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Robin R Knight,
Deborah Kronenberg,
Min Zhao,
Guo Cai Huang,
Martin Eichmann,
Anna Bulek,
Linda Wooldridge,
David K Cole, Andrew K Sewell,
Mark Peakman,
Ania Skowera
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Anna M Bulek,
David K Cole,
Ania Skowera,
Garry Dolton,
Stephanie Gras,
Florian Madura,
Anna Fuller,
John J Miles,
Emma Gostick,
David A Price, [......],
Robin R Knight,
Guo C Huang,
Nikolai Lissin,
Peter E Molloy,
Linda Wooldridge,
Bent K Jakobsen,
Jamie Rossjohn,
Mark Peakman,
Pierre J Rizkallah, Andrew K Sewell
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Ania Skowera,
Richard J Ellis,
Ruben Varela-Calviño,
Sefina Arif,
Guo Cai Huang,
Cassie Van-Krinks,
Anna Zaremba,
Chloe Rackham,
Jennifer S Allen,
Timothy I M Tree,
Min Zhao,
Colin M Dayan, Andrew K Sewell,
Wendy W Unger,
Wendy Unger,
Jan W Drijfhout,
Ferry Ossendorp,
Bart O Roep,
Mark Peakman
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Weidong Xu,
Douglas M Watts,
Margaret C Costanzo,
Xiaolei Tang,
Leon A Venegas,
Feng Jiao,
Alessandro Sette,
John Sidney, Andrew K Sewell,
Linda Wooldridge,
Shinji Makino,
John C Morrill,
Clarence J Peters,
June Kan-Mitchell
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ABSTRACT: There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8(+) T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8(+) T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N121-129) and IL9 (ILDAHSLYL, N165-173), were defined. VT9- and IL9-specific CD8(+) T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8(+) T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8(+) T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8(+) T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.
PLoS ONE 01/2013; 8(3):e59210. · 4.09 Impact Factor
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ABSTRACT: Multiple endogenous mechanisms that regulate immune and inflammatory processes contribute to the maintenance of peripheral tolerance and prevent chronic inflammation in mammals. Yet pathogens and tumors are able to exploit these homeostatic pathways to foster immunosuppressive microenvironments and evade immune surveillance. The release of adenosine in the extra-cellular space contributes to these phenomena by exerting a broad range of immunomodulatory effects. Here we document the influence of adenosine receptor triggering on human dendritic cell differentiation and functions. We show that the expression of several immunomodulatory proteins and myeloid/monocytic lineage markers was affected by adenosine receptors and the cAMP pathway. These changes were reminiscent of the phenotype associated with tolerogenic dendritic cells and, functionally, translated into a defective capacity to prime CD8(+) T-cells with a common tumor antigen in vitro. These results establish a novel mechanism by which adenosine hampers CD8(+) T-cell immunity via dendritic cells that may contribute to peripheral tolerance as well as to the establishment of immunosuppressive microenvironments relevant to tumour biology. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.
Immunology 12/2012; · 3.32 Impact Factor
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Julia Ekeruche-Makinde,
John J Miles,
Hugo A van den Berg,
Ania Skowera,
David K Cole,
Garry Dolton,
Andrea J A Schauenburg,
Mai Ping Tan,
Johanne M Pentier,
Sian Llewellyn-Lacey, [......],
Tamsin Williams,
Andrew Trimby,
Mick Bailey,
Pierre Rizkallah,
Jamie Rossjohn,
Mark Peakman,
David A Price,
Scott R Burrows, Andrew K Sewell,
Linda Wooldridge
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ABSTRACT: The αβ T-cell receptors (TCRs) expressed at the CD8(+) T-cell surface interact with short peptide fragments (p), bound to major histocompatibility complex class I (MHCI) molecules. The TCR/pMHCI interaction is pivotal in all aspects of CD8(+) T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood which is a major barrier to understanding the requirements for both effective immunity and vaccination. Here, we have discovered an unexpected feature of the TCR/pMHCI interaction by showing that any given TCR exhibits an explicit preference for a single MHCI-peptide length. Agonists of non-preferred length were extremely rare, suboptimal and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This novel finding demonstrates that the outcome of TCR/pMHCI engagement is determined by peptide length in addition to the sequence identity of the MHCI-bound peptide. Accordingly, the effective recognition of pMHCI antigen, which is a prerequisite for successful CD8(+) T-cell immunity and protective vaccination, can only be achieved by "length-matched" antigen-specific CD8(+) T-cell clonotypes.
