Abstract
The T cell receptor repertoire provides a window to the cellular adaptive immune response within a tumor, and has the potential to identify specific and personalized biomarkers for tracking host responses during cancer therapy, including immunotherapy. We describe a protocol for amplifying, sequencing, and analyzing T cell receptors which is economical, robust, sensitive, and versatile. The key experimental step is the ligation of a single-stranded oligonucleotide to the 3′ end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus producing a more accurate measure of the true T cell receptor frequency within the sample. This method has been applied to the analysis of unfractionated human tumor lysates, subpopulations of tumor-infiltrating lymphocytes, and peripheral blood samples from patients with a variety of solid tumors.
TRAcking Cancer Evolution through therapy (Rx). Full list of members in Appendix.
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Acknowledgments
This research was funded by grants from CancerResearchUK, the UK MRC, the Rosetrees Trust, the Stoneygate Foundation, a grant to BC from Unilever PLC, and supported by the National Institute for Health Research UCL Hospitals Biomedical Research. JH was supported by an MRC studentship. K.J. is funded by the Sam Keen Foundation and UCL/Royal Marsden Hospital NIHR Biomedical Research Centres. We are grateful to the TRACERx consortium, funded by Cancer Research UK (http://www.cruklungcentre.org/Research/TRACERx) for giving us access to the tumor samples which the amplified TCR libraries illustrated in Figs. 5 and 6 were prepared.
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Appendix: The TRACERx Consortium
Appendix: The TRACERx Consortium
Name | Site/Institution |
---|---|
Jason Lester | Velindre Cancer Centre, Cardiff, Wales |
Fiona Morgan | Cardiff & Vale University Health Board, Cardiff, Wales |
Malgorzata Kornaszewska | Cardiff & Vale University Health Board, Cardiff, Wales |
Richard Attanoos | Cardiff & Vale University Health Board, Cardiff, Wales |
Haydn Adams | Cardiff & Vale University Health Board, Cardiff, Wales |
Helen Davies | Cardiff & Vale University Health Board, Cardiff, Wales |
Dean Fennell | Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK & Leicester University Hospitals, Leicester, UK |
John Le Quesne | Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK |
Apostolos Nakas | Leicester University Hospitals, Leicester, UK |
Sridhar Rathinam | Leicester University Hospitals, Leicester, UK |
William Monteiro | National Institute for Health Research Leicester Respiratory Biomedical Research Unit, Leicester, UK |
Hilary Marshall | National Institute for Health Research Leicester Respiratory Biomedical Research Unit, Leicester, UK |
Louise Nelson | Leicester University Hospitals, Leicester, UK |
Kim Ryanna | Leicester University Hospitals, Leicester, UK |
Alan Dawson | Leicester University Hospitals, Leicester, UK |
Mohamad Tuffail | Leicester University Hospitals, Leicester, UK |
Amrita Bajaj | Leicester University Hospitals, Leicester, UK |
Jan Brozik | Leicester University Hospitals, Leicester, UK |
Harriet Walter | Leicester University Hospitals, Leicester, UK |
Jacqui Shaw | Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK |
Joan Riley | Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK |
Lindsay Primrose | Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK |
Luke Martinson | Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK |
Nicolas Carey | Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK |
Girija Anand | Barnet Hospital, Barnet, UK |
Sajid Khan | Barnet Hospital, Barnet, UK |
Gillian Price | Aberdeen Royal Infirmary, Aberdeen, UK |
Marianne Nicolson | Aberdeen Royal Infirmary, Aberdeen, UK |
Keith Kerr | Aberdeen Royal Infirmary, Aberdeen, UK |
Shirley Palmer | Aberdeen Royal Infirmary, Aberdeen, UK |
Hardy Remmen | Aberdeen Royal Infirmary, Aberdeen, UK |
Joy Miller | Aberdeen Royal Infirmary, Aberdeen, UK |
Keith Buchan | Aberdeen Royal Infirmary, Aberdeen, UK |
Mahendran Chetty | Aberdeen Royal Infirmary, Aberdeen, UK |
Lesley Gomersall | Aberdeen Royal Infirmary, Aberdeen, UK |
