45 results on '"Holland, Christopher J"'
Search Results
2. Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA
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Holland, Christopher J., Crean, Rory M., Pentier, Johanne M., de WeAngharad Lloyd, Ben, Srikannathasan, Velupillai, Lissin, Nikolai, Lloyd, Katy A., Blicher, Thomas H., Conroy, Paul J., Hock, Miriam, Pengelly, Robert J., Spinner, Thomas E., Cameron, Brian, Potter, Elizabeth A., Jeyanthan, Anitha, Molloy, Peter E., Sami, Malkit, Aleksic, Milos, Liddy, Nathaniel, Robinson, Ross A., Harper, Stephen, Lepore, Marco, Pudney, Chris R., van der Kamp, Marc W., Rizkallah, Pierre J., Jakobsen, Bent K., and Cole, Annelise Vuidepoand David K.
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Peptides -- Health aspects ,Antibodies -- Health aspects ,Antigenic determinants -- Health aspects ,HLA antigens -- Health aspects ,T cells -- Health aspects ,Antigens ,Cancer treatment ,Cancer ,Reagents ,Tumors ,Health care industry - Abstract
Tumor-associated peptide-human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface-expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents., Introduction The ability to selectively target tumor-specific antigens holds great promise for the development of specific cancer treatments, but their identification remains a key challenge. Peptide fragments presented on the [...]
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- 2020
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3. TIM3 is a context-dependent co-regulator of cytotoxic T cell function
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Alamir, Hanin, primary, Wong, Carissa C.W., additional, Alsubaiti, Amal, additional, Edmunds, Grace L., additional, Grant, Tressan, additional, Alsulaimani, Safaa, additional, Boyd, James, additional, Holland, Christopher J., additional, Morgan, David J., additional, Gallimore, Awen M., additional, and Wülfing, Christoph, additional
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- 2023
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4. Individual differences in the effects of baby images on attitudes toward getting married
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Lord, Charles G., Holland, Christopher J., and Hill, Sarah E.
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- 2018
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5. Heterogeneous yet stable Vδ2 (+) T-cell profiles define distinct cytotoxic effector potentials in healthy human individuals
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Ryan, Paul L., Sumaria, Nital, Holland, Christopher J., Bradford, Claire M., Izotova, Natalia, Grandjean, Capucine L., Jawad, Ali S., Bergmeier, Lesley A., and Pennington, Daniel J.
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- 2016
6. Attitudes: An Evolutionary Perspective
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Lord, Charles G., Hill, Sarah E., Holland, Christopher J., Yoke, Kristin, Lu, Tong, Shackelford, Todd K., Series editor, Weekes-Shackelford, Viviana A., Series editor, Zeigler-Hill, Virgil, editor, and Welling, Lisa L. M., editor
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- 2015
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7. Communicating beyond the information given can make the communicator’s attitudes toward a social group more extreme
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Decker, Kaleigh A., primary, Lord, Charles G., additional, and Holland, Christopher J., additional
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- 2022
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8. CD8+ T-cell specificity is compromised at a defined MHCI/CD8 affinity threshold
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Dockree, Tamsin, Holland, Christopher J, Clement, Mathew, Ladell, Kristin, McLaren, James E, van den Berg, Hugo A, Gostick, Emma, Miners, Kelly L, Llewellyn-Lacey, Sian, Bridgeman, John S, Man, Stephen, Bailey, Mick, Burrows, Scott R, Price, David A, and Wooldridge, Linda
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- 2017
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9. More tricks with tetramers: a practical guide to staining T cells with peptide–MHC multimers
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Dolton, Garry, Tungatt, Katie, Lloyd, Angharad, Bianchi, Valentina, Theaker, Sarah M., Trimby, Andrew, Holland, Christopher J., Donia, Marco, Godkin, Andrew J., Cole, David K., Thor Straten, Per, Peakman, Mark, Svane, Inge Marie, and Sewell, Andrew K.