Blood 12/2012; · 9.90 Impact Factor
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ABSTRACT: T cells that express clonally distributed αβ T-cell receptors (TCRs) corecognize antigenic peptides (p) bound to major histocompatibility complex class I (MHC-I) and class II molecules (MHC-II). Extensive human leukocyte antigen (HLA) polymorphism enables HLA molecules from different haplotypes to capture an array of self- and microbe-derived peptide antigens that is fundamental to adaptive immunity. T cells developing in the thymus are selected for weak binding to self-peptide-HLA complexes generating a vast repertoire of clonally distinct T cells in the periphery. Indeed, diversity within germline loci and the finally assembled TCR genes, coupled with inherent TCR cross-reactivity, enables CD8(+) T cells to survey the multitude of pHLA-I landscapes. Precisely how does the TCR ligate to pHLA-I, and how does knowledge of the detailed structural interactions inform immunobiology? A recent number of our structural studies concerning the TCR-pMHC-I axis, alongside others in the field, have provided insight into HLA-I polymorphism, pMHC-I flexibility, TCR bias, TCR polymorphism, maintenance of self-tolerance, T-cell cross-reactivity, and alloreactivity. Collectively, the data also provide an opportunity to address the structural correlates of MHC-I restriction. Here, we provide our perspective concerning these advances in the field. Although much key information has been gleaned, the structural data show that some of the key concepts surrounding the TCR-pMHC-I interaction remain controversial and unresolved.
Immunological Reviews 11/2012; 250(1):61-81. · 11.15 Impact Factor
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Philippa C Matthews,
Madoka Koyanagi,
Henrik N Kløverpris,
Mikkel Harndahl,
Anette Stryhn,
Tomohiro Akahoshi,
Hiroyuki Gatanaga,
Shinichi Oka,
Claudia Juarez Molina,
Humberto Valenzuela Ponce, [......],
Roger Shapiro,
Christian Brander,
Bruce Walker, Andrew K Sewell,
Gustavo Reyes Teran,
David Heckerman,
Eric Hunter,
Søren Buus,
Masafumi Takiguchi,
Philip J R Goulder
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ABSTRACT: The strongest genetic influence on immune control in HIV-1 infection is HLA class I genotype. Rapid disease progression in B-clade infection has been linked to HLA-B*35 expression, in particular to the less common HLA-B*3502 and HLA-B*3503 subtypes, but also to the most prevalent subtype, HLA-B*3501. In these studies we first demonstrate that, whereas HLA-B*3501 is associated with high viral setpoint in two further B-clade infected cohorts, in Japan and Mexico, this association does not hold in two large C-clade infected African cohorts. We tested the hypothesis that clade-specific differences in HLA associations with disease outcomes may be related to distinct targeting of critical CD8+ T-cell epitopes. We observed that only one epitope was significantly targeted differentially, namely, the Gag-specific epitope, NPPIPVGDIY ('NY10' , Gag 253-262, p=2×10(-5)). In common with two other HLA-B*3501-restricted epitopes, in Gag and Nef, that were not targeted differentially, a response towards NY10 was associated with a significantly lower viral setpoint. Non-immunogenicity of NY10 in B-clade infected subjects derives from the Gag-D260E polymorphism present in ∼90% of B-clade sequences, which critically reduces recognition of the Gag-NY10 epitope. These data suggest that, in spite of any inherent HLA-linked T-cell receptor repertoire differences that may exist, maximizing the breadth of the Gag-specific CD8+ T-cell response, by the addition of even a single epitope, may be of over-riding importance in achieving immune control of HIV infection. This distinction is of direct relevance to development of vaccines designed to optimize the anti-HIV CD8+ T-cell response in all individuals, irrespective of HLA type.
Journal of Virology 09/2012; · 5.40 Impact Factor
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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 J Rizkallah,
David A Price,
Linda Wooldridge, Andrew K Sewell
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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, due to a specific increase in T-cell receptor (TCR) binding affinity for the cognate antigen. Structural analysis revealed the potential for new contacts between the MEL5 TCR 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.
Journal of Biological Chemistry 09/2012; · 4.77 Impact Factor
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Robin R Knight,
Deborah Kronenberg,
Min Zhao,
Guo Cai Huang,
Martin Eichmann,
Anna Bulek,
Linda Wooldridge,
David K Cole, Andrew K Sewell,
Mark Peakman,
Ania Skowera
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ABSTRACT: The end-stage immunopathology of type 1 diabetes resulting in β-cell destruction appears to be strongly dominated by cytotoxic CD8 T lymphocytes (CD8 T cells). However, the mechanism of cytotoxicity used by autoreactive CD8 T cells in the human setting remains unknown. Using type 1 diabetes patient-derived preproinsulin-specific CD8 T-cell clones recognizing either an HLA-A2 (A*0201) or HLA-A24 (A*2402)-restricted epitope (peptide of preproinsulin [PPI](15-24), ALWGPDPAAA; or PPI(3-11), LWMRLLPLL), we assessed the use of conventional mediators of cytotoxicity in the destruction of human β-cells in vitro compared with virus-specific cytotoxic CD8 T-cell clones. We show that PPI-specific CD8 T-cell clones are mainly reliant upon cytotoxic degranulation for inducing β-cell death. Furthermore, we find that in comparison with virus-specific CD8 T cells, there are differences in the killing potency of PPI-specific CD8 T cells that are not due to cell-intrinsic differences, but rather are mediated by differences in strength of signaling by peptide-HLA ligands. The study highlights the regulation of β-cell killing as a potential point for therapeutic control, including the possibility of blocking autoreactive CD8 T-cell function without impacting upon general immune competence.
Diabetes 08/2012; · 8.29 Impact Factor
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British Journal of Haematology 08/2012; 159(3):370-3. · 4.94 Impact Factor
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ABSTRACT: CD8(+) T cells respond to signals mediated through a specific interaction between the T-cell receptor (TCR) and a composite antigen in the form of an epitopic peptide bound between the polymorphic α1 and α2 helices of an MHC class I (MHCI) molecule. The CD8 glycoprotein 'co-receives' antigen by binding to an invariant region of the MHCI molecule and can enhance ligand recognition by up to 1 million-fold. In recent years, a number of structural and biophysical investigations have shed light on the role of the CD8 co-receptor during T-cell antigen recognition. Here, we provide a collated resource for these data, and discuss how the structural and biophysical parameters governing CD8 co-receptor function further our understanding of T-cell cross-reactivity and the productive engagement of low-affinity antigenic ligands.
Immunology 07/2012; 137(2):139-48. · 3.32 Impact Factor
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ABSTRACT: The interaction between the clonotypic αβ T cell receptor (TCR), expressed on the T cell surface, and peptide-major histocompatibility complex (pMHC) molecules, expressed on the target cell surface, governs T cell mediated autoimmunity and immunity against pathogens and cancer. Structural investigations of this interaction have been limited because of the challenges inherent in the production of good quality TCR/pMHC protein crystals. Here, we report the development of an 'intelligently designed' crystallization screen that reproducibly generates high quality TCR/pMHC complex crystals suitable for X-ray crystallographic studies, thereby reducing protein consumption. Over the last 2 years, we have implemented this screen to produce 32 T cell related protein structures at high resolution, substantially contributing to the current immune protein database. Protein crystallography, used to study this interaction, has already extended our understanding of the molecular rules that govern T cell immunity. Subsequently, these data may help to guide the intelligent design of T cell based therapies that target human diseases, underlining the importance of developing optimized approaches for crystallizing novel TCR/pMHC complexes.
Journal of immunological methods 06/2012; 382(1-2):203-10. · 2.35 Impact Factor
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Nathaniel Liddy,
Giovanna Bossi,
Katherine J Adams,
Anna Lissina,
Tara M Mahon,
Namir J Hassan,
Jessie Gavarret,
Frayne C Bianchi,
Nicholas J Pumphrey,
Kristin Ladell, [......],
Andy Johnson,
Yvonne McGrath,
Gabriela Plesa,
Carl H June,
Michael Kalos,
David A Price,
Annelise Vuidepot,
Daniel D Williams,
Deborah H Sutton,
Bent K Jakobsen
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ABSTRACT: T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.
Nature medicine 05/2012; 18(6):980-7. · 27.14 Impact Factor
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Anna M Bulek,
David K Cole,
Ania Skowera,
Garry Dolton,
Stephanie Gras,
Florian Madura,
Anna Fuller,
John J Miles,
Emma Gostick,
David A Price, [......],
Robin R Knight,
Guo C Huang,
Nikolai Lissin,
Peter E Molloy,
Linda Wooldridge,
Bent K Jakobsen,
Jamie Rossjohn,
Mark Peakman,
Pierre J Rizkallah, Andrew K Sewell
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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.
Nature Immunology 01/2012; 13(3):283-9. · 26.01 Impact Factor
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David K Cole,
Kathleen Gallagher,
Brigitte Lemercier,
Christopher J Holland,
Sayed Junaid,
James P Hindley,
Katherine K Wynn,
Emma Gostick, Andrew K Sewell,
Awen M Gallimore,
Kristin Ladell,
David A Price,
Marie-Lise Gougeon,
Andrew Godkin
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ABSTRACT: Human CD4(+) αβ T cells are activated via T-cell receptor recognition of peptide epitopes presented by major histocompatibility complex (MHC) class II (MHC-II). The open ends of the MHC-II binding groove allow peptide epitopes to extend beyond a central nonamer core region at both the amino- and carboxy-terminus. We have previously found that these non-bound C-terminal residues can alter T cell activation in an MHC allele-transcending fashion, although the mechanism for this effect remained unclear. Here we show that modification of the C-terminal peptide-flanking region of an influenza hemagglutinin (HA(305-320)) epitope can alter T-cell receptor binding affinity, T-cell activation and repertoire selection of influenza-specific CD4(+) T cells expanded from peripheral blood. These data provide the first demonstration that changes in the C-terminus of the peptide-flanking region can substantially alter T-cell receptor binding affinity, and indicate a mechanism through which peptide flanking residues could influence repertoire selection.
Nature Communications 01/2012; 3:665. · 7.40 Impact Factor
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ABSTRACT: Successful immunity requires that a limited pool of αβ T-cell receptors (TCRs) provide cover for a vast number of potential foreign peptide antigens presented by 'self' major histocompatibility complex (pMHC) molecules. Structures of unligated and ligated MHC class-I-restricted TCRs with different ligands, supplemented with biophysical analyses, have revealed a number of important mechanisms that govern TCR mediated antigen recognition. HA1.7 TCR binding to the influenza hemagglutinin antigen (HA(306-318)) presented by HLA-DR1 or HLA-DR4 represents an ideal system for interrogating pMHC-II antigen recognition. Accordingly, we solved the structure of the unligated HA1.7 TCR and compared it to both complex structures. Despite a relatively rigid binding mode, HA1.7 T-cells could tolerate mutations in key contact residues within the peptide epitope. Thermodynamic analysis revealed that limited plasticity and extreme favorable entropy underpinned the ability of the HA1.7 T-cell clone to cross-react with HA(306-318) presented by multiple MHC-II alleles.
Scientific Reports 01/2012; 2:629.
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ABSTRACT: The molecular rules that govern MHC restriction, and allow T-cells to differentiate between peptides derived from healthy cells and those from diseased cells, remain poorly understood. Here we provide an overview of the structural constraints that enable the T-cell receptor (TCR) to discriminate between self and non-self peptides, and summarize studies that have attempted to correlate the biophysical parameters of TCR/peptide–major histocompatibility complex (pMHC) binding with T-cell activation. We further review how the antigenic origin of peptide epitopes affects TCR binding parameters and the ‘quality’ of a T-cell response. Understanding the principles that govern pMHC recognition by T-cells will unlock pathways to the rational development of immunotherapeutic approaches for the treatment of infectious disease, cancer and autoimmunity.
Immunology 12/2011; 135(1):9 - 18. · 3.32 Impact Factor
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Linda Wooldridge,
Julia Ekeruche-Makinde,
Hugo A van den Berg,
Anna Skowera,
John J Miles,
Mai Ping Tan,
Garry Dolton,
Mathew Clement,
Sian Llewellyn-Lacey,
David A Price,
Mark Peakman, Andrew K Sewell
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ABSTRACT: The T cell receptor (TCR) orchestrates immune responses by binding to foreign peptides presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Effective immunity requires that all possible foreign peptide-MHC molecules are recognized or risks leaving holes in immune coverage that pathogens could quickly evolve to exploit. It is unclear how a limited pool of <10(8) human TCRs can successfully provide immunity to the vast array of possible different peptides that could be produced from 20 proteogenic amino acids and presented by self-MHC molecules (>10(15) distinct peptide-MHCs). One possibility is that T cell immunity incorporates an extremely high level of receptor degeneracy, enabling each TCR to recognize multiple peptides. However, the extent of such TCR degeneracy has never been fully quantified. Here, we perform a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8(+) T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule. A large number of peptides that acted as substantially better agonists than the wild-type "index" preproinsulin-derived peptide (ALWGPDPAAA) were identified. The RQFGPDFPTI peptide (sampled from >10(8) peptides) was >100-fold more potent than the index peptide despite differing from this sequence at 7 of 10 positions. Quantification of this previously unappreciated high level of CD8(+) T cell cross-reactivity represents an important step toward understanding the system requirements for adaptive immunity and highlights the enormous potential of TCR degeneracy to be the causative factor in autoimmune disease.
Journal of Biological Chemistry 11/2011; 287(2):1168-77. · 4.77 Impact Factor
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ABSTRACT: Short peptide fragments generated by intracellular protein cleavage are presented on the surface of most nucleated cells bound to highly polymorphic MHCI molecules. These pMHCI complexes constitute an interface that allows the immune system to identify and eradicate anomalous cells, such as those that harbor infectious agents, through the activation of CTLs. Molecular recognition of pMHCI complexes is mediated primarily by clonally distributed TCRs expressed on the surface of CTLs. The coreceptor CD8 contributes to this antigen-recognition process by binding to a largely invariant region of the MHCI molecule and by promoting intracellular signaling, the effects of which serve to enhance TCR stimuli triggered by cognate ligands. Recent investigations have shed light on the role of CD8 in the activation of MHCI-restricted, antigen-experienced T cells and in the processes of T cell selection and lineage commitment in the thymus. Here, we review these data and discuss their implications for the development of potential therapeutic strategies that selectively target pathogenic CTL responses erroneously directed against self-derived antigens.
Journal of leukocyte biology 09/2011; 90(6):1089-99. · 4.99 Impact Factor
