Dissecting the role of TGF-beta+ regulatory CD4+ T cells in pathogenesis of Marek's disease in chickens

Lead Research Organisation: The Pirbright Institute
Department Name: Molecular Immunology

Abstract

White blood cells are a key part of the body's immune system to combat infections by viruses, bacteria and other pathogens. Our research is focused on a type of white blood cells called T cells. There are different types of T cells with distinct functional abilities, for example, killer T cells recognize and lyse cancer cells or infected cells, while suppressor T cells dampen immunity and suppress inflammatory responses. Many pathogens have learned to co-exist with the host by manipulating suppressor T cells to their advantage and thus they can escape immune control. We know very little about suppressor T cells in chicken, because we do not have specific and stable markers to identify these cells in chickens.

A novel chicken suppressor T cell subpopulation has been recently identified, by our group, based on the expression of a suppressive molecule called TGF-beta. We have demonstrated that these cells may be involved in the pathogenesis of an economically important infectious disease called "Marek's disease" in chickens. Similar to humans, many animals including chickens develop cancer with the distinction that cancers in animals are mainly induced by viruses. Marek's disease (MD) is a common disease of chickens, causing transformation of the infected T cells and tumour growth in various tissues and eventually death. Our data show that cancer cells resemble the recently identified suppressor T cells and Marek disease infection increases the number of the suppressor T cells in the birds. We propose to determine whether the recently identified suppressor T cells are the precursor of cancer cells and if these cells are specifically targeted by the virus. We will also explore whether differences in molecular signature of the recently identified suppressor T cells can explain the susceptibility to MDV infection.

Technical Summary

Using FACS and confocal microscopy, we have discovered that chicken CD4+CD25+ Treg cells can be divided into two subpopulations; TGF-beta positive and negative Treg cells. Using FACS, we have observed that: (1) MDV-susceptible chicken lines have significantly higher percentages of TGF-beta+ Treg cells than that in the resistant lines. (2) Oncogenic MDV strain, but not vaccine strain, increased the percentages of TGF-beta+ Treg. Using confocal microscopy, we have shown that MDV-lymphoma cells are phenotypically similar to TGF-beta+ Treg cells. Taken together, we hypothesize that TGF-beta+ Treg are involved in pathogenesis of MDV infection.

To examine this hypothesis and determine whether TGF-beta+ Treg are the precursor of lymphoma, CDR3 spectratype analysis of the TCR of the expanded TGF-beta+ Treg and MDV-lymphoma from the same birds will be utilized. Five approaches will be used to attribute TGF-beta+ Treg to MDV-susceptibility. First, the frequencies of these cells will be determined in all the available resistant and susceptible lines using FACS. Second, adoptive Treg cell transfer from susceptible lines to resistant lines prior to MDV infection will determine whether the cell transfer renders recipients to develop lymphoma after MDV challenge. Third, it will be examined whether the Treg are preferentially targeted or have survival advantage by detecting copy of viral genes during different stages of infection in different CD4+ T cell subsets using qPCR. Fourth, the differential biological effects of vIL-8 on the Treg from susceptible and resistant lines will be studied in vitro and in vivo. Fifth, ploysome and ribosome profiling will determine differential gene signature of the Treg from susceptible and resistant lines. The results from gene signature will also help us identify the potential inhibitory genes involved in the function of these cells and discover unique molecules/ transcription factors for further classification of these cells.

Planned Impact

The beneficiaries of this research are academic scientists, the poultry industry including the poultry breeding companies (e.g. Aviagen), vaccine production companies (such as CEVA), farmers and the general public. Poultry and poultry products are the cheapest and most accessible source of proteins and demands for these products have increased exponentially. Compared to the other livestock sectors, the modern poultry production methods have the most efficient feed-to-meat conversion ratios with lowest global warming potential. It is estimated that poultry will be the major source of meat by 2018 and will account for 46% of meat consumed by 2022 around the world (http://oecd.org/site/oecd-faoagriculturaloutlook/ O-FAO). The poultry meat is important not just because of the food it provides to our nation, it also contributes £ 3.3 billion to UK GDP, with every £1 billion generating another £1.3 billion in the rest of UK economy. The industry supports 35,400 direct and a further 37,900 indirect jobs in the wider supply chain (Oxford Economics, 2013).
Infectious diseases are a continuous threat to poultry industry through losses or reduction in production and animal welfare. Marek's disease (MD) is one of the major diseases of poultry which causes serious economic losses and the global estimate of losses from Marek's disease is approximately $2,000 million annually. The proposed work will result in the identification of a cell subset associated with the suppression of immune response to Marek's disease and modulation of vaccinal immunity. The outcome will provide information to select and breed birds, which are resistant to Marek's disease and can generate more potent and protective immune responses after vaccination. Although the focus in this proposal is on MD, our results will have a wider impact by providing underpinning knowledge for similar studies with other economically important avian pathogens. The results of the project will be communicated to the Pirbri Institute stakeholders, such as BBSRC and DEFRA. The reagents and scientific knowledge generated will be published in peer-reviewed journals, and in presentations at meetings for scientists, and the impact of the project will also be publicized by communication with the farming community and the public through our website, workshops, training courses and exhibitions. The Pirbright Institute is committed to the development of highly skilled researchers and the postdoctoral scientists who will be recruited for this post will receive excellent training in confocal microscopy, flow cytometry, cellular and molecular techniques, thereby contributing to the wider training, innovation, skills and capability of the UK science with consequent boosting to science-based industries and the UK economy.

Publications

10 25 50
 
Description We have discovered that chickens have two different types of suppressor lymphocytes which can be identified in their blood and their various organs such as spleen, lung and cecal tonsils. We were able to isolate these cells and show that they can suppress the function of other immune cells that are important in the control of viral infections. Interestingly, we could observe that the number of these suppressor lymphocytes are much higher in chickens lines that are genetically more susceptible to infection with Marek's Disease virus. Moreover, we found out that MDV-induced tumour cells in chickens have similar characteristics as suppressor T cells, suggesting that suppressor T cells might be precursor of tumour cells.
Exploitation Route Education: The information from this research will provide better understanding of immune system cells in chickens which can be important for scientists working on poultry disease, immunologists and even could be incorporated into text book for undergraduate and postgraduate students.

Generating more resistant chickens: A reverse association between lower frequencies of suppressor lymphocytes and susceptibility to Marek's Disease in chickens may lead to the development or identification of chickens lines that may be more resistant to the disease.
Sectors Agriculture, Food and Drink,Education

URL https://bbsrc.ukri.org/news/fundamental-bioscience/2018/180103-n-new-chicken-immune-cell-increases-susceptibility-to-mareks-disease/
 
Description The Project started in August 2016.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Healthcare
Impact Types Economic

 
Description Professor Benedikt Kaufer 
Organisation Free University of Berlin
Country Germany 
Sector Academic/University 
PI Contribution Providing knowledge and methodologies.
Collaborator Contribution Providing reagents and methodologies to perform experiments.
Impact Two publications from this collaboration: Gurung A, Kamble N, Kaufer BB, Pathan A, Shahriar Behboudi. Association of Marek's Disease induced immunosuppression with activation of a novel regulatory T cells in chickens, PLoS Pathogens, 2017, 13 (12), e1006745. Boodhoo N, Kamble N, Kaufer BB, Shahriar Behboudi. Replication of Marek's disease virus is dependent on de novo synthesis of fatty acid and Prostaglandin E2. J Virol. 2019 Apr 10
Start Year 2019
 
Description Professor Shayan Sharif, University of Guelph, Canada 
Organisation University of Guelph
Department Department of Pathobiology
Country Canada 
Sector Academic/University 
PI Contribution Collaboration between my team and Professor Sharif lead to new finding in Avian immunology, specifically in understanding the effects of nutrients on antigen presenting cells and T cells of chicken. We took the lead in studying the effects of some nutrients (Vitamin D) on the function of chicken T cells, performed most of the experiments in our laboratory. Meanwhile, we participated in studying the effects of Vitamin D on antigen presenting cells of chicken by performing some key experiments because we had access to some specific reagents (monoclonal antibodies) recognizing molecules expressed on chicken antigen presenting cells.
Collaborator Contribution Professor Sharif team took the lead in studying the effects of Vitamin D on antigen presenting cells of chicken, and contributed to experimental plan for studying the effects on chicken T cells.
Impact Two publications in 2015 and 2016: 1. Nitish Boodhoo, Shayan Sharif, Shahriar Behboudi. 1a,25(OH)2 Vitamin D3 Modulates Avian T Lymