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11 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	TCR‐induced alteration of primary MHC peptide anchor residue Florian Madura , Pierre J. Rizkallah , Mateusz Legut , Christopher J. Holland , Anna Fuller , Anna Bulek , Andrea J. Schauenburg , Andrew Trimby , Jade R. Hopkins , Stephen Wells , Andrew Godkin , John J. Miles , Malkit Sami , Yi Li , Nathaniel Liddy , Bent K. Jakobsen , E. Joel Loveridge , David K. Cole , Andrew K. Sewell European Journal Of Immunology DOI: 10.1002/eji.201948085 Diamond Proposal Number(s): [4352, 6232] Open Access Show Abstract ▼ Abstract: The HLA‐A02:01‐restricted decapeptide EAAGIGILTV, derived from Melanoma Antigen Recognized by T‐cells‐1 (MART‐1) protein, represents one of the best‐studied tumor associated T‐cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptides are presented in distinct conformations by HLA‐A02:01 and TCRs from clinically relevant T‐cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5‐HLA‐A*02:01‐AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide‐MHC anchoring. This ‘flexing’ at the TCR‐peptide‐MHC interface to accommodate the peptide antigen explains previously‐observed incongruences in this well‐studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T‐cells.	May 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig Katie Tungatt , Garry Dolton , Sophie B. Morgan , Meriem Attaf , Anna Fuller , Thomas Whalley , Johanneke D. Hemmink , Emily Porter , Barbara Szomolay , Maria Montoya , John A. Hammond , John J. Miles , David K. Cole , Alain Townsend , Mick Bailey , Pierre Rizkallah , Bryan Charleston , Elma Tchilian , Andrew K. Sewell Plos Pathogens 14 DOI: 10.1371/journal.ppat.1007017 Diamond Proposal Number(s): [10462, 14843, 20147] Open Access Show Abstract ▼ Abstract: There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens.	May 2018
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Dual molecular mechanisms govern escape at immunodominant HLA A2-restricted HIV epitope David K. Cole , Anna Fuller , Garry Dolton , Efthalia Zervoudi , Mateusz Legut , Kim Miles , Lori Blanchfield , Florian Madura , Christopher J. Holland , Anna M. Bulek , John S. Bridgeman , John J. Miles , Andrea J. A. Schauenburg , Konrad Beck , Brian D. Evavold , Pierre Rizkallah , Andrew K. Sewell Frontiers In Immunology 8 DOI: 10.3389/fimmu.2017.01503 Diamond Proposal Number(s): [4532, 6232] Show Abstract ▼ Abstract: Serial accumulation of mutations to fixation in the SLYNTVATL (SL9) immunodominant, HIV p17 Gag-derived, HLA A2-restricted cytotoxic T lymphocyte epitope produce the SLFNTIAVL triple mutant “ultimate” escape variant. These mutations in solvent-exposed residues are believed to interfere with TCR recognition, although confirmation has awaited structural verification. Here, we solved a TCR co-complex structure with SL9 and the triple escape mutant to determine the mechanism of immune escape in this eminent system. We show that, in contrast to prevailing hypotheses, the main TCR contact residue is 4N and the dominant mechanism of escape is not via lack of TCR engagement. Instead, mutation of solvent-exposed residues in the peptide destabilise the peptide–HLA and reduce peptide density at the cell surface. These results highlight the extraordinary lengths that HIV employs to evade detection by high-affinity TCRs with a broad peptide-binding footprint and necessitate re-evaluation of this exemplar model of HIV TCR escape.	Nov 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
B23-Circular Dichroism I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity David Cole , Anna Bulek , Garry Dolton , Andrea J. Schauenberg , Barbara Szomolay , William Rittase , Andrew Trimby , Prithiviraj Jothikumar , Anna Fuller , Ania Skowera , Jamie Rossjohn , Cheng Zhu , John Miles , Mark Peakman , Linda Wooldridge , Pierre Rizkallah , Andrew K. Sewell Journal Of Clinical Investigation 126, 2191 - 2204 DOI: 10.1172/JCI85679 Diamond Proposal Number(s): [7687] Open Access Show Abstract ▼ Abstract: The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease.	May 2016
