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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	CD4+ T cells recognize conserved influenza a epitopes through shared patterns of V-gene usage and complementary biochemical features Alexander Greenshields-Watson , Meriem Attaf , Bruce J. Maclachlan , Thomas Whalley , Cristina Rius , Aaron Wall , Angharad Lloyd , Hywel Hughes , Kathryn E. Strange , Georgina H. Mason , Andrea J. Schauenburg , Sarah L. Hulin-Curtis , James Geary , Yuan Chen , Sarah N. Lauder , Kathryn Smart , Dhanasekaran Vijaykrishna , Miguel L. Grau , Mikhail Shugay , Robert Andrews , Garry Dolton , Pierre J. Rizkallah , Awen M. Gallimore , Andrew K. Sewell , Andrew J. Godkin , David K. Cole Cell Reports 32 DOI: 10.1016/j.celrep.2020.107885 Diamond Proposal Number(s): [10462, 14843] Open Access Show Abstract ▼ Abstract: T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies.	Jul 2020
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	T cell receptor interactions with human leukocyte antigen govern indirect peptide selectivity for the cancer testis antigen MAGE-A4 Charlotte Coles , Catriona Mcmurran , Angharad Lloyd , Miriam Hock , Linda Hibbert , Marine C. C. Raman , Conor Hayes , Patrick Lupardus , David K. Cole , Stephen Harper Journal Of Biological Chemistry DOI: 10.1074/jbc.RA120.014016 Diamond Proposal Number(s): [17077] Open Access Show Abstract ▼ Abstract: T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide human leukocyte antigen complexes (pHLA) and is essential for immunosurveillance and disease control. This interaction is generally stabilised 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 Trp167, which acted as a tuneable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp167 sidechain 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.	Jun 2020
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	TCRs with distinct specificity profiles use different binding modes to engage an identical peptide–HLA complex Charlotte Coles , Rachel M. Mulvaney , Sunir Malla , Andrew Walker , Kathrine J. Smith , Angharad Lloyd , Kate L. Lowe , Michelle L. Mccully , Ruth Martinez Hague , Milos Aleksic , Jane Harper , Samantha J. Paston , Zoe Donnellan , Fiona Chester , Katrin Wiederhold , Ross A. Robinson , Andrew Knox , Andrew R. Stacey , Joseph Dukes , Emma Baston , Sue Griffin , Bent K. Jakobsen , Annelise Vuidepot , Stephen Harper Journal Of Immunology 204, 1943-1953 DOI: 10.4049/jimmunol.1900915 Open Access Show Abstract ▼ Abstract: The molecular rules driving TCR cross-reactivity are poorly understood and, consequently, it is unclear the extent to which TCRs targeting the same Ag recognize the same off-target peptides. We determined TCR–peptide–HLA crystal structures and, using a single-chain peptide–HLA phage library, we generated peptide specificity profiles for three newly identified human TCRs specific for the cancer testis Ag NY-ESO-1157–165–HLA-A2. Two TCRs engaged the same central peptide feature, although were more permissive at peripheral peptide positions and, accordingly, possessed partially overlapping peptide specificity profiles. The third TCR engaged a flipped peptide conformation, leading to the recognition of off-target peptides sharing little similarity with the cognate peptide. These data show that TCRs specific for a cognate peptide recognize discrete peptide repertoires and reconciles how an individual’s limited TCR repertoire following negative selection in the thymus is able to recognize a vastly larger antigenic pool.	Apr 2020
I04-1-Macromolecular Crystallography (fixed wavelength)	Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA Christopher J. Holland , Rory M. Crean , Johanne M. Pentier , Ben De Wet , Angharad Lloyd , Velupillai Srikannathasan , Nikolai Lissin , Katy A. Lloyd , Thomas H. Blicher , Paul J. Conroy , Miriam Hock , Robert J. Pengelly , Thomas E. Spinner , Brian Cameron , Elizabeth A. Potter , Anitha Jeyanthan , Peter E. Molloy , Malkit Sami , Milos Aleksic , Nathaniel Liddy , Ross A. Robinson , Stephen Harper , Marco Lepore , Chris R. Pudney , Marc W. Van Der Kamp , Pierre J. Rizkallah , Bent K. Jakobsen , Annelise Vuidepot , David K. Cole Journal Of Clinical Investigation 130, 2673 - 2688 DOI: 10.1172/JCI130562 Diamond Proposal Number(s): [17077, 14843] Show Abstract ▼ Abstract: Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.	Apr 2020
