33 results on '"Lloyd, Angharad"'
Search Results
2. Gene editing in T-cells and T-cell targets
- Author
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Lloyd, Angharad
- Subjects
616.07 ,QR180 Immunology ,R Medicine (General) - Abstract
Recent years have witnessed a rapid proliferation of gene editing in mammalian cells due to the increasing ease and reduced cost of targeted gene knockout. There has been much excitement about the prospect of engineering T-cells by gene editing in order to provide these cells with optimal attributes prior to adoptive cell therapy for cancer and autoimmune disease. I began by attempting to compare short hairpin RNA (shRNA) and zinc finger nuclease (ZFN) approaches using the CD8A gene as a target for proof of concept of gene editing in Molt3 cells. During the course of my studies the clustered regularly interspaced short palindromic repeats (CRISPR) mechanism for gene editing was discovered so I also included CRISPR/Cas9 in my studies. A direct comparison of the three gene editing tools indicated that the CRISPR/Cas9 system was superior in terms of ease, efficiency of knockout and cost. As the use of gene editing tools increases there are concerns about the inherent risks associated with the use of nuclease based gene editing tools prior to cellular therapy. Expression of nucleases can lead to off target mutagenesis and malignancy. To circumvent this problem I generated a non-nuclease based gene silencing system using the CD8A zinc finger (ZF) fused to a Krüppel associated box (KRAB) repressor domain. The ZF-KRAB fusion resulted in effective silencing of the CD8A gene in both the Molt3 cell line and in primary CD8+ T-cells. Importantly, unlike CRISPR/Cas9 based gene editing, the ZF-KRAB fusion was small enough to be transferred in a single lentiviral vector with a TCR allowing simultaneous redirection of patient T-cell specificity and alteration of T-cell function in a single construct. To improve the efficiency of gene editing with CRISPR/Cas9 I developed an ‘all in one’ CRISPR/Cas9 system which incorporated all elements of the CRISPR/Cas9 gene editing system in a single plasmid. The ‘all in one’ system was utilised to derive MHC-related protein 1 (MR1) deficient clones from the A549 lung carcinoma and THP-1 monocytic cell lines in order to study MR1 biology. Mucosal-associated invariant T-cell (MAIT) clones were not activated by MR1 deficient A549 or THP-1 clones infected with bacteria.
- Published
- 2016
3. 362 Allelic variants of MR1 drive cancer and allo-reactivity by MR1-restricted T cells
- Author
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Howie, Duncan, primary, Cornforth, Terri V, additional, Moyo, Nathifa, additional, Cole, Suzanne, additional, Lam, Emily, additional, Lobry, Tatiana, additional, Wolchinsky, Ron, additional, Lloyd, Angharad, additional, Ward, Katarzyna, additional, Denham, Eleanor M, additional, Besra, Gurdyal S, additional, Illing, Patricia, additional, MacLachlan, Bruce, additional, Vivian, Julian, additional, Raynes, Jeremy, additional, Nours, Jerome Le, additional, Purcell, Anthony, additional, Kundu, Samit, additional, Silk, Jonathan D, additional, Williams, Luke, additional, Papa, Sophie, additional, Rossjohn, Jamie, additional, and Dukes, Joseph, additional
- Published
- 2023
- Full Text
- View/download PDF
4. Genome-wide CRISPR–Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1
- Author
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Crowther, Michael D., Dolton, Garry, Legut, Mateusz, Caillaud, Marine E., Lloyd, Angharad, Attaf, Meriem, Galloway, Sarah A. E., Rius, Cristina, Farrell, Colin P., Szomolay, Barbara, Ager, Ann, Parker, Alan L., Fuller, Anna, Donia, Marco, McCluskey, James, Rossjohn, Jamie, Svane, Inge Marie, Phillips, John D., and Sewell, Andrew K.