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- 2015
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10. Molecular rules underpinning enhanced affinity binding of human T cell receptors engineered 2 for immunotherapy
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Crean, Rory M, MacLachlan, Bruce J, Madura, Florian, Whalley, Thomas, Rizkallah, Pierre J, Holland, Christopher J, McMurran, Catriona, Harper, Stephen, Godkin, Andrew, Sewell, Andrew K, Pudney, Christopher R, Van Der Kamp, Marc W, and Cole, David K
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cancer immunotherapy ,peptide-human leukocyte antigen (pHLA) ,T cells ,hemic and immune systems ,chemical and pharmacologic phenomena ,T cell receptor (TCR) ,molecular dynamics (MD) simulations ,X-ray crystallography - Abstract
Immuno-oncology approaches that utilize T cell receptors (TCRs) are becoming highly attractive because of their potential to target virtually all cellular proteins, including cancer-specific epitopes, via the recognition of peptide-human leukocyte antigen (pHLA) complexes presented at the cell surface. However, because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities, efforts have been made to enhance their binding strength, in some cases by several million-fold. In this study, we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors. Additionally, we performed molecular dynamics simulations to better understand the energetic mechanisms driving the affinity enhancements. These data demonstrate that supra-physiological binding affinities can be achieved without altering native TCR-pHLA binding modes via relatively subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. Individual energetic components of the TCR-pHLA interaction governing affinity enhancements were distinct and highly variable for each TCR, often resulting from additive, or knock-on, effects beyond the mutated residues. This comprehensive analysis of affinity-enhanced TCRs has important implications for the future rational design of engineered TCRs as efficacious and safe drugs for cancer treatment.
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- 2020
11. Molecular Rules Underpinning Enhanced Affinity Binding of Human T Cell Receptors Engineered for Immunotherapy
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Crean, Rory M., primary, MacLachlan, Bruce J., additional, Madura, Florian, additional, Whalley, Thomas, additional, Rizkallah, Pierre J., additional, Holland, Christopher J., additional, McMurran, Catriona, additional, Harper, Stephen, additional, Godkin, Andrew, additional, Sewell, Andrew K., additional, Pudney, Christopher R., additional, van der Kamp, Marc W., additional, and Cole, David K., additional
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- 2020
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12. Abstract 4554: Functionally distinct T cell subsets contribute to ImmTAC-mediated anti-tumor response
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Benlahrech, Adel, primary, Cole, David K., additional, Holland, Christopher J., additional, Kenefeck, Rupert, additional, Khanolkar, Rahul C., additional, McAlpine, Cheryl, additional, Lewis, Sion, additional, Valerio-Fernandes, Angela, additional, Vardeu, Mariantonella, additional, Berman, David, additional, and Lepore, Marco, additional
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- 2020
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13. Acute Effects of Increased Joint Mobilization Treatment Duration on Ankle Function and Dynamic Postural Control in Female Athletes With Chronic Ankle Instability
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Holland, Christopher J., primary, Hughes, Jonathan D., additional, and De Ste Croix, Mark B.A., additional
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- 2020
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14. The metabolic demand of external load carriage in golfers: a comparison of a single versus double-strap golf bag
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Holland, Christopher J., primary and Godwin, Mark S., additional
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- 2020
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15. TCR-induced alteration of primary MHC peptide anchor residue
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Madura, Florian, Rizkallah, Pierre J., Legut, Mateusz, Holland, Christopher J., Fuller, Anna, Bulek, Anna, Schauenburg, Andrea J., Trimby, Andrew, Hopkins, Jade R., Wells, Stephen A., Godkin, Andrew, Miles, John J., Sami, Malkit, Li, Yi, Liddy, Nathaniel, Jakobsen, Bent K., Loveridge, E. Joel, Cole, David K., and Sewell, Andrew K.
- 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 nonapeptide 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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- 2019
16. Enhanced Detection of Antigen-Specific CD4+ T Cells Using Altered Peptide Flanking Residue Peptide–MHC Class II Multimers
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Holland, Christopher J., Dolton, Garry, Scurr, Martin, Ladell, Kristin, Schauenburg, Andrea J., Miners, Kelly, Madura, Florian, Sewell, Andrew K., Price, David A., Cole, David K., and Godkin, Andrew J.
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CD4-Positive T-Lymphocytes ,Histocompatibility Antigens Class II ,Receptors, Antigen, T-Cell ,chemical and pharmacologic phenomena ,T-Cell Antigen Receptor Specificity ,Cell Separation ,Lymphocyte Activation ,Peptide Fragments ,Cell Line ,HLA Antigens ,Novel Immunological Methods ,QR180 ,Humans ,Antigens ,Protein Binding - Abstract
Fluorochrome-conjugated peptide-MHC (pMHC) class I multimers are staple components of the immunologist's toolbox, enabling reliable quantification and analysis of Ag-specific CD8(+) T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4(+) T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II-bound peptides, can enhance TCR-pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4(+) T cells, highlighting an unappreciated feature of TCR-pMHC-II interactions.
