I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[10462, 14843, 20147]
Open Access
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.
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May 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[4532, 6232]
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.
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Nov 2017
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B23-Circular Dichroism
I03-Macromolecular Crystallography
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Amandine
Bovay
,
Vincent
Zoete
,
Garry
Dolton
,
Anna M.
Bulek
,
David K.
Cole
,
Pierre J.
Rizkallah
,
Anna
Fuller
,
Konrad
Beck
,
Olivier
Michielin
,
Daniel E.
Speiser
,
Andrew K.
Sewell
,
Silvia A.
Fuertes Marraco
Diamond Proposal Number(s):
[10462, 10049, 12332]
Abstract: The repertoire of human αβ T cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen-specific T cells and/or exhibit bias towards a TCR gene segment. Here, we studied the TCR repertoire of the HLA-A*0201-restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8+ T cell response to the highly effective YF-17D vaccine. We discover that these A2/LLW-specific CD8+ T cells are highly biased for the TCR α chain TRAV12-2. This bias is already present in A2/LLW-specific naïve T cells before vaccination with YF-17D. Using CD8+ T cell clones, we show that TRAV12-2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline-encoded complementarity determining region (CDR) 1α loop of TRAV12-2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T-cell responses specific for A2/LLW epitope.
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Oct 2017
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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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B23-Circular Dichroism
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[7687]
Open Access
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.
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May 2016
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8096, 10462]
Open Access
Abstract: Human CD8+ cytotoxic T lymphocytes (CTLs) can mediate tumour regression in melanoma through the specific recognition of human leukocyte antigen (HLA)-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCR), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumour immunity capable of breaking tolerance towards these self-antigens. Previous studies have shown that these immunogenic designer peptides are not always effective and detailed structural analyses will be required in order to improve future success rates. The melanocyte differentiation protein, glycoprotein (gp)100, encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope, and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280-288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3>Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition through knock-on effects. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination.
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Feb 2016
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I03-Macromolecular Crystallography
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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
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.
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Dec 2012
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I03-Macromolecular Crystallography
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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
Open Access
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 A*0201-Melan A27–35 (HLA A*0201-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 A*0201-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 A*0201+ 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.
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Oct 2012
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I04-1-Macromolecular Crystallography (fixed wavelength)
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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
Open Access
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-A*0201–restricted glucose-sensitive preproinsulin peptide by the autoreactive TCR 1E6. Rigid 'lock-and-key' binding underpinned the 1E6–HLA-A*0201–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.
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Jan 2012
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I03-Macromolecular Crystallography
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John J.
Miles
,
Anna M.
Bulek
,
David K.
Cole
,
Emma
Gostick
,
Andrea J.
Schauenburg
,
Garry
Dolton
,
Vanessa
Venturi
,
Miles
Daveport
,
Mai
Ping Tan
,
Scott
Burrows
,
Linda
Woolridge
,
David A.
Price
,
Pierre J.
Rizkallah
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[1788]
Open Access
Abstract: Despite the ∼1018 αβ T cell receptor (TCR) structures that can be randomly manufactured by the human thymus, some surface more frequently than others. The pinnacles of this distortion are public TCRs, which exhibit amino acid-identical structures across different individuals. Public TCRs are thought to result from both recombinatorial bias and antigen-driven selection, but the mechanisms that underlie inter-individual TCR sharing are still largely theoretical. To examine this phenomenon at the atomic level, we solved the co-complex structure of one of the most widespread and numerically frequent public TCRs in the human population. The archetypal AS01 public TCR recognizes an immunodominant BMLF1 peptide, derived from the ubiquitous Epstein-Barr virus, bound to HLA-A*0201. The AS01 TCR was observed to dock in a diagonal fashion, grasping the solvent exposed peptide crest with two sets of complementarity-determining region (CDR) loops, and was fastened to the peptide and HLA-A*0201 platform with residue sets found only within TCR genes biased in the public response. Computer simulations of a random V(D)J recombination process demonstrated that both TCRα and TCRβ amino acid sequences could be manufactured easily, thereby explaining the prevalence of this receptor across different individuals. Interestingly, the AS01 TCR was encoded largely by germline DNA, indicating that the TCR loci already comprise gene segments that specifically recognize this ancient pathogen. Such pattern recognition receptor-like traits within the αβ TCR system further blur the boundaries between the adaptive and innate immune systems.
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Nov 2010
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