Sara Lock | The Whittington Hospital NHS Trust, London, UK |
Kayleigh Gilbert | The Whittington Hospital NHS Trust, London, UK |
Babu Naidu | Heart of England NHS Foundation Trust, Birmingham, UK |
Gerald Langman | Heart of England NHS Foundation Trust, Birmingham, UK |
Simon Trotter | Heart of England NHS Foundation Trust, Birmingham, UK |
Hollie Bancroft | Heart of England NHS Foundation Trust, Birmingham, UK |
Amy Kerr | Heart of England NHS Foundation Trust, Birmingham, UK |
Salma Kadiri | Heart of England NHS Foundation Trust, Birmingham, UK |
Joanne Webb | Heart of England NHS Foundation Trust, Birmingham, UK |
Gary Middleton | Heart of England NHS Foundation Trust, Birmingham, UK |
Madava Djearaman | Heart of England NHS Foundation Trust, Birmingham, UK |
Akshay Patel | Heart of England NHS Foundation Trust, Birmingham, UK |
Yvonne Summers | University Hospital of South Manchester, Manchester, UK |
Raffaele Califano | University Hospital of South Manchester, Manchester, UK |
Paul Taylor | University Hospital of South Manchester, Manchester, UK |
Rajesh Shah | University Hospital of South Manchester, Manchester, UK |
Piotr Krysiak | University Hospital of South Manchester, Manchester, UK |
Kendadai Rammohan | University Hospital of South Manchester, Manchester, UK |
Eustace Fontaine | University Hospital of South Manchester, Manchester, UK |
Richard Booton | University Hospital of South Manchester, Manchester, UK |
Matthew Evison | University Hospital of South Manchester, Manchester, UK |
Phil Crosbie | University Hospital of South Manchester, Manchester, UK |
Stuart Moss | University Hospital of South Manchester, Manchester, UK |
Juliette Novasio | University Hospital of South Manchester, Manchester, UK |
Leena Joseph | University Hospital of South Manchester, Manchester, UK |
Paul Bishop | University Hospital of South Manchester, Manchester, UK |
Anshuman Chaturvedi | University Hospital of South Manchester, Manchester, UK |
Anne Marie Quinn | University Hospital of South Manchester, Manchester, UK |
Helen Doran | University Hospital of South Manchester, Manchester, UK |
Felice Granato | University Hospital of South Manchester, Manchester, UK |
Angela leek | Manchester Cancer Research Centre Biobank, Manchester, UK |
Phil Harrison | Manchester Cancer Research Centre Biobank, Manchester, UK |
Katrina Moore | Manchester Cancer Research Centre Biobank, Manchester, UK |
Rachael Waddington | Manchester Cancer Research Centre Biobank, Manchester, UK |
Fiona Blackhall | Christie NHS Foundation Trust, Manchester, UK |
Jane Rogan | Manchester Cancer Research Centre Biobank, Manchester, UK |
Elaine Smith | University Hospital of South Manchester, Manchester, UK |
Katie Baker | Christie NHS Foundation Trust, Manchester, UK |
Mathew Carter | Christie NHS Foundation Trust, Manchester, UK |
Lynsey Priest | Christie NHS Foundation Trust, Manchester, UK |
Matthew G Krebs | Christie NHS Foundation Trust, Manchester, UK |
Colin Lindsay | Christie NHS Foundation Trust, Manchester, UK |
Fabio Gomes | Christie NHS Foundation Trust, Manchester, UK |
Caroline Dive | Manchester labs |
Ged Brady | Manchester labs |
Dominic Rothwell | Manchester labs |
Sakshi Gulati | Manchester labs |
Francesca Chemie | Manchester labs |
Jonathan Tugwood | Manchester labs |
Jackie Pierce | Manchester labs |
David Lawrence | University College London Hospitals, London, UK |
Martin Hayward | University College London Hospitals, London, UK |
Nikolaos Panagiotopoulos | University College London Hospitals, London, UK |
Robert George | University College London Hospitals, London, UK |
Davide Patrini | University College London Hospitals, London, UK |
Mary Falzon | University College London Hospitals, London, UK |
Elaine Borg | University College London Hospitals, London, UK |
Reena Khiroya | University College London Hospitals, London, UK |
Charles Swanton | Translational Cancer Therapeutics Laboratory, University College London Cancer Institute, London, UK; Department of Medical Oncology, University College London Hospitals, London, UK; Translational Cancer Therapeutics Laboratory, London Research Institute, London, UK |