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- 2015
17. In Silico and Structural Analyses Demonstrate That Intrinsic Protein Motions Guide T Cell Receptor Complementarity Determining Region Loop Flexibility
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Holland, Christopher J., Maclachlan, Bruce J., Bianchi, Valentina, Hesketh, Sophie J., Morgan, Richard, Vickery, Owen, Bulek, Anna M., Fuller, Anna, Godkin, Andrew, Sewell, Andrew K., Rizkallah, Pierre J., Stephen Wells, and Cole, David K.
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complementarity determining regions loops ,T cell receptor ,T-cells ,X-ray crystallography ,computational simulations ,protein flexibility ,Immunology ,Immunology and Allergy ,chemical and pharmacologic phenomena ,Original Research - 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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- 2018
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18. Dual molecular mechanisms govern escape at immunodominant HLA A2-restricted HIV epitope
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Cole, David K., Fuller, Anna, Dolton, Garry, Zervoudi, Efthalia, Legut, Mateusz, Miles, Kim, Blanchfield, Lori, Madura, Florian, Holland, Christopher J., Bulek, Anna M., Bridgeman, John S., Miles, John J., Schauenburg, Andrea J. A., Beck, Konrad, Evavold, Brian D., Rizkallah, Pierre J., and Sewell, Andrew K.
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chemical and pharmacologic phenomena ,R1 - Abstract
Serial accumulation of mutations to fixation in the SLYNTVATL (SL9) immunodominant, HIV p17 Gag-derived, HLA A2-restricted CTL 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 destabilize 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 reevaluation of this exemplar model of HIV TCR escape.
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- 2017
19. CD8+ T-cell specificity is compromised at a defined major histocompatibility complex class I/CD8 affinity threshold\ud
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Dockree, Tamsin, Holland, Christopher J., Clement, Mathew, Ladell, Kristin, McLaren, James E., Berg, Hugo van den, Gostick, Emma, Llewellyn-Lacey, Sian, Man, Stephen, Bailey, Mick, Burrows, Scott R., Price, D. A., and Wooldridge, Linda
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QR180 - Abstract
The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity.\ud \ud
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- 2016
20. CD8+ T‐cell specificity is compromised at a defined MHCI/CD8 affinity threshold
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Dockree, Tamsin, primary, Holland, Christopher J, additional, Clement, Mathew, additional, Ladell, Kristin, additional, McLaren, James E, additional, van den Berg, Hugo A, additional, Gostick, Emma, additional, L Miners, Kelly, additional, Llewellyn‐Lacey, Sian, additional, Bridgeman, John S, additional, Man, Stephen, additional, Bailey, Mick, additional, Burrows, Scott R, additional, Price, David A, additional, and Wooldridge, Linda, additional
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- 2016
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21. Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs
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Tungatt, Katie, Bianchi, Valentina, Crowther, Michael D., Powell, Wendy, Schauenburg, Andrea J. A., Trimby, Andrew R., Donia, M., Miles, John James, Holland, Christopher J., Cole, David, Godkin, Andrew James, Peakman, M., Straten, P. T., Svane, I. M., Sewell, Andrew K., and Dolton, Garry Michael
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chemical and pharmacologic phenomena ,R1 - Abstract
Fluorochrome-conjugated peptide–MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II–restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochromeconjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR–pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents. The Journal of Immunology, 2015, 194: 000–000.
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- 2015
22. More tricks with tetramers:a practical guide to staining T cells with peptide-MHC multimers
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Dolton, Garry, Tungatt, Katie, Lloyd, Angharad, Bianchi, Valentina, Theaker, Sarah M, Trimby, Andrew, Holland, Christopher J, Donia, Marco, Godkin, Andrew J, Cole, David K, Straten, Per Thor, Peakman, Mark, Svane, Inge Marie, Sewell, Andrew K, Dolton, Garry, Tungatt, Katie, Lloyd, Angharad, Bianchi, Valentina, Theaker, Sarah M, Trimby, Andrew, Holland, Christopher J, Donia, Marco, Godkin, Andrew J, Cole, David K, Straten, Per Thor, Peakman, Mark, Svane, Inge Marie, and Sewell, Andrew K
- Abstract
Analysis of antigen-specific T-cell populations by flow cytometry with peptide-MHC (pMHC) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of 'tricks' that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of 'boosting' staining by inclusion of an antibody against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T-cell receptor is over 20-fold weaker (K(D) > 1 mm) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anti-cancer or autoimmune T-cell populations, which tend to bear lower affinity T-cell receptors. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log-fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them.
