely against HLA residue Trp-167 (estimated ΔG = −1.9 kcal mol), which in turn packed against peptide position Gly-1 (estimated ΔG = −2.5 kcal mol), forming an indirect bridge between the TCR and peptide residue Gly-1 (Fig. 3A). This relatively strong indirect TCR-peptide interaction opened the possibility that the TCR might be able to sense the chemical properties of the peptide residue in this position. Reanalysis of the trimolecular complex structure of another N-terminally shifted TCR, CF34 (26Gras S. Burrows S.R. Kjer-Nielsen L. Clements C.S. Liu Y.C. Sullivan L.C. Bell M.J. Brooks A.G. Purcell A.W. McCluskey J. Rossjohn J. The shaping of T cell receptor recognition by self-tolerance.Immunity. 2009; 30 (19167249): 193-20310.1016/j.immuni.2008.11.011Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), demonstrated a similar indirect interaction between the TCR CDR1α residue Pro-30 and peptide residue Phe-1 via HLA residue Trp-167, despite Trp-167 being shifted back toward the HLAα2 helices to accommodate the larger peptide P1 side chain in this structure (Fig. 3B). Thus, this observation is not isolated to the GVY01 TCR and may represent a more general TCR mechanism to sense peptide and achieve pHLA binding specificity. We reanalyzed the structures of several other peptide-HLA-A*02:01 complexes with different amino acids at peptide residue 1 (Fig. 3C and Fig. S2). This analysis revealed that Trp-167 acts as a molecular gateway, altering its angle with respect to the HLAα2 helices, depending on the identity of peptide residue 1. Peptides with Gly-1 formed the first distinct family with respect to the position of Trp-167. For peptides with Ala at position 1, Trp-167 substantially altered its position to accommodate the slightly larger side chain, representing a second family. Ser, Thr, and Cys at peptide position 1 represented a third mechanism of distinct positioning of Trp-167, and three further families were apparent; the fourth with Val, Ile, Leu, and Pro; the fifth with Phe and Tyr; and the sixth with Glu, Asp, Arg, and Lys (Fig. S2). These observations suggested that the GVY01 TCR might be sensitive to peptide alterations at position 1, despite making no direct contact with this peptide residue. The refolded soluble GVY01 TCR extracellular domain bound to A2-GVY with a KD of 160 μm as measured by surface plasmon resonance (Fig. 3D). This is within the binding affinity range of other tumor-specific TCRs (7Bridgeman J.S. Sewell A.K. Miles J.J. Price D.A. Cole D.K. Structural and biophysical determinants of αβ T-cell antigen recognition.Immunology. 2012; 135 (22044041): 9-1810.1111/j.1365-2567.2011.03515.xCrossref PubMed Scopus (80) Google Scholar, 8Aleksic M. Liddy N. Molloy P.E. Pumphrey N. Vuidepot A. Chang K.-M. Jakobsen B.K. Different affinity windows for virus and cancer-specific T-cell receptors: implications for therapeutic strategies.Eur. J. Immunol. 2012; 42 (22949370): 3174-317910.1002/eji.201242606Crossref PubMed Scopus (163) Google Scholar, 38Cole D.K. Pumphrey N.J. Boulter J.M. Sami M. Bell J.I. Gostick E. Price D.A. Gao G.F. Sewell A.K. Jakobsen B.K. Human TCR-binding affinity is governed by MHC class restriction.J. Immunol. 2007; 178 (17442956): 5727-573410.4049/jimmunol.178.9.5727Crossref PubMed Scopus (154) Google Scholar). Binding affinity measurements of the GVY01 TCR to A2-GVY containing a G1A peptide mutation (the most conservative mutation possible) demonstrated a complete loss of binding (Fig. 3E). To explore this finding further, we solved the co-complex structure of A2-AVY at 1.35 Å (Table 1 and Fig. S1). A superposition of A2-GVY in the GVY01-A2-GVY TCR-pHLA trimolecular complex with the A2-AVY co-complex demonstrated that an Ala in position 1 would result in the CH2 of HLA residue Trp-167 shifting 1.6 Å toward TCR CDR1α loop residue Pro-29, disrupting the indirect interaction between the GVY01 TCR and the peptide through steric hindrance (Fig. 3F). A2-GVY is an important therapeutic target for a broad range of tumors. Thus, we used our previously described methods (39Li Y. Moysey R. Molloy P.E. Vuidepot A.-L. Mahon T. Baston E. Dunn S. Liddy N. Jacob J. Jakobsen B.K. Boulter J.M. Directed evolution of human T-cell receptors with picomolar affinities by phage display.Nat. Biotechnol. 2005; 23 (15723046): 349-35410.1038/nbt1070Crossref PubMed Scopus (309) Google Scholar) to engineer an affinity-enhanced version of the GVY01 TCR, which contained four mutations in the CDR3β loop (Fig. 4, A and B). The selectivity of this affinity-enhanced GVY01 TCR (GVY01_αwtβ1) was determined by measuring its binding affinity to pHLA variants with individual peptide alanine substitutions. As with the GVY01 TCR, the GVY01_αwtβ1 TCR was also highly sensitive to changes at peptide position 1 (Fig. 4B). However, compared with the GVY01 TCR, the GVY01_αwtβ1 TCR bound to all of the peptide alanine mutants, including G1A, with a measurable affinity, thereby enabling a better comparison of the effects of point mutations in the TCR on the bridge between peptide residue 1 and Trp-167. For these analyses, we generated an alternative version of the GVY01_αwtβ1 TCR in which Pro-29 (the main TCR residue contacting HLA residue Trp-167) was mutated to Ala (GVY01_αwtβ1_P29A). For the GVY01_αwtβ1 TCR, a G1A peptide substitution reduced the binding affinity by 115-fold, whereas the GVY01_αwtβ1_P29A mutated TCR bound with only a 7-fold loss in affinity (Fig. 4C). Full analysis of Ala mutations along the peptide also confirmed these differences, in that, for the GVY01_αwtβ1 TCR, a G1A peptide substitution was the most detrimental to binding, whereas for the GVY01_αwtβ1_P29A TCR, G1A substitution made much less of an impact (Fig. 4D and Fig. S3). These data d