- Published
- 2020
- Full Text
- View/download PDF
5. Conserved allomorphs of MR1 drive specificity of MR1-restricted TCRs
- Author
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Cornforth, Terri V, primary, Moyo, Nathifa A, additional, Cole, Suzanne, additional, Lam, Emily, additional, Lobry, Tatiana, additional, Wolchinsky, Ron, additional, Lloyd, Angharad, additional, Ward, Katarzyna, additional, Denham, Eleanor M, additional, Besra, Gurdyal S, additional, Veerapen, Natacha, additional, Illing, Patricia T, additional, Vivian, Julian P, additional, Raynes, Jeremy M, additional, Le Nours, Jerome, additional, Purcell, Anthony W, additional, Kundu, Samit, additional, Silk, Jonathan D, additional, Williams, Luke, additional, Papa, Sophie, additional, Rossjohn, Jamie, additional, Howie, Duncan, additional, and Dukes, Joseph, additional
- Published
- 2023
- Full Text
- View/download PDF
6. Promiscuous recognition of MR1 drives self-reactive mucosal-associated invariant T cell responses
- Author
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Chancellor, Andrew, primary, Alan Simmons, Robert, additional, Khanolkar, Rahul C., additional, Nosi, Vladimir, additional, Beshirova, Aisha, additional, Berloffa, Giuliano, additional, Colombo, Rodrigo, additional, Karuppiah, Vijaykumar, additional, Pentier, Johanne M., additional, Tubb, Vanessa, additional, Ghadbane, Hemza, additional, Suckling, Richard J., additional, Page, Keith, additional, Crean, Rory M., additional, Vacchini, Alessandro, additional, De Gregorio, Corinne, additional, Schaefer, Verena, additional, Constantin, Daniel, additional, Gligoris, Thomas, additional, Lloyd, Angharad, additional, Hock, Miriam, additional, Srikannathasan, Velupillai, additional, Robinson, Ross A., additional, Besra, Gurdyal S., additional, van der Kamp, Marc W., additional, Mori, Lucia, additional, Calogero, Raffaele, additional, Cole, David K., additional, De Libero, Gennaro, additional, and Lepore, Marco, additional
- Published
- 2023
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- View/download PDF
7. Promiscuous recognition of MR1 drives self-reactive mucosal-associated invariant T cell responses
- Author
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Chancellor, Andrew, Alan Simmons, Robert, Khanolkar, Rahul C, Nosi, Vladimir, Beshirova, Aisha, Berloffa, Giuliano, Colombo, Rodrigo, Karuppiah, Vijaykumar, Pentier, Johanne M, Tubb, Vanessa, Ghadbane, Hemza, Suckling, Richard J, Page, Keith, Crean, Rory M, Vacchini, Alessandro, De Gregorio, Corinne, Schaefer, Verena, Constantin, Daniel, Gligoris, Thomas, Lloyd, Angharad, Hock, Miriam, Srikannathasan, Velupillai, Robinson, Ross A, Besra, Gurdyal S, van der Kamp, Marc W, Mori, Lucia, Calogero, Raffaele, Cole, David K, De Libero, Gennaro, and Lepore, Marco
- Published
- 2023
8. Author Correction: Genome-wide CRISPR–Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1
- Author
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Crowther, Michael D., Dolton, Garry, Legut, Mateusz, Caillaud, Marine E., Lloyd, Angharad, Attaf, Meriem, Galloway, Sarah A. E., Rius, Cristina, Farrell, Colin P., Szomolay, Barbara, Ager, Ann, Parker, Alan L., Fuller, Anna, Donia, Marco, McCluskey, James, Rossjohn, Jamie, Svane, Inge Marie, Phillips, John D., and Sewell, Andrew K.
- Published
- 2020
- Full Text
- View/download PDF
9. More tricks with tetramers: a practical guide to staining T cells with peptide–MHC multimers
- Author
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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
- Full Text
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10. T cell receptor interactions with human leukocyte antigen govern indirect peptide selectivity for the cancer testis antigen MAGE-A4
- Author
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Coles, Charlotte H., primary, McMurran, Catriona, additional, Lloyd, Angharad, additional, Hock, Miriam, additional, Hibbert, Linda, additional, Raman, Marine C.C., additional, Hayes, Conor, additional, Lupardus, Patrick, additional, Cole, David K., additional, and Harper, Stephen, additional
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- 2020
- Full Text
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11. CD4+ T Cells Recognize Conserved Influenza A Epitopes through Shared Patterns of V-Gene Usage and Complementary Biochemical Features
- Author
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Greenshields-Watson, Alexander, primary, Attaf, Meriem, additional, MacLachlan, Bruce J., additional, Whalley, Thomas, additional, Rius, Cristina, additional, Wall, Aaron, additional, Lloyd, Angharad, additional, Hughes, Hywel, additional, Strange, Kathryn E., additional, Mason, Georgina H., additional, Schauenburg, Andrea J., additional, Hulin-Curtis, Sarah L., additional, Geary, James, additional, Chen, Yuan, additional, Lauder, Sarah N., additional, Smart, Kathryn, additional, Vijaykrishna, Dhanasekaran, additional, Grau, Miguel L., additional, Shugay, Mikhail, additional, Andrews, Robert, additional, Dolton, Garry, additional, Rizkallah, Pierre J., additional, Gallimore, Awen M., additional, Sewell, Andrew K., additional, Godkin, Andrew J., additional, and Cole, David K., additional
- Published
- 2020
- Full Text
- View/download PDF
12. Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA
- Author
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Holland, Christopher J., primary, Crean, Rory M., additional, Pentier, Johanne M., additional, de Wet, Ben, additional, Lloyd, Angharad, additional, Srikannathasan, Velupillai, additional, Lissin, Nikolai, additional, Lloyd, Katy A., additional, Blicher, Thomas H., additional, Conroy, Paul J., additional, Hock, Miriam, additional, Pengelly, Robert J., additional, Spinner, Thomas E., additional, Cameron, Brian, additional, Potter, Elizabeth A., additional, Jeyanthan, Anitha, additional, Molloy, Peter E., additional, Sami, Malkit, additional, Aleksic, Milos, additional, Liddy, Nathaniel, additional, Robinson, Ross A., additional, Harper, Stephen, additional, Lepore, Marco, additional, Pudney, Chris R., additional, van der Kamp, Marc W., additional, Rizkallah, Pierre J., additional, Jakobsen, Bent K., additional, Vuidepot, Annelise, additional, and Cole, David K., additional
- Published
- 2020
- Full Text
- View/download PDF
13. TCRs with Distinct Specificity Profiles Use Different Binding Modes to Engage an Identical Peptide–HLA Complex
- Author
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Coles, Charlotte H., primary, Mulvaney, Rachel M., additional, Malla, Sunir, additional, Walker, Andrew, additional, Smith, Kathrine J., additional, Lloyd, Angharad, additional, Lowe, Kate L., additional, McCully, Michelle L., additional, Martinez Hague, Ruth, additional, Aleksic, Milos, additional, Harper, Jane, additional, Paston, Samantha J., additional, Donnellan, Zoe, additional, Chester, Fiona, additional, Wiederhold, Katrin, additional, Robinson, Ross A., additional, Knox, Andrew, additional, Stacey, Andrea R., additional, Dukes, Joseph, additional, Baston, Emma, additional, Griffin, Sue, additional, Jakobsen, Bent K., additional, Vuidepot, Annelise, additional, and Harper, Stephen, additional
- Published
- 2020
- Full Text
- View/download PDF
14. Peptide Super-Agonist Enhances T-Cell Responses to Melanoma
- Author
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Galloway, Sarah A E, Dolton, Garry, Attaf, Meriem, Wall, Aaron, Fuller, Anna, Rius, Cristina, Bianchi, Valentina, Theaker, Sarah, Lloyd, Angharad, Caillaud, Marine E, Svane, Inge Marie, Donia, Marco, Cole, David K, Szomolay, Barbara, Rizkallah, Pierre, Sewell, Andrew K, Galloway, Sarah A E, Dolton, Garry, Attaf, Meriem, Wall, Aaron, Fuller, Anna, Rius, Cristina, Bianchi, Valentina, Theaker, Sarah, Lloyd, Angharad, Caillaud, Marine E, Svane, Inge Marie, Donia, Marco, Cole, David K, Szomolay, Barbara, Rizkallah, Pierre, and Sewell, Andrew K