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	T-cell receptor specificity maintained by altered thermodynamics Florian Madura , Pierre J. Rizkallah , Kim M. Miles , Christopher J. Holland , Anna M. Bulek , Anna Fuller , Andrea J. A. Schauenburg , John Miles , Nathaniel Liddy , Malkit Sami , Yi Li , Moushumi Hossain , Brian M. Baker , Bent K. Jakobsen , Andrew K. Sewell , David K. Cole Journal Of Biological Chemistry DOI: 10.1074/jbc.M113.464560 PMID: 23698002 Open Access Show Abstract ▼ Abstract: The T-cell receptor (TCR) recognizes peptides bound to major histocompatibility molecules (MHC) and allows T-cells to interrogate the cellular proteome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterized by weak affinity (KD = 100 nm to 270 μm). We used phage-display to produce a melanoma-specific TCR (α24β17) with a 30,000-fold enhanced binding affinity (KD = 0.6 nm) to aid our exploration of the molecular mechanisms utilized to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, α24β17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition.	May 2013
I03-Macromolecular Crystallography	Peptide length determines the outcome of T cell receptor/peptide-MHCI engagement J. Ekeruche-Makinde , J. J. Miles , H. A. Van Den Berg , A. Skowera , D. K. Cole , G. Dolton , A. J. A. Schauenburg , M. P. Tan , J. M. Pentier , S. Llewellyn-Lacey , K. M. Miles , A. M. Bulek , M. Clement , T. Williams , A. Trimby , M. Bailey , P. Rizkallah , J. Rossjohn , M. Peakman , D. A. Price , S. R. Burrows , A. K. Sewell , L. Wooldridge Blood 121 (7), 1112-1123 DOI: 10.1182/blood-2012-06-437202 PMID: 23255554 Show Abstract ▼ Abstract: MHCI-restricted TCRs exhibit an explicit preference for a single MHCI-peptide length. Effective CD8+ T-cell immunity can only be achieved by length-matched Ag-specific T-cell clonotypes.	Dec 2012
I03-Macromolecular Crystallography	T-cell receptor-optimized peptide skewing of the T-cell repertoire can enhance antigen targeting Julia Ekeruche-Makinde , Mathew Clement , David K. Cole , Emily S. J. Edwards , Kristin Ladell , John J. Miles , Katherine K. Matthews , Anna Fuller , Katy A. Lloyd , Florian Madura , Garry M. Dolton , Johanne Pentier , Anna Lissina , Emma Gostick , Tiffany K. Baxter , Brian M. Baker , Pierre Rizkallah , David A. Price , Linda Wooldridge , Andrew K. Sewell Journal Of Biological Chemistry 287 (44), 37269 - 37281 DOI: 10.1074/jbc.M112.386409 PMID: 22952231 Open Access Show Abstract ▼ Abstract: Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8+ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A0201-Melan A27–35 (HLA A0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8+ T-cell clones that recognized HLA A0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8+ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A0201+ individuals that were capable of efficient HLA A*0201+ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.	Oct 2012
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis for the killing of human beta cells by CD8+ T cells in type 1 diabetes Anna Bulek , David Cole , Ania Skowera , Garry Dolton , Stephanie Gras , Florian Madura , Anna Fuller , John Miles , Emma Gostick , David A. Price , Jan W. Drijfhout , Robin R. Knight , Guo C. Huang , Nikolai Lissin , Peter E. Molloy , Linda Fothergill-Gilmore , Bent K. Jakobsen , Jamie Rossjohn , Mark Peakman , Pierre J. Rizkallah , Andrew K. Sewell Nature Immunology DOI: 10.1038/ni.2206 PMID: 22245737 Open Access Show Abstract ▼ Abstract: The structural characteristics of the engagement of major histocompatibility complex (MHC) class II–restricted self antigens by autoreactive T cell antigen receptors (TCRs) is established, but how autoimmune TCRs interact with complexes of self peptide and MHC class I has been unclear. Here we examined how CD8+ T cells kill human islet beta cells in type 1 diabetes via recognition of a human leukocyte antigen HLA-A0201–restricted glucose-sensitive preproinsulin peptide by the autoreactive TCR 1E6. Rigid 'lock-and-key' binding underpinned the 1E6–HLA-A0201–peptide interaction, whereby 1E6 docked similarly to most MHC class I–restricted TCRs. However, this interaction was extraordinarily weak because of limited contacts with MHC class I. TCR binding was highly peptide centric, dominated by two residues of the complementarity-determining region 3 (CDR3) loops that acted as an 'aromatic-cap' over the complex of peptide and MHC class I (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8+ T cell–mediated autoreactivity.	Jan 2012
I03-Macromolecular Crystallography	TOPSORT: TCR optimized peptide skewing of the repertoire of T-cells J Ekeruche , M Clement , David Cole , K Ladell , E Edwards , John Miles , K Wynn , A Fuller , A Lissina , E Gostick Annual Congress of the British Society for Immunology Diamond Proposal Number(s): [4352]	Dec 2010

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