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- 2015
23. T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions
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Cole, David, Miles, Kim Michelle, Madura, Florian, Holland, Christopher J., Schauenburg, Andrea J. A., Godkin, Andrew James, Bulek, Anna Marta, Fuller, Anna, Akpovwa, Hephzibah, Pymm, Phillip G., Liddy, N., Sami, M., Li, Y., Rizkallah, Pierre, Jakobsen, B. K., and Sewell, Andrew K.
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hemic and immune systems ,chemical and pharmacologic phenomena ,R1 - Abstract
αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.
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- 2013
24. Structural basis for ineffective T‐cell responses to MHC anchor residue‐improved “heteroclitic” peptides
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Madura, Florian, primary, Rizkallah, Pierre J., additional, Holland, Christopher J., additional, Fuller, Anna, additional, Bulek, Anna, additional, Godkin, Andrew J., additional, Schauenburg, Andrea J., additional, Cole, David K., additional, and Sewell, Andrew K., additional
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- 2014
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25. CD8+ T-cell specificity is compromised at a defined MHCI/CD8 affinity threshold.
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Dockree, Tamsin, Holland, Christopher J, Clement, Mathew, Ladell, Kristin, McLaren, James E, Berg, Hugo A, Gostick, Emma, L Miners, Kelly, Llewellyn‐Lacey, Sian, Bridgeman, John S, Man, Stephen, Bailey, Mick, Burrows, Scott R, Price, David A, and Wooldridge, Linda
- Abstract
The CD8 co‐receptor engages peptide‐major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T‐cell receptor (TCR)‐binding platform and enhances the sensitivity of antigen‐driven activation to promote effective CD8+ T‐cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5‐fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10‐fold). In this study, we used a panel of MHCI mutants with altered CD8‐binding properties to show that TCR‐mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T‐cell transfer irrespective of antigen specificity. [ABSTRACT FROM AUTHOR]
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- 2017
- Full Text
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26. Heterogeneous yet stable Vδ2(+) T-cell profiles define distinct cytotoxic effector potentials in healthy human individuals.
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Sumaria, Nital, Holland, Christopher J., Bradford, Claire M., Izotova, Natalia, Grandjean, Capucine L., Pennington, Daniel J., Ryan, Paul L., Jawad, Ali S., and Bergmeier, Lesley A.
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T cells , *CYTOTOXINS , *PATHOGENIC microorganisms , *CANCER , *IMMUNOTHERAPY , *PHYSIOLOGY - Abstract
Human γδ T cells display potent responses to pathogens and malignancies. Of particular interest are those expressing a γδ T-cell receptor (TCR) incorporating TCRδ-chain variable-region-2 [Vδ2(+)], which are activated by pathogen-derived phosphoantigens (pAgs), or host-derived pAgs that accumulate in transformed cells or in cells exposed to aminobisphosphonates. Once activated, Vδ2(+) T cells exhibit multiple effector functions that have made them attractive candidates for immunotherapy. Despite this, clinical trials have reported mixed patient responses, highlighting a need for better understanding of Vδ2(+) T-cell biology. Here, we reveal previously unappreciated functional heterogeneity between the Vδ2(+) T-cell compartments of 63 healthy individuals. In this cohort, we identify distinct “Vδ2 profiles” that are stable over time; that do not correlate with age, gender, or history of phosphoantigen activation; and that develop after leaving the thymus. Multiple analyses suggest these Vδ2 profiles consist of variable proportions of two dominant but contrasting Vδ2(+) T-cell subsets that have divergent transcriptional programs and that display mechanistically distinct cytotoxic potentials. Importantly, an individual’s Vδ2(+) profile predicts defined effector capacities, demonstrated by contrasting mechanisms and efficiencies of killing of a range of tumor cell lines. In short, these data support patient stratification to identify individuals with Vδ2 profiles that have effector mechanisms compatible with tumor killing and suggest that tailored Vδ2-profile–specific activation protocols may maximize the chances of future treatment success. [ABSTRACT FROM AUTHOR]
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- 2016
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27. T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions
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Cole, David K., primary, Miles, Kim M., additional, Madura, Florian, additional, Holland, Christopher J., additional, Schauenburg, Andrea J.A., additional, Godkin, Andrew J., additional, Bulek, Anna M., additional, Fuller, Anna, additional, Akpovwa, Hephzibah J.E., additional, Pymm, Phillip G., additional, Liddy, Nathaniel, additional, Sami, Malkit, additional, Li, Yi, additional, Rizkallah, Pierre J., additional, Jakobsen, Bent K., additional, and Sewell, Andrew K., additional
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- 2014
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28. T-cell Receptor Specificity Maintained by Altered Thermodynamics
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Madura, Florian, primary, Rizkallah, Pierre J., additional, Miles, Kim M., additional, Holland, Christopher J., additional, Bulek, Anna M., additional, Fuller, Anna, additional, Schauenburg, Andrea J.A., additional, Miles, John J., additional, Liddy, Nathaniel, additional, Sami, Malkit, additional, Li, Yi, additional, Hossain, Moushumi, additional, Baker, Brian M., additional, Jakobsen, Bent K., additional, Sewell, Andrew K., additional, and Cole, David K., additional
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- 2013
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29. Re-Directing CD4+ T Cell Responses with the Flanking Residues of MHC Class II-Bound Peptides: The Core is Not Enough
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Holland, Christopher J., primary, Cole, David K., additional, and Godkin, Andrew, additional
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- 2013
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30. Enhanced Detection of Antigen-Specific CD4+ T Cells Using Altered Peptide Flanking Residue Peptide-MHC Class II Multimers.