- Abstract
Recent immunotherapeutic approaches using adoptive cell therapy, or checkpoint blockade, have demonstrated the powerful anti-cancer potential of CD8 cytotoxic T-lymphocytes (CTL). While these approaches have shown great promise, they are only effective in some patients with some cancers. The potential power, and relative ease, of therapeutic vaccination against tumour associated antigens (TAA) present in different cancers has been a long sought-after approach for harnessing the discriminating sensitivity of CTL to treat cancer and has seen recent renewed interest following cancer vaccination successes using unique tumour neoantigens. Unfortunately, results with TAA-targeted "universal" cancer vaccines (UCV) have been largely disappointing. Infectious disease models have demonstrated that T-cell clonotypes that recognise the same antigen should not be viewed as being equally effective. Extrapolation of this notion to UCV would suggest that the quality of response in terms of the T-cell receptor (TCR) clonotypes induced might be more important than the quantity of the response. Unfortunately, there is little opportunity to assess the effectiveness of individual T-cell clonotypes in vivo. Here, we identified effective, persistent T-cell clonotypes in an HLA A2+ patient following successful tumour infiltrating lymphocyte (TIL) therapy. One such T-cell clone was used to generate super-agonist altered peptide ligands (APLs). Further refinement produced an APL that was capable of inducing T-cells in greater magnitude, and with improved effectiveness, from the blood of all 14 healthy donors tested. Importantly, this APL also induced T-cells from melanoma patient blood that exhibited superior recognition of the patient's own tumour compared to those induced by the natural antigen sequence. These results suggest that use of APL to skew the clonotypic quality of T-cells induced by cancer vaccination could provide a promising avenue in the hunt for the UCV "magic bullet."
- Published
- 2019
15. Engineering of Isogenic Cells Deficient for MR1 with a CRISPR/Cas9 Lentiviral System: Tools To Study Microbial Antigen Processing and Presentation to Human MR1-Restricted T Cells
- Author
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Laugel, Bruno, Lloyd, Angharad, Meermeier, Erin W., Crowther, Michael D., Connor, Thomas R., Dolton, Garry, Miles, John J., Burrows, Scott R., Gold, Marielle C., Lewinsohn, David M., and Sewell, Andrew K.
- Subjects
Gene Editing ,Antigen Presentation ,T-Lymphocytes ,Genetic Vectors ,Histocompatibility Antigens Class I ,Lentivirus ,Flow Cytometry ,Lymphocyte Activation ,Polymerase Chain Reaction ,Cell Line ,Minor Histocompatibility Antigens ,T-Lymphocyte Subsets ,Novel Immunological Methods ,QR180 ,Mutagenesis, Site-Directed ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats - Abstract
The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1(-/-) clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1(-/-) clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells.
- Published
- 2016
16. Peptide Super-Agonist Enhances T-Cell Responses to Melanoma
- Author
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Galloway, Sarah A. E., primary, Dolton, Garry, additional, Attaf, Meriem, additional, Wall, Aaron, additional, Fuller, Anna, additional, Rius, Cristina, additional, Bianchi, Valentina, additional, Theaker, Sarah, additional, Lloyd, Angharad, additional, Caillaud, Marine E., additional, Svane, Inge Marie, additional, Donia, Marco, additional, Cole, David K., additional, Szomolay, Barbara, additional, Rizkallah, Pierre, additional, and Sewell, Andrew K., additional
- Published
- 2019
- Full Text
- View/download PDF
17. Structural mechanism underpinning cross-reactivity of a CD8+ T-cell clone that recognises a peptide derived from human telomerase reverse transcriptase
- Author
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Cole, David K., Berg, Hugo van den, Lloyd, Angharad, Crowther, Michael D., Beck, Konrad, Ekeruche-Makinde, Julia, Miles, John J., Bulek, Anna M., Dolton, Garry, Schauenburg, Andrea J., Wall, Aaron, Fuller, Anna, Clement, Mathew, Laugel, Bruno, Rizkallah, Pierre J., Wooldridge, Linda, and Sewell, Andrew K.
