Comprehensive analysis of T-cell receptor degeneracy and T-cell crossreactivity

Lead Research Organisation: Cardiff University
Department Name: School of Medicine

Abstract

T-cells are white blood cells designed to protect our bodies from infection. The devastating effects of low numbers of just one type of T-cell are all too evident in HIV-AIDS. T-cells perform extremely important roles because: (1) They orchestrate immunity and are key elements in the control of infection; (2) They are important for the natural eradication of cancer; (3) They hold the key to successful vaccination; (4) They mediate many allergic reactions; (5) They play a substantial role in transplant rejection; and, (6) When they go wrong, they are the cells that cause autoimmune diseases such as diabetes, arthritis and multiple sclerosis. Our T-cells spring into action when the molecules on their surface called T-cell receptors (TCRs) recognize bits of microbes and cancer molecules called antigens. It is estimated that we have T-cells with about 25 million different TCRs in our bodies. TCRs are extremely important molecules because they are at the very focal point of all the above roles. In addition, TCRs are largely responsible for how our immune systems 'learn' and therefore protect us from subsequent exposures to the same germs. In order to deal with all possible infections, our T-cells have to be able to recognize more than 1,000,000,000,000,000 different 'foreign' antigens that we could encounter. It is clear that the immune system has to cover many more foreign antigens than it has different T-cells. To achieve this, each individual TCR molecule may recognize more than a million different possible antigens. As a result, T-cells are said to be extremely 'crossreactive'. This essential T-cell crossreactivity is permissible because the TCR molecule on the T-cell surface can be tremendously promiscuous and recognize many similar 'shapes'. While TCR promiscuity allows our T-cells to control infection, it is also thought to be responsible for the harmful effects these cells can sometimes cause. Autoimmunity is believed to arise when a TCR that is raised to fight infection is inadvertently promiscuous enough to recognize our own tissue. This promiscuous TCR recognition can also result in allergic reactions and is responsible for why our immune cells attack a 'foreign' organ in the first week after it is transplanted. Thus, TCR promiscuity sits at the very heart of most human disease. Despite its obvious importance, there has never yet been a proper attempt to examine or assess TCR promiscuity and the T-cell crossreactivity it enables. Study of TCR promiscuity will require a longer-term effort by an experienced and interdisciplinary team. In this application, a biochemist (Professor Andy Sewell) an infectious diseases clinician and cellular immunologist (Professor David Price), a veterinarian (Dr. Linda Wooldridge), a structure biologist (Dr. Pierre Rizkallah) and a mathematician (Dr. Hugo van den Berg) will apply their collective expertise in T-cell research to undertake a comprehensive analysis of TCR promiscuity for the first time. New tools that this team has developed have finally provided the keys to unlock this study and make this application especially timely. The potential applications and benefits of this work are immense. We have already built TCRs that are promiscuous enough to see all known immune escape variants of the HIV virus. We have further built TCRs that have better 'shapes' for detecting and eliminating cancer. In addition, we expect that this work will revolutionize vaccination and provide insights into the blight of autoimmune disease. In short, this work represents one of those rare examples of basic biological research that has obvious and numerous potentials for translation to clinical practice. As such, we anticipate that this work will generate valuable spin-offs that will improve clinical practice in addition to furthering our understanding of the very interaction that orchestrates human immunity.

Technical Summary

The 25 million T-cell receptors (TCRs) in the human naïve T-cell pool enable T-cell responses to almost any 'foreign' peptide bound to a 'self' MHC molecule. This complete immune coverage of the array of possible permutations generated from 20 amino acid residues that could bind to the self repertoire of MHC molecules theoretically requires that each TCR must crossreact with over a million different peptides. However, such levels of TCR binding degeneracy encompass the potential for inadvertent recognition of 'self' peptide by TCRs raised against a pathogen; indeed, this is thought to be the root cause of all autoimmunity. Promiscuous TCR recognition can also result in allergic reactions and acute transplant rejection. Despite its obvious importance, there has never been a real attempt to assess TCR degeneracy and T-cell crossreactivity. This paucity of knowledge reflects a lack of methodological tools. Our new technologies and interdisciplinary team now enable the timely fulfilment of this critical research gap. We will use an approach that incorporates T-cell culture/assay, soluble TCR and pMHC manufacture, biophysical/structural analysis of TCR/pMHC interactions, polychromatic flow cytometric dissection of antigen-specific T-cell populations, high throughput direct ex vivo quantitative TCR clonotyping, positional scanning peptide library analysis and bioinformatics to measure TCR degeneracy for the first time. This basic biological research will further our understanding of the very interaction that orchestrates human immunity and will transform the way biologists view T-cell immunity. The study of these pivotal interactions in health will almost certainly be beneficial to understanding their role in disease. This work has obvious and numerous potentials for translation to clinical practice. Our results could influence TCR-based therapies, revolutionize vaccination and illuminate our understanding of autoimmunity.

Publications

10 25 50
 
Description We have proved that individual T-cells can recognise millions of different peptides. The resultant JBC paper has been cited over 250 time already.