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Holland, Christopher J., Dolton, Garry, Scurr, Martin, Ladell, Kristin, Schauenburg, Andrea J., Miners, Kelly, Madura, Florian, Sewell, Andrew K., Price, David A., Cole, David K., and Godkin, Andrew J.
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FLUOROPHORES , *PEPTIDES , *T cells , *CELL populations , *CELL proliferation , *COOPERATIVE binding (Biochemistry) , *BINDING sites , *LIGAND binding (Biochemistry) - Abstract
Fluorochrome-conjugated peptide-MHC (pMHC) class I multimers are staple components of the immunologist's toolbox, enabling reliable quantification and analysis of Ag-specific CD8+ T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4+ T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-IIbound peptides, can enhance TCR-pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4+ T cells, highlighting an unappreciated feature of TCR-pMHC-II interactions. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
31. Minimal conformational plasticity enables TCR cross-reactivity to different MHC class II heterodimers
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Holland, Christopher J., primary, Rizkallah, Pierre J., additional, Vollers, Sabrina, additional, Calvo-Calle, J. Mauricio, additional, Madura, Florian, additional, Fuller, Anna, additional, Sewell, Andrew K., additional, Stern, Lawrence J., additional, Godkin, Andrew, additional, and Cole, David K., additional
- Published
- 2012
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32. TCR/pMHC Optimized Protein crystallization Screen
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Bulek, Anna M., primary, Madura, Florian, additional, Fuller, Anna, additional, Holland, Christopher J., additional, Schauenburg, Andrea J.A., additional, Sewell, Andrew K., additional, Rizkallah, Pierre J., additional, and Cole, David K., additional
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- 2012
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33. Modification of the carboxy-terminal flanking region of a universal influenza epitope alters CD4+ T-cell repertoire selection
- Author
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Cole, David K., primary, Gallagher, Kathleen, additional, Lemercier, Brigitte, additional, Holland, Christopher J., additional, Junaid, Sayed, additional, Hindley, James P., additional, Wynn, Katherine K., additional, Gostick, Emma, additional, Sewell, Andrew K., additional, Gallimore, Awen M., additional, Ladell, Kristin, additional, Price, David A., additional, Gougeon, Marie-Lise, additional, and Godkin, Andrew, additional
- Published
- 2012
- Full Text
- View/download PDF
34. Structural basis for ineffective T-cell responses to MHC anchor residue-improved 'heteroclitic' peptides.
- Author
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Madura, Florian, Rizkallah, Pierre J., Holland, Christopher J., Fuller, Anna, Bulek, Anna, Godkin, Andrew J., Schauenburg, Andrea J., Cole, David K., and Sewell, Andrew K.
- Abstract
MHC anchor residue-modified 'heteroclitic' peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild-type peptide. The best-studied system to date is the decamer MART-1/Melan-A
26-35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)-A*0201 anchoring. The resulting E LAGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the E LAGIGILTV peptide can fail to recognize the natural tumor-expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA-A*0201-EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA-A*0201-E LAGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to 'pull' the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced-fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation. [ABSTRACT FROM AUTHOR]- Published
- 2015
- Full Text
- View/download PDF
35. Re-directing CD4+ T cell responses with the flanking residues of MHC class II-bound peptides: the core is not enough.