- Subjects
Biochemistry & Molecular Biology ,Immunology ,T cells ,Receptors, Antigen, T-Cell ,chemical and pharmacologic phenomena ,CD8-Positive T-Lymphocytes ,Cross Reactions ,telomerase ,BETA-CELLS ,CLASS-I ,BINDING ,Humans ,QD ,tumor immunology ,ANTIGEN RECOGNITION ,SPECIFICITY ,Cells, Cultured ,AFFINITY ,X-ray crystallography ,CARDIOVASCULAR TOXICITY ,Science & Technology ,RECEPTOR RECOGNITION ,11 Medical And Health Sciences ,06 Biological Sciences ,T cell degeneracy ,R1 ,MAJOR HISTOCOMPATIBILITY COMPLEX ,peptides ,T cell receptor ,03 Chemical Sciences ,Life Sciences & Biomedicine ,surface plasmon resonance (SPR) ,TCR - 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 focussed 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 (hTERT). 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.
- Published
- 2017
18. A molecular switch abrogates glycoprotein 100 (gp100) T-cell Receptor (TCR) targeting of a human melanoma antigen
- Author
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Bianchi, Valentina, Bulek, Anna, Fuller, Anna, Lloyd, Angharad, Attaf, Meriem, Rizkallah, Pierre J., Dolton, Garry, Sewell, Andrew K., and Cole, David K.
- Subjects
chemical and pharmacologic phenomena - Abstract
Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), 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. 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.
- Published
- 2016
19. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones
- Author
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Theaker, Sarah M., Rius, Cristina, Greenshields-Watson, Alexander, Lloyd, Angharad, Trimby, Andrew, Fuller, Anna, Miles, John J., Cole, David K., Peakman, Mark, Sewell, Andrew K., and Dolton, Garry
- Subjects
Library ,Peptide-specific ,Type 1 diabetes ,Immunology ,Ebola ,T-cell clone ,QR180 ,Immunology and Allergy ,Tumour - Abstract
Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer.
- Published
- 2016
20. Confirmation of mutation landscape of NF1‐associated malignant peripheral nerve sheath tumors
- Author
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Sohier, Pierre, primary, Luscan, Armelle, additional, Lloyd, Angharad, additional, Ashelford, Kevin, additional, Laurendeau, Ingrid, additional, Briand‐Suleau, Audrey, additional, Vidaud, Dominique, additional, Ortonne, Nicolas, additional, Pasmant, Eric, additional, and Upadhyaya, Meena, additional
- Published
- 2017
- Full Text
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21. Thermal Stability of Heterotrimeric pMHC Proteins as Determined by Circular Dichroism Spectroscopy
- Author
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Fuller, Anna, primary, Wall, Aaron, additional, Crowther, Michael, additional, Lloyd, Angharad, additional, Zhurov, Alexei, additional, Sewell, Andrew, additional, Cole, David, additional, and Beck, Konrad, additional
- Published
- 2017
- Full Text
- View/download PDF
22. More tricks with tetramers:a practical guide to staining T cells with peptide-MHC multimers
- Author
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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.
- Published
- 2015
23. Beyond the Antigen Receptor: Editing the Genome of T-Cells for Cancer Adoptive Cellular Therapies
- Author
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Lloyd, Angharad, primary, Vickery, Owen N., additional, and Laugel, Bruno, additional
- Published
- 2013
- Full Text
- View/download PDF
24. CD4+T Cells Recognize Conserved Influenza A Epitopes through Shared Patterns of V-Gene Usage and Complementary Biochemical Features
- Author
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Greenshields-Watson, Alexander, Attaf, Meriem, MacLachlan, Bruce J., Whalley, Thomas, Rius, Cristina, Wall, Aaron, Lloyd, Angharad, Hughes, Hywel, Strange, Kathryn E., Mason, Georgina H., Schauenburg, Andrea J., Hulin-Curtis, Sarah L., Geary, James, Chen, Yuan, Lauder, Sarah N., Smart, Kathryn, Vijaykrishna, Dhanasekaran, Grau, Miguel L., Shugay, Mikhail, Andrews, Robert, Dolton, Garry, Rizkallah, Pierre J., Gallimore, Awen M., Sewell, Andrew K., Godkin, Andrew J., and Cole, David K.