This programme is directly associated with over 50 publications, many in high impact journals. There are still a few publications to come from the award. In particular, the technology developed as part of the award has enabled us to build T-cell epitope discovery platforms that we have used to discover exciting new cancer targets. It has also allowed us to discover the underlying cause of type 1 diabetes (unpublished). The Google search engine finds well over a thousand mentions of the grant number (BB/H001085/1). There is much more to come. We recently used the findings from this award to develop a technique we termed T-cell receptor (TCR)-Optimised Peptide Skewing of the Repertoire of T-cells (TOPSORT). This technique was successfully used to prime improved anticancer T-cells from all 17 donors tested. Indeed, we were able to show that the technique could prime T-cells from cancer patient blood that exhibited vastly improved killing of the patient's own cancer (in vitro). The melanoma results were published in Frontiers in Immunology (https://www.frontiersin.org/articles/10.3389/fimmu.2019.00319/full). We have since successfully used the approach in four other cancers. We have recently raised funding via Ervaxx to take this approach to clinic. Safety testing permitting, we hope to trial the approach in cancer patients in 2021.
Exploitation Route In the long run we believe that studies of this nature will be useful to understanding autoimmune disease and for predicting such diseases before the onset of symptoms.

We also hope to use our approach to build synthetic edible vaccines that can be added to livestock feed. We have submitted a follow up grant to the BBSRC on this aspect.
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description This programme is directly associated with over 50 publications, many in high impact journals. There are still a few publications to come from the award. In particular, the technology developed as part of the award has enabled us to build T-cell epitope discovery platforms that we have used to discover exciting new cancer targets. It has also allowed us to discover the underlying cause of type 1 diabetes (unpublished). The Google search engine finds well over a thousand mentions of the grant number (BB/H001085/1). There is much more to come. We recently used the findings from this award to develop a technique we termed T-cell receptor (TCR)-Optimised Peptide Skewing of the Repertoire of T-cells (TOPSORT). This technique was successfully used to prime improved anticancer T-cells from all 17 donors tested. Indeed, we were able to show that the technique could prime T-cells from cancer patient blood that exhibited vastly improved killing of the patient's own cancer (in vitro). The melanoma results were published in Frontiers in Immunology (https://www.frontiersin.org/articles/10.3389/fimmu.2019.00319/full). We have since successfully used the approach in four other cancers. We have recently raised funding via Ervaxx to take this approach to clinic. Safety testing permitting, we hope to trial the approach in cancer patients in 2021.
First Year Of Impact 2020
Sector Healthcare
Impact Types Economic

 
Title Method for finding cross-reactive peptides for a T-cell receptor 
Description Method published in: Wooldridge, L., J. Ekeruche-Makinde, H.A. van den Berg, A. Skowera, J.J. Miles, M.P. Tan, G. Dolton, M. Clement, S. Llewellyn-Lacey, D.A. Price, M. Peakman and A.K. Sewell (2012) A single autoimmune T-cell receptor recognises over a million different peptides. Journal of Biological Chemistry, 287, 1168-77. This paper has been highly cited and was featured in Nature Immunology (volume 13 p197-8) 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact Above paper cited >60 times already. Methodology now used for screening TCRs prior to TCR gene transfer therapy with enhanced TCRs to ensure that there self-reactivity 
 
Title TCR/pMHC optimized protein crystallisation screen 
Description Method published: Bulek, A.M. F. Madura, A. Fuller, C.J. Holland, A.J.A. Schauenburg, A.K. Sewell. P.J. Rizkallah and D.K. Cole (2012). TCR/pMHC optimized protein crystallisation screen. Journal of Immunological Methods, 382, 203-210. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact We have used this method to double the database of TCR/pMHC structures. 
 
Description Collaboration with the Pirbright Institute 
Organisation The Pirbright Institute
Department Arbovirus Molecular Research
Country United Kingdom 
Sector Private 
PI Contribution The Babraham pig is the main porcine model at the Pirbright Institute. We have opened up the study of T-cells in pigs using the Babraham. Specifically we have: 1. Cultured and clones pig T-cells for the first time 2. Defined immunodominat influenza A virus T-cell epitopes in the Babraham 3. Produced MHC (SLA-1 and SLA-2) structures of Babraham MHC class I alleles 4. Defined the primary MHC anchor positions for SLA-1 and SLA-2 5. Defined peptide binding motifs for SLA-1 and SLA-2 in Babraham pigs 6. Produced peptide-SLA tetramers for T-cell monitoring
Collaborator Contribution Our efforts in Cardiff are pivotal to the IAV and African Swine Fever vaccine programmes at Pirbright (See Pirbright Director Professor Bryan Charleston for details).
Impact See contributions listed above.
Start Year 2013
 
Description Professor John Phillips, University of Utah 
Organisation University of Utah
Department School of Medicine Utah
Country United States 
Sector Academic/University 
PI Contribution Prof Phillips runs the University of Utah Core services. We regularly collaborate with John especially for nucleic acid sequencing and CRIPSR work
Collaborator Contribution Prof Phillips runs the University of Utah Core services. We regularly collaborate with John especially for nucleic acid sequencing and CRIPSR work
Impact https://www.nature.com/articles/s41590-019-0578-8/metrics
 
Description Protein structure and biophysics collaboration with Professor Jamie Rossjohn 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution We collaborate with Monash on T-cell receptor/antigen interactions.
Collaborator Contribution Prof Rossjohn brings world-class expertise in TCR/antigen interactions.
Impact We have coauthored many high impact publications with Monash
Start Year 2006