- Author
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Holland, Christopher J., Cole, David K., and Godkin, Andrew
- Subjects
PEPTIDES ,HISTOCOMPATIBILITY ,T-cell receptor genes ,CRYSTAL structure ,CELL membranes ,CYTOSOL ,EPITOPES - Abstract
Recombinant αβT cell receptors, expressed onT cell membranes, recognize short peptides presented at the cell surface in complex with MHC molecules.There are two main subsets of αβ T cells: CD8+ T cells that recognize mainly cytosol-derived peptides in the context of MHC class I (pMHC-I), and CD4+ T cells that recognize peptides usually derived from exogenous proteins presented by MHC class II (pMHC-II). Unlike the more uniform peptide lengths (usually 8-13mers) bound in the MHC-I closed groove, MHC-II presented peptides are of a highly variable length.The bound peptides consist of a core bound 9mer (reflecting the binding motif for the particular MHC-II type) but with variable peptide flanking residues (PFRs) that can extend from both the N- and C-terminus of the MHC-II binding groove. Although pMHC-I and pMHC-II play a virtually identical role during T cell responses (T cell antigen presentation) and are very similar in overall conformation, there exist a number of subtle but important differences that may govern the functional dichotomy observed between CD8+ and CD4+T cells. Here, we provide an overview of the impact of structural differences between pMHC-I and pMHC-II and the molecular interactions with the T cell receptor including the functional importance of MHC-II PFRs. We consider how factors such as anatomical location, inflammatory milieu, and particular types of antigen presenting cell might, in theory, contribute to the quantitative (i.e., pMHC ligand frequency) as well as qualitative (i.e., variable PFR) nature of peptide epitopes, and hence offer a means of control and influence of a CD4+ T cell response. Lastly, we review our recent findings showing how modifications to MHC-II PFRs can modify CD4+ T cell antigen recognition. These findings may have novel applications for the development of CD4+ T cell peptide vaccines and diagnostics. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
36. Honors Living-Learning Communities: A Model of Success and Collaboration.
- Author
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Daffron, Eric and Holland, Christopher J.
- Subjects
LIVING learning centers ,COMMUNITY-school relationships ,LEARNING ,COMMUNITY & college ,EDUCATION ,STUDENTS ,COLLEGE students ,PLACE-based education ,TRADITIONAL knowledge - Abstract
The article provides information on the significance of living-learning communities to the college students' learning development and welfare. It mentions the benefits of living-learning communities to the student's areas of development, particularly the psychosocial and cognitive and cites that these students would someday ready to face the challenges of the modern world, due to this innovative learning. It highlights the relevance of implementing honors programs for the betterment of the students. Furthermore, the background of living-learning communities implementation is also elucidated.
- Published
- 2009
37. TCR‐induced alteration of primary MHC peptide anchor residue
- Author
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Madura, Florian, Rizkallah, Pierre J., Legut, Mateusz, Holland, Christopher J., Fuller, Anna, Bulek, Anna, Schauenburg, Andrea J., Trimby, Andrew, Hopkins, Jade R., Wells, Stephen A., Godkin, Andrew, Miles, John J., Sami, Malkit, Li, Yi, Liddy, Nathaniel, Jakobsen, Bent K., Loveridge, E. Joel, Cole, David K., and Sewell, Andrew K.
- Full Text
- View/download PDF
38. Molecular Rules Underpinning Enhanced Affinity Binding of Human T Cell Receptors Engineered for Immunotherapy.
- Author
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Crean RM, MacLachlan BJ, Madura F, Whalley T, Rizkallah PJ, Holland CJ, McMurran C, Harper S, Godkin A, Sewell AK, Pudney CR, van der Kamp MW, and Cole DK
- Abstract
Immuno-oncology approaches that utilize T cell receptors (TCRs) are becoming highly attractive because of their potential to target virtually all cellular proteins, including cancer-specific epitopes, via the recognition of peptide-human leukocyte antigen (pHLA) complexes presented at the cell surface. However, because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities, efforts have been made to enhance their binding strength, in some cases by several million-fold. In this study, we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors. Additionally, we performed molecular dynamics simulations to better understand the energetic mechanisms driving the affinity enhancements. These data demonstrate that supra-physiological binding affinities can be achieved without altering native TCR-pHLA binding modes via relatively subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. Individual energetic components of the TCR-pHLA interaction governing affinity enhancements were distinct and highly variable for each TCR, often resulting from additive, or knock-on, effects beyond the mutated residues. This comprehensive analysis of affinity-enhanced TCRs has important implications for the future rational design of engineered TCRs as efficacious and safe drugs for cancer treatment., (Crown Copyright © 2020.)