- Abstract
T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+T cells. Here, we investigate CD4+T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+T cells in five HLA-DR1+subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies.
- Published
- 2020
- Full Text
- View/download PDF
25. Gene editing in T-cells and T-cell targets
- Author
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Lloyd, Angharad
- Subjects
QR180 ,R1 - Abstract
Recent years have witnessed a rapid proliferation of gene editing in mammalian cells due to the increasing ease and reduced cost of targeted gene knockout. There has been much excitement about the prospect of engineering T-cells by gene editing in order to provide these cells with optimal attributes prior to adoptive cell therapy for cancer and autoimmune disease. I began by attempting to compare short hairpin RNA (shRNA) and zinc finger nuclease (ZFN) approaches using the CD8A gene as a target for proof of concept of gene editing in Molt3 cells. During the course of my studies the clustered regularly interspaced short palindromic repeats (CRISPR) mechanism for gene editing was discovered so I also included CRISPR/Cas9 in my studies. A direct comparison of the three gene editing tools indicated that the CRISPR/Cas9 system was superior in terms of ease, efficiency of knockout and cost.\ud As the use of gene editing tools increases there are concerns about the inherent risks associated with the use of nuclease based gene editing tools prior to cellular therapy. Expression of nucleases can lead to off target mutagenesis and malignancy. To circumvent this problem I generated a non-nuclease based gene silencing system using the CD8A zinc finger (ZF) fused to a Krüppel associated box (KRAB) repressor domain. The ZF-KRAB fusion resulted in effective silencing of the CD8A gene in both the Molt3 cell line and in primary CD8+ T-cells. Importantly, unlike CRISPR/Cas9 based gene editing, the ZF-KRAB fusion was small enough to be transferred in a single lentiviral vector with a TCR allowing simultaneous redirection of patient T-cell specificity and alteration of T-cell function in a single construct.\ud To improve the efficiency of gene editing with CRISPR/Cas9 I developed an ‘all in one’ CRISPR/Cas9 system which incorporated all elements of the CRISPR/Cas9 gene editing system in a single plasmid. The ‘all in one’ system was utilised to derive MHC-related protein 1 (MR1) deficient clones from the A549 lung carcinoma and THP-1 monocytic cell lines in order to study MR1 biology. Mucosal-associated invariant T-cell (MAIT) clones were not activated by MR1 deficient A549 or THP-1 clones infected with bacteria.
26. Gene editing in T-cells and T-cell targets
- Author
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Lloyd, Angharad and Lloyd, Angharad
- Abstract
Recent years have witnessed a rapid proliferation of gene editing in mammalian cells due to the increasing ease and reduced cost of targeted gene knockout. There has been much excitement about the prospect of engineering T-cells by gene editing in order to provide these cells with optimal attributes prior to adoptive cell therapy for cancer and autoimmune disease. I began by attempting to compare short hairpin RNA (shRNA) and zinc finger nuclease (ZFN) approaches using the CD8A gene as a target for proof of concept of gene editing in Molt3 cells. During the course of my studies the clustered regularly interspaced short palindromic repeats (CRISPR) mechanism for gene editing was discovered so I also included CRISPR/Cas9 in my studies. A direct comparison of the three gene editing tools indicated that the CRISPR/Cas9 system was superior in terms of ease, efficiency of knockout and cost. As the use of gene editing tools increases there are concerns about the inherent risks associated with the use of nuclease based gene editing tools prior to cellular therapy. Expression of