I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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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
Diamond Proposal Number(s):
[4352, 6232]
Open Access
Abstract: The HLA‐A*02:01‐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‐A*02:01 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.
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May 2019
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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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Christopher J.
Holland
,
Bruce J.
Maclachlan
,
Valentina
Bianchi
,
Sophie J.
Hesketh
,
Richard
Morgan
,
Owen
Vickery
,
Anna M.
Bulek
,
Anna
Fuller
,
Andrew
Godkin
,
Andrew K.
Sewell
,
Pierre
Rizkallah
,
Stephen
Wells
,
David
Cole
Open Access
Abstract: T-cell immunity is controlled by T cell receptor (TCR) binding to peptide major histocompatibility complexes (pMHCs). The nature of the interaction between these two proteins has been the subject of many investigations because of its central role in immunity against pathogens, cancer, in autoimmunity, and during organ transplant rejection. Crystal structures comparing unbound and pMHC-bound TCRs have revealed flexibility at the interaction interface, particularly from the perspective of the TCR. However, crystal structures represent only a snapshot of protein conformation that could be influenced through biologically irrelevant crystal lattice contacts and other factors. Here, we solved the structures of three unbound TCRs from multiple crystals. Superposition of identical TCR structures from different crystals revealed some conformation differences of up to 5 Å in individual complementarity determining region (CDR) loops that are similar to those that have previously been attributed to antigen engagement. We then used a combination of rigidity analysis and simulations of protein motion to reveal the theoretical potential of TCR CDR loop flexibility in unbound state. These simulations of protein motion support the notion that crystal structures may only offer an artifactual indication of TCR flexibility, influenced by crystallization conditions and crystal packing that is inconsistent with the theoretical potential of intrinsic TCR motions.
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Apr 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
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Diamond Proposal Number(s):
[12332]
Open Access
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.
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Jul 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6232]
Open Access
Abstract: Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.
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Mar 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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