I24-Microfocus Macromolecular Crystallography	Peptide super-agonist enhances T-cell responses to melanoma Sarah A. E. Galloway , Garry Dolton , Meriem Attaf , Aaron Wall , Anna Fuller , Cristina Rius , Valentina Bianchi , Sarah Theaker , Angharad Lloyd , Marine E. Caillaud , Inge Marie Svane , Marco Donia , David K. Cole , Barbara Szomolay , Pierre Rizkallah , Andrew K. Sewell Frontiers In Immunology 10 DOI: 10.3389/fimmu.2019.00319 Diamond Proposal Number(s): [14843] Open Access Show Abstract ▼ Abstract: Recent immunotherapeutic approaches using adoptive cell therapy, or checkpoint blockade, have demonstrated the powerful anti-cancer potential of CD8 cytotoxic T-lymphocytes (CTL). While these approaches have shown great promise, they are only effective in some patients with some cancers. The potential power, and relative ease, of therapeutic vaccination against tumour associated antigens (TAA) present in different cancers has been a long sought-after approach for harnessing the discriminating sensitivity of CTL to treat cancer and has seen recent renewed interest following cancer vaccination successes using unique tumour neoantigens. Unfortunately, results with TAA-targeted “universal” cancer vaccines (UCV) have been largely disappointing. Infectious disease models have demonstrated that T-cell clonotypes that recognise the same antigen should not be viewed as being equally effective. Extrapolation of this notion to UCV would suggest that the quality of response in terms of the T-cell receptor (TCR) clonotypes induced might be more important than the quantity of the response. Unfortunately, there is little opportunity to assess the effectiveness of individual T-cell clonotypes in vivo. Here, we identified effective, persistent T-cell clonotypes in an HLA A2+ patient following successful tumour infiltrating lymphocyte (TIL) therapy. One such T-cell clone was used to generate super-agonist altered peptide ligands (APLs). Further refinement produced an APL that was capable of inducing T-cells in greater magnitude, and with improved effectiveness, from the blood of all 14 healthy donors tested. Importantly, this APL also induced T-cells from melanoma patient blood that exhibited superior recognition of the patient's own tumour compared to those induced by the natural antigen sequence. These results suggest that use of APL to skew the clonotypic quality of T-cells induced by cancer vaccination could provide a promising avenue in the hunt for the UCV “magic bullet.”	Mar 2019
B23-Circular Dichroism	Thermal stability of heterotrimeric pMHC proteins as determined by circular dichroism spectroscopy Anna Fuller , Aaron Wall , Michael D. Crowther , Angharad Lloyd , Alexei Zhurov , Andrew K. Sewell , David Cole , Konrad Beck Bio-Protocol 7 DOI: 10.21769/BioProtoc.2366 Diamond Proposal Number(s): [12332] Open Access Show Abstract ▼ Abstract: T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting cells. These factors are particularly relevant for the peptide vaccination field, in which more stable pMHC interactions could enable more effective protection against disease. Here, we discuss a method for the determination of pMHC stability that we have used to investigate HIV immune escape, T-cell sensitivity to cancer antigens and mechanisms leading to autoimmunity.	Jul 2017
B23-Circular Dichroism I02-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural mechanism underpinning cross-reactivity of a CD8+ T-cell clone that recognizes a peptide derived from human telomerase reverse transcriptase David K. Cole , Hugo A. Van Den Berg , Angharad Lloyd , Michael D. Crowther , Konrad Beck , Julia Ekeruche-Makinde , John J. Miles , Anna M. Bulek , Garry Dolton , Andrea J. Schauenburg , Aaron Wall , Anna Fuller , Mathew Clement , Bruno Laugel , Pierre J. Rizkallah , Linda Wooldridge , Andrew K. Sewell Journal Of Biological Chemistry 292, 802 - 813 DOI: 10.1074/jbc.M116.741603 Diamond Proposal Number(s): [6232, 8096, 10462, 10049, 9308, 12332] Open Access Show Abstract ▼ Abstract: T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8 T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection.	Jan 2017

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