- Published
- 2020
- Full Text
- View/download PDF
39. The metabolic demand of external load carriage in golfers: a comparison of a single versus double-strap golf bag.
- Author
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Holland CJ and Godwin MS
- Subjects
- Adult, Athletes statistics & numerical data, Energy Metabolism, Exercise Test, Heart Rate, Humans, Male, Middle Aged, Oxygen Consumption, Respiratory Function Tests, Walking, Equipment and Supplies, Golf physiology
- Abstract
Background: A golf bag filled with a set of clubs provides a substantial load. When carried over distance this can increase the demands placed upon the golfer, leading to discomfort, fatigue and injuries. This study aimed to compare the metabolic demands of 2 methods of golf bag carriage., Methods: A total of 16 healthy male recreational golfers participated in the study. Participants were given an initial familiarization session in which their self-selected walking speed was determined. This was utilized as the treadmill speed for all subsequent trials. The testing protocol consisted of 3 randomized trials of treadmill walking for 5 minutes in each of three conditions: unloaded, single-strap bag and double-strap bag. Equipment consisted of a double-strap golf bag with a standard set of clubs weighing 12.5kg. For all trials oxygen consumption (L·min-1), VO2 (mL·kg·min-1) respiratory minute volume (VE) (L·min-1), and heart rate (HR) were measured., Results: Results showed that the double-strap bag required significantly less oxygen consumption (1.19±0.19 vs. 1.31±0.16 L·min-1, P<0.01) relative oxygen consumption (14.49±2.06 vs. 15.93±2.25 mL·kg·min-1, P<0.01), reduced respiratory minute volume (29.95±4.19 vs. 32.47±4.26 L·min-1, P<0.05), and lower heart rates (100.14±11.05 vs. 106.96±9.33 BPM, P<0.001) than the single-strap bag. Both methods of carriage showed significantly greater metabolic demands than the unloaded condition (P<0.05)., Conclusions: The decreased metabolic cost of carrying a double-strap golf bag may facilitate a reduction in fatigue and reduced mechanical stress. Golf bag transportation must therefore be recognized as a factor in reducing the risk of injury and improving playing performance.
- Published
- 2019
- Full Text
- View/download PDF
40. TCR-induced alteration of primary MHC peptide anchor residue.
- Author
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Madura F, Rizkallah PJ, Legut M, Holland CJ, Fuller A, Bulek A, Schauenburg AJ, Trimby A, Hopkins JR, Wells SA, Godkin A, Miles JJ, Sami M, Li Y, Liddy N, Jakobsen BK, Loveridge EJ, Cole DK, and Sewell AK
- Subjects
- Amino Acids, Antigen Presentation, Binding Sites, Cells, Cultured, Clone Cells, HLA-A2 Antigen chemistry, HLA-A2 Antigen metabolism, Humans, Lymphocyte Activation, MART-1 Antigen chemistry, Melanoma therapy, Peptides chemistry, Protein Binding, Protein Conformation, Receptors, Antigen, T-Cell genetics, T-Lymphocytes transplantation, Immunodominant Epitopes metabolism, Immunotherapy, Adoptive methods, MART-1 Antigen metabolism, Melanoma immunology, Peptides metabolism, Receptors, Antigen, T-Cell metabolism, T-Lymphocytes immunology
- 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 nonapeptide 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., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
- Full Text
- View/download PDF
41. Dual Molecular Mechanisms Govern Escape at Immunodominant HLA A2-Restricted HIV Epitope.
- Author
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Cole DK, Fuller A, Dolton G, Zervoudi E, Legut M, Miles K, Blanchfield L, Madura F, Holland CJ, Bulek AM, Bridgeman JS, Miles JJ, Schauenburg AJA, Beck K, Evavold BD, Rizkallah PJ, and Sewell AK
- 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.
- Published
- 2017
- Full Text
- View/download PDF
42. CD8 + T-cell specificity is compromised at a defined MHCI/CD8 affinity threshold.
- Author
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Dockree T, Holland CJ, Clement M, Ladell K, McLaren JE, van den Berg HA, Gostick E, L Miners K, Llewellyn-Lacey S, Bridgeman JS, Man S, Bailey M, Burrows SR, Price DA, and Wooldridge L
- Subjects
- Cell Membrane metabolism, Humans, Lymphocyte Activation immunology, Mutation genetics, Peptides metabolism, CD8 Antigens metabolism, CD8-Positive T-Lymphocytes immunology, Histocompatibility Antigens Class I immunology
- Abstract
The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8
+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity., Competing Interests: The authors have no conflicts of interest with the contents of this article.- Published
- 2017
- Full Text
- View/download PDF
43. Heterogeneous yet stable Vδ2(+) T-cell profiles define distinct cytotoxic effector potentials in healthy human individuals.