Mariam Jamal-Hanjani | Translational Cancer Therapeutics Laboratory, University College London Cancer Institute, London, UK; Department of Medical Oncology, University College London Hospitals, London, UK |
Gareth Wilson | The Francis Crick Institute, London, UK |
Nicolai Juul Birkbak | The Francis Crick Institute, London, UK |
Thomas Watkins | The Francis Crick Institute, London, UK |
Nicholas McGranahan | The Francis Crick Institute, London, UK |
Christopher Abbosh | Translational Cancer Therapeutics Laboratory, University College London Cancer Institute, London, UK |
Rachel Rosenthal | The Francis Crick Institute, London, UK |
Mickael Escudero | The Francis Crick Institute, London, UK |
Aengus Stewart | The Francis Crick Institute, London, UK |
Andrew Rowan | The Francis Crick Institute, London, UK |
Crispin Hiley | The Francis Crick Institute, London, UK; Cancer Research UK Lung Cancer Centre of Excellence, UCL Cancer Institute, London, UK |
Jacki Goldman | The Francis Crick Institute, London, UK |
Asia Ahmed | University College London Hospitals, London, UK |
Magali Taylor | University College London Hospitals, London, UK |
Junaid Choudhary | University College London Hospitals, London, UK |
Penny Shaw | |
Raju Veeriah | Translational Cancer Therapeutics Laboratory, University College London Cancer Institute, London, UK |
Justyna Czyzewska-Khan | Translational Cancer Therapeutics Laboratory, University College London Cancer Institute, London, UK |
Diana Johnson | Translational Cancer Therapeutics Laboratory, University College London Cancer Institute, London, UK |
Joanne Laycock | Translational Cancer Therapeutics Laboratory, University College London Cancer Institute, London, UK |
Marcin Skrzypski | |
Robert Hynds | |
Kroopa Joshi | |
Assma Ben Aissa | |
Yien Ning Sophia Wong | |
Andy Georgiou | |
Mariana Werner Sunderland | |
James Reading | |
Sergio Quezada | |
Carmella Beastall | University College London Cancer Institute, London, UK |
Nagina Mangal | University College London Cancer Institute, London, UK |
Karl Peggs | |
Teresa Marafioti | |
Sam Janes | University College London Hospitals, London, UK |
Martin Forster | University College London Hospitals, London, UK |
Tanya Ahmad | University College London Hospitals, London, UK |
Siow Ming Lee | University College London Hospitals, London, UK |
Peter van Loo | |
Javier Herrero | The Francis Crick Institute, London, UK |
John Hartley | |
Richard Kevin Stone | The Francis Crick Institute, London, UK |
Tamara Denner | The Francis Crick Institute, London, UK |
Emma Nye | The Francis Crick Institute, London, UK |
Sophia Ward | The Francis Crick Institute, London, UK |
Emilia Lim | The Francis Crick Institute, London, UK |
Maise Al-Bakir | |
Dawn Carnell | University College London Hospitals, London, UK |
Ruheena Mendes | University College London Hospitals, London, UK |
Jeremy George | University College London Hospitals, London, UK |
Neal Navani | University College London Hospitals, London, UK |
Dionysis Papadatos-Pastos | University College London Hospitals, London, UK |
Marco Scarci | University College London Hospitals, London, UK |
Pat Gorman | |
Helen Lowe | |
Leah Ensell | |
David Moore | |
Dhruva Biswas | University College London Cancer Institute, London, UK |
Roberto Salgado | Department of Pathology, GZA-ZNA Antwerp, Belgium |
Maryam Razaq | |
Jerome Nicod | |
Stephan Beck | |
Saioa Lopez | Nickys lab |
Ariana Huebner | Nickys lab |
Michelle Dietzen | Nickys lab |
Thanos Mourikis | Nickys lab |
Mairead MacKenzie | Independent Cancer Patients Voice |
Maggie Wilcox | Independent Cancer Patients Voice |
Harriet Bell | Cancer Research UK & UCL Cancer Trials Centre, London, UK |
Allan Hackshaw | Cancer Research UK & UCL Cancer Trials Centre, London, UK |
Yenting Ngai | Cancer Research UK & UCL Cancer Trials Centre, London, UK |
Sean Smith | Cancer Research UK & UCL Cancer Trials Centre, London, UK |
Nicole Gower | Cancer Research UK & UCL Cancer Trials Centre, London, UK |
Christian Ottensmeier | University Hospital Southampton NHS Foundation Trust |
Serena Chee | University Hospital Southampton NHS Foundation Trust |
Benjamin Johnson | University Hospital Southampton NHS Foundation Trust |
