I04-Macromolecular Crystallography
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Takeshi
Nakama
,
Aaron
Wall
,
Garry
Dolton
,
Li-Rong
Tan
,
Hannah
Thomas
,
Hiroshi
Hamana
,
Yoshiki
Aritsu
,
Toong Seng
Tan
,
Mako
Toyoda
,
Yoshihiko
Goto
,
Huanyu
Li
,
Mizuki
Kitamatsu
,
Keiko
Udaka
,
Yusuke
Miyashita
,
Hiroyuki
Oshiumi
,
Kimitoshi
Nakamura
,
Yoji
Nagasaki
,
Rumi
Minami
,
Hirotomo
Nakata
,
Pierre J.
Rizkallah
,
Hiroyuki
Kishi
,
Takamasa
Ueno
,
Andrew K.
Sewell
,
Chihiro
Motozono
Open Access
Abstract: Cytotoxic T lymphocytes form a critical component of SARS-CoV-2 immunity by recognizing viral peptides bound to HLA class I molecules. Here, we identified the Spike-derived peptide NYNYLYRLF448-456 (NF9) as the immunodominant HLA-A*24:02-restricted epitope in both convalescent and vaccinated donors. Across cohorts, A24/NF9-specific responses were dominated by public TCR motifs featuring TRAV12-1 (or TRAV6-1) paired with TRBJ2-7 and a conserved CDR3β sequence (CASSXXXGYEQYF). Using a panel of thirteen TCRs, we mapped recognition of single amino acid substitutions within NF9 and identified residue 5 (L452) as the principal determinant of escape. The L452R substitution, characteristic of the Delta variant, abolished recognition across all tested TCRs despite preserved HLA binding. Crystallography of a representative public TCR (P1-15) revealed that mutation at position 5 reoriented the peptide within HLA-A*24:02, flipping the adjacent Y453 side chain into the peptide-binding groove and eliminating the dominant TCR contact. This position-5-driven conformational switch provided a structural mechanism for universal loss of NF9 recognition by HLA-A*24:02-restricted T-cells. Consistent with this, Delta-infected convalescents failed to mount de novo NF9-5R-specific responses while retaining responses to the conserved A24/QI9 Spike epitope. Together, these findings defined the basis of A24/NF9 recognition and showed how one mutation remodelled peptide presentation to abrogate TCR responses.
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Mar 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Garry
Dolton
,
Anna
Bulek
,
Aaron
Wall
,
Hannah
Thomas
,
Jade R.
Hopkins
,
Cristina
Rius
,
Sarah A. E.
Galloway
,
Thomas
Whalley
,
Li Rong
Tan
,
Théo
Morin
,
Nader
Omidvar
,
Anna
Fuller
,
Katie
Topley
,
Md Samiul
Hasan
,
Shikha
Jain
,
Nirupa
D’souza
,
Thomas
Hodges-Hoyland
,
Owen B.
Spiller
,
Deborah
Kronenberg-Versteeg
,
Barbara
Szomolay
,
Hugo A.
Van Den Berg
,
Lucy C.
Jones
,
Mark
Peakman
,
David K.
Cole
,
Pierre J.
Rizkallah
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[10462, 18812]
Open Access
Abstract: CD8+ T cells destroy insulin-producing pancreatic β cells in type 1 diabetes through HLA class I–restricted presentation of self-antigens. Combinatorial peptide library screening was used to produce a preferred peptide recognition landscape for a patient-derived T cell receptor (TCR) that recognized the preproinsulin-derived (PPI-derived) peptide sequence LWMRLLPLL in the context of disease risk allele HLA A*24:02. Data were used to generate a strong superagonist peptide, enabling production of an autoimmune HLA A*24:02–peptide–TCR structure by crystal seeding. TCR binding to the PPI epitope was strongly focused on peptide residues Arg4 and Leu5, with more flexibility at other positions, allowing the TCR to strongly engage many peptides derived from pathogenic bacteria. We confirmed an epitope from Klebsiella that was recognized by PPI-reactive T cells from 3 of 3 HLA A*24:02+ patients. Remarkably, the same epitope selected T cells from 7 of 8 HLA A*24+ healthy donors that cross-reacted with PPI, leading to recognition and killing of HLA A*24:02+ cells expressing PPI. These data provide a mechanism by which molecular mimicry between pathogen and self-antigens could have resulted in the breaking of self-tolerance to initiate disease.
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Sep 2024
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I03-Macromolecular Crystallography
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Sarah
Hulin-Curtis
,
James K.
Geary
,
Bruce J.
Maclachlan
,
Danny M.
Altmann
,
Laury
Baillon
,
David K.
Cole
,
Alexander
Greenshields-Watson
,
Sophie J.
Hesketh
,
Ian R.
Humphreys
,
Ian M.
Jones
,
Sarah N.
Lauder
,
Georgina H.
Mason
,
Kathryn
Smart
,
D. Oliver
Scourfield
,
Jake
Scott
,
Ksenia
Sukhova
,
Richard J.
Stanton
,
Aaron
Wall
,
Pierre J.
Rizkallah
,
Wendy S.
Barclay
,
Awen
Gallimore
,
Andrew
Godkin
Diamond Proposal Number(s):
[10462]
Open Access
Abstract: CD4+ T cells are central to adaptive immunity. Their role in cross-protection in viral infections such as influenza and severe acute respiratory syndrome (SARS) is well documented; however, molecular rules governing T cell receptor (TCR) engagement of peptide-human leukocyte antigen (pHLA) class II are less understood. Here, we exploit an aspect of HLA class II presentation, the peptide-flanking residues (PFRs), to “tune” CD4+ T cell responses within an in vivo model system of influenza. Using a recombinant virus containing targeted substitutions at immunodominant HLA-DR1 epitopes, we demonstrate limited weight loss and improved clinical scores after heterosubtypic re-challenge. We observe enhanced protection linked to lung-derived influenza-specific CD4+ and CD8+ T cells prior to re-infection. Structural analysis of the ternary TCR:pHLA complex identifies that flanking amino acids influence side chains in the core 9-mer peptide, increasing TCR affinity. Augmentation of CD4+ T cell immunity is achievable with a single mutation, representing a strategy to enhance adaptive immunity that is decoupled from vaccine modality.
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Jun 2024
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Garry
Dolton
,
Cristina
Rius
,
Md Samiul
Hasan
,
Aaron
Wall
,
Barbara
Szomolay
,
Enas
Behiry
,
Thomas
Whalley
,
Joel
Southgate
,
Anna
Fuller
,
Théo
Morin
,
Katie
Topley
,
Li Rong
Tan
,
Philip J. R.
Goulder
,
Owen B.
Spiller
,
Pierre J.
Rizkallah
,
Lucy C.
Jones
,
Thomas R.
Connor
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[29502]
Open Access
Abstract: We studied the prevalent cytotoxic CD8 T-cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent Human Leukocyte Antigen (HLA) class I worldwide, HLA A∗02. The Spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘variants of concern’, was not recognised by the large CD8 T-cell response seen across cohorts of HLA A∗02+ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T-cell receptors. Viral escape at prevalent T-cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focussed on a single protein such as SARS-CoV-2 Spike providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T-cell escape in new SARS-CoV-2 variants.
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Jul 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[10462, 14843]
Open Access
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.
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Jul 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Harley L.
Worthy
,
Husam Sabah
Auhim
,
W. David
Jamieson
,
Jacob R.
Pope
,
Aaron
Wall
,
Robert
Batchelor
,
Rachel L.
Johnson
,
Daniel W.
Watkins
,
Pierre
Rizkallah
,
Oliver K.
Castell
,
D. Dafydd
Jones
Diamond Proposal Number(s):
[14843]
Open Access
Abstract: Construction of artificial higher order protein complexes allows sampling of structural architectures and functional features not accessible by classical monomeric proteins. Here, we combine in silico modelling with expanded genetic code facilitated strain promoted azide-alkyne cycloaddition to construct artificial complexes that are structurally integrated protein dimers and demonstrate functional synergy. Using fluorescent proteins sfGFP and Venus as models, homodimers and heterodimers are constructed that switched ON once assembled and display enhanced spectral properties. Symmetrical crosslinks are found to be important for functional enhancement. The determined molecular structure of one artificial dimer shows that a new long-range polar network comprised mostly of organised water molecules links the two chromophores leading to activation and functional enhancement. Single molecule analysis reveals the dimer is more resistant to photobleaching spending longer times in the ON state. Thus, genetically encoded bioorthogonal chemistry can be used to generate truly integrated artificial protein complexes that enhance function.
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Jul 2019
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I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[14843]
Open Access
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.”
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Mar 2019
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B23-Circular Dichroism
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[6232, 8096, 10462, 10049, 9308, 12332]
Open Access
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.
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Jan 2017
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