- Author
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Ryan PL, Sumaria N, Holland CJ, Bradford CM, Izotova N, Grandjean CL, Jawad AS, Bergmeier LA, and Pennington DJ
- Subjects
- Adolescent, Adult, Aged, CX3C Chemokine Receptor 1 metabolism, Child, Child, Preschool, Cytotoxicity, Immunologic, Female, Gene Expression Profiling, Genes, T-Cell Receptor delta, Healthy Volunteers, Humans, Immunophenotyping, Lymphocyte Activation immunology, Male, Middle Aged, Receptors, Antigen, T-Cell, gamma-delta genetics, Receptors, CCR6 metabolism, Young Adult, Receptors, Antigen, T-Cell, gamma-delta metabolism, T-Lymphocyte Subsets immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Human γδ T cells display potent responses to pathogens and malignancies. Of particular interest are those expressing a γδ T-cell receptor (TCR) incorporating TCRδ-chain variable-region-2 [Vδ2
(+) ], which are activated by pathogen-derived phosphoantigens (pAgs), or host-derived pAgs that accumulate in transformed cells or in cells exposed to aminobisphosphonates. Once activated, Vδ2(+) T cells exhibit multiple effector functions that have made them attractive candidates for immunotherapy. Despite this, clinical trials have reported mixed patient responses, highlighting a need for better understanding of Vδ2(+) T-cell biology. Here, we reveal previously unappreciated functional heterogeneity between the Vδ2(+) T-cell compartments of 63 healthy individuals. In this cohort, we identify distinct "Vδ2 profiles" that are stable over time; that do not correlate with age, gender, or history of phosphoantigen activation; and that develop after leaving the thymus. Multiple analyses suggest these Vδ2 profiles consist of variable proportions of two dominant but contrasting Vδ2(+) T-cell subsets that have divergent transcriptional programs and that display mechanistically distinct cytotoxic potentials. Importantly, an individual's Vδ2 profile predicts defined effector capacities, demonstrated by contrasting mechanisms and efficiencies of killing of a range of tumor cell lines. In short, these data support patient stratification to identify individuals with Vδ2 profiles that have effector mechanisms compatible with tumor killing and suggest that tailored Vδ2-profile-specific activation protocols may maximize the chances of future treatment success., Competing Interests: The authors declare no conflict of interest.- Published
- 2016
- Full Text
- View/download PDF
44. Enhanced Detection of Antigen-Specific CD4+ T Cells Using Altered Peptide Flanking Residue Peptide-MHC Class II Multimers.
- Author
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Holland CJ, Dolton G, Scurr M, Ladell K, Schauenburg AJ, Miners K, Madura F, Sewell AK, Price DA, Cole DK, and Godkin AJ
- Subjects
- Cell Line, Histocompatibility Antigens Class II metabolism, Humans, Lymphocyte Activation, Protein Binding, Receptors, Antigen, T-Cell immunology, T-Cell Antigen Receptor Specificity, Antigens metabolism, CD4-Positive T-Lymphocytes immunology, Cell Separation methods, HLA Antigens immunology, Peptide Fragments metabolism
- Abstract
Fluorochrome-conjugated peptide-MHC (pMHC) class I multimers are staple components of the immunologist's toolbox, enabling reliable quantification and analysis of Ag-specific CD8(+) T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4(+) T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II-bound peptides, can enhance TCR-pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4(+) T cells, highlighting an unappreciated feature of TCR-pMHC-II interactions., (Copyright © 2015 The Authors.)
- Published
- 2015
- Full Text
- View/download PDF
45. Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs.
- Author
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Tungatt K, Bianchi V, Crowther MD, Powell WE, Schauenburg AJ, Trimby A, Donia M, Miles JJ, Holland CJ, Cole DK, Godkin AJ, Peakman M, Straten PT, Svane IM, Sewell AK, and Dolton G
- Subjects
- Antibodies chemistry, Antibodies immunology, Cells, Cultured, Fluorescent Dyes chemistry, Humans, Phycocyanin chemistry, Protein Binding immunology, Protein Kinase Inhibitors pharmacology, T-Lymphocytes cytology, Flow Cytometry methods, Fluorescent Antibody Technique methods, Receptors, Antigen, T-Cell immunology, Staining and Labeling methods, T-Lymphocytes immunology
- Abstract
Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II-restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR-pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents., (Copyright © 2014 The Authors.)
- Published
- 2015
- Full Text
- View/download PDF
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