Aiman Alzetani | University Hospital Southampton NHS Foundation Trust |
Emily Shaw | University Hospital Southampton NHS Foundation Trust |
Eric Lim | Royal Brompton and Harefield NHS Foundation Trust |
Paulo De Sousa | Royal Brompton and Harefield NHS Foundation Trust |
Monica Tavares Barbosa | Royal Brompton and Harefield NHS Foundation Trust |
Andrew Nicholson | Royal Brompton and Harefield NHS Foundation Trust |
Alex Bowman | Royal Brompton and Harefield NHS Foundation Trust |
Simon Jordan | Royal Brompton and Harefield NHS Foundation Trust |
Alexandra Rice | Royal Brompton and Harefield NHS Foundation Trust |
Hilgardt Raubenheimer | Royal Brompton and Harefield NHS Foundation Trust |
Harshil Bhayani | Royal Brompton and Harefield NHS Foundation Trust |
Morag Hamilton | Royal Brompton and Harefield NHS Foundation Trust |
Natalie Mensah | Royal Brompton and Harefield NHS Foundation Trust |
Lyn Ambrose | Royal Brompton and Harefield NHS Foundation Trust |
Anand Devaraj | Royal Brompton and Harefield NHS Foundation Trust |
Hemangi Chavan | Royal Brompton and Harefield NHS Foundation Trust |
Toyin Adefila-Ideozu | Royal Brompton and Harefield NHS Foundation Trust |
Sofina Begum | Royal Brompton and Harefield NHS Foundation Trust |
Henriette Klein | Royal Brompton and Harefield NHS Foundation Trust |
Aleksander Mani | Royal Brompton and Harefield NHS Foundation Trust |
Soraia Carvalho | Royal Brompton and Harefield NHS Foundation Trust |
Daniel Kaniu | Royal Brompton and Harefield NHS Foundation Trust |
Cristina Realingo | Royal Brompton and Harefield NHS Foundation Trust |
Mpho Malima | Royal Brompton and Harefield NHS Foundation Trust |
Sarah Booth | Royal Brompton and Harefield NHS Foundation Trust |
Kelvin Lau | Barts Health NHS Trust |
Michael Sheaff | Barts Health NHS Trust |
Peter Schmid | Barts Health NHS Trust |
Louise Lim | Barts Health NHS Trust |
John Conibear | Barts Health NHS Trust |
Veni Ezhil | Ashford and St. Peter's Hospitals NHS Foundation Trust |
Vineet Prakash | Ashford and St. Peter's Hospitals NHS Foundation Trust |
Sarah Danson | Sheffield Teaching Hospitals NHS Foundation Trust |
Jonathan Bury | Sheffield Teaching Hospitals NHS Foundation Trust |
John Edwards | Sheffield Teaching Hospitals NHS Foundation Trust |
Jennifer Hill | Sheffield Teaching Hospitals NHS Foundation Trust |
Sue Matthews | Sheffield Teaching Hospitals NHS Foundation Trust |
Yota Kitsanta | Sheffield Teaching Hospitals NHS Foundation Trust |
Jagan Rao | Sheffield Teaching Hospitals NHS Foundation Trust |
Sara Tenconi | Sheffield Teaching Hospitals NHS Foundation Trust |
Laura Socci | Sheffield Teaching Hospitals NHS Foundation Trust |
Kim Suvarna | Sheffield Teaching Hospitals NHS Foundation Trust |
Faith Kibutu | Sheffield Teaching Hospitals NHS Foundation Trust |
Michael Agyemang | Sheffield Teaching Hospitals NHS Foundation Trust |
Patricia Fisher | Sheffield Teaching Hospitals NHS Foundation Trust |
Robin Young | Sheffield Teaching Hospitals NHS Foundation Trust |
Michael Shackcloth | Liverpool Heart and Chest Hospital NHS Foundation Trust |
John Gosney | Liverpool Heart and Chest Hospital NHS Foundation Trust |
Sarah Feeney | Liverpool Heart and Chest Hospital NHS Foundation Trust |
Julius Asante-Siaw | Liverpool Heart and Chest Hospital NHS Foundation Trust |
Teresa Light | The Princess Alexandra Hospital NHS Trust |
Tracey Horey | The Princess Alexandra Hospital NHS Trust |
Dr. Dionysis Papadatos-Pastos | The Princess Alexandra Hospital NHS Trust |
Dr. Peter Russell | The Princess Alexandra Hospital NHS Trust |
Kevin G. Blyth | NHS Greater Glasgow and Clyde |
Craig Dick | NHS Greater Glasgow and Clyde |
Alan Kirk | Golden Jubilee National Hospital |
Andrew Kidd | NHS Greater Glasgow and Clyde |
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Uddin, I. et al. (2019). An Economical, Quantitative, and Robust Protocol for High-Throughput T Cell Receptor Sequencing from Tumor or Blood. In: López-Soto, A., Folgueras, A. (eds) Cancer Immunosurveillance. Methods in Molecular Biology, vol 1884. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8885-3_2
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