nucleases can lead to off target mutagenesis and malignancy. To circumvent this problem I generated a non-nuclease based gene silencing system using the CD8A zinc finger (ZF) fused to a Krüppel associated box (KRAB) repressor domain. The ZF-KRAB fusion resulted in effective silencing of the CD8A gene in both the Molt3 cell line and in primary CD8+ T-cells. Importantly, unlike CRISPR/Cas9 based gene editing, the ZF-KRAB fusion was small enough to be transferred in a single lentiviral vector with a TCR allowing simultaneous redirection of patient T-cell specificity and alteration of T-cell function in a single construct. To improve the efficiency of gene editing with CRISPR/Cas9 I developed an ‘all in one’ CRISPR/Cas9 system which incorporated all elements of the CRISPR/Cas9 gene editing system in a single plasmid. The ‘all in one’ system was utilised to derive MHC-related protein 1 (MR1) deficient clo
27. Gene editing in T-cells and T-cell targets
- Author
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Lloyd, Angharad and Lloyd, Angharad
- Abstract
Recent years have witnessed a rapid proliferation of gene editing in mammalian cells due to the increasing ease and reduced cost of targeted gene knockout. There has been much excitement about the prospect of engineering T-cells by gene editing in order to provide these cells with optimal attributes prior to adoptive cell therapy for cancer and autoimmune disease. I began by attempting to compare short hairpin RNA (shRNA) and zinc finger nuclease (ZFN) approaches using the CD8A gene as a target for proof of concept of gene editing in Molt3 cells. During the course of my studies the clustered regularly interspaced short palindromic repeats (CRISPR) mechanism for gene editing was discovered so I also included CRISPR/Cas9 in my studies. A direct comparison of the three gene editing tools indicated that the CRISPR/Cas9 system was superior in terms of ease, efficiency of knockout and cost. As the use of gene editing tools increases there are concerns about the inherent risks associated with the use of nuclease based gene editing tools prior to cellular therapy. Expression of nucleases can lead to off target mutagenesis and malignancy. To circumvent this problem I generated a non-nuclease based gene silencing system using the CD8A zinc finger (ZF) fused to a Krüppel associated box (KRAB) repressor domain. The ZF-KRAB fusion resulted in effective silencing of the CD8A gene in both the Molt3 cell line and in primary CD8+ T-cells. Importantly, unlike CRISPR/Cas9 based gene editing, the ZF-KRAB fusion was small enough to be transferred in a single lentiviral vector with a TCR allowing simultaneous redirection of patient T-cell specificity and alteration of T-cell function in a single construct. To improve the efficiency of gene editing with CRISPR/Cas9 I developed an ‘all in one’ CRISPR/Cas9 system which incorporated all elements of the CRISPR/Cas9 gene editing system in a single plasmid. The ‘all in one’ system was utilised to derive MHC-related protein 1 (MR1) deficient clo
28. Structural Mechanism Underpinning Cross-reactivity of a CD8+ T-cell Clone That Recognizes a Peptide Derived from Human Telomerase Reverse Transcriptase.
- Author
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Cole, David K., van den Berg, Hugo A., Lloyd, Angharad, Crowther, Michael D., Beck, Konrad, Ekeruche-Makinde, Julia, Miles, John J., Bulek, Anna M., Dolton, Garry, Schauenburg, Andrea J., Wall, Aaron, Fuller, Anna, Clement, Mathew, Laugel, Bruno, Rizkallah, Pierre J., Wooldridge, Linda, and Sewell, Andrew K.
- Subjects
- *
T cells , *TELOMERASE reverse transcriptase , *AUTOIMMUNITY , *PROTEIN expression , *MAJOR histocompatibility complex - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
29. CD4 + T Cells Recognize Conserved Influenza A Epitopes through Shared Patterns of V-Gene Usage and Complementary Biochemical Features.
- Author
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Greenshields-Watson A, Attaf M, MacLachlan BJ, Whalley T, Rius C, Wall A, Lloyd A, Hughes H, Strange KE, Mason GH, Schauenburg AJ, Hulin-Curtis SL, Geary J, Chen Y, Lauder SN, Smart K, Vijaykrishna D, Grau ML, Shugay M, Andrews R, Dolton G, Rizkallah PJ, Gallimore AM, Sewell AK, Godkin AJ, and Cole DK
- Subjects
- Adult, Amino Acid Motifs, Amino Acid Sequence, Animals, Birds virology, Complementarity Determining Regions chemistry, Conserved Sequence, Epitopes chemistry, Female, Germ Cells metabolism, HLA-DR1 Antigen immunology, Humans, Immunodominant Epitopes chemistry, Immunodominant Epitopes immunology, Male, Middle Aged, Peptides chemistry, Peptides immunology, Receptors, Antigen, T-Cell metabolism, Swine virology, Tissue Donors, Viral Proteins immunology, Young Adult, Zoonoses immunology, Zoonoses virology, CD4-Positive T-Lymphocytes immunology, Epitopes immunology, Immunoglobulin Variable Region genetics, Influenza A virus immunology
- Abstract
T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8
+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
30. Structural Mechanism Underpinning Cross-reactivity of a CD8+ T-cell Clone That Recognizes a Peptide Derived from Human Telomerase Reverse Transcriptase.
- Author
-
Cole DK, van den Berg HA, Lloyd A, Crowther MD, Beck K, Ekeruche-Makinde J, Miles JJ, Bulek AM, Dolton G, Schauenburg AJ, Wall A, Fuller A, Clement M, Laugel B, Rizkallah PJ, Wooldridge L, and Sewell AK
- Subjects
- Cells, Cultured, Cross Reactions, Humans, CD8-Positive T-Lymphocytes chemistry, CD8-Positive T-Lymphocytes immunology, Peptides chemistry, Peptides immunology, Receptors, Antigen, T-Cell chemistry, Receptors, Antigen, T-Cell immunology, Telomerase chemistry, Telomerase immunology
- 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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
- Full Text
- View/download PDF
31. Engineering of Isogenic Cells Deficient for MR1 with a CRISPR/Cas9 Lentiviral System: Tools To Study Microbial Antigen Processing and Presentation to Human MR1-Restricted T Cells.
- Author
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Laugel B, Lloyd A, Meermeier EW, Crowther MD, Connor TR, Dolton G, Miles JJ, Burrows SR, Gold MC, Lewinsohn DM, and Sewell AK
- Subjects
- Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats genetics, Flow Cytometry, Genetic Vectors, Humans, Lentivirus, Mutagenesis, Site-Directed, Polymerase Chain Reaction, T-Lymphocyte Subsets immunology, Antigen Presentation immunology, Gene Editing methods, Histocompatibility Antigens Class I immunology, Lymphocyte Activation immunology, Minor Histocompatibility Antigens immunology, T-Lymphocytes immunology
- Abstract
The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1(-/-) clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1(-/-) clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells., (Copyright © 2016 The Authors.)
- Published
- 2016
- Full Text
- View/download PDF
32. A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen.
- Author
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Bianchi V, Bulek A, Fuller A, Lloyd A, Attaf M, Rizkallah PJ, Dolton G, Sewell AK, and Cole DK
- Subjects
- CD8-Positive T-Lymphocytes pathology, Humans, Melanoma genetics, Melanoma pathology, Protein Structure, Quaternary, Receptors, Antigen, T-Cell genetics, gp100 Melanoma Antigen genetics, CD8-Positive T-Lymphocytes immunology, Melanoma immunology, Receptors, Antigen, T-Cell chemistry, Receptors, Antigen, T-Cell immunology, gp100 Melanoma Antigen chemistry, gp100 Melanoma Antigen immunology
- Abstract
Human CD8(+) cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), 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 Glu(3) 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 gp100(280-288) peptide showed that Glu(3) was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu(3) → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. 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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
33. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones.
- Author
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Theaker SM, Rius C, Greenshields-Watson A, Lloyd A, Trimby A, Fuller A, Miles JJ, Cole DK, Peakman M, Sewell AK, and Dolton G
- Subjects
- Antigens, Viral immunology, Clone Cells immunology, Cytotoxicity, Immunologic, Ebolavirus immunology, Enzyme-Linked Immunospot Assay methods, Herpesvirus 4, Human, Humans, Receptors, Antigen, T-Cell, alpha-beta immunology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Peptides genetics, Peptides immunology
- Abstract
Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8(+) or CD4(+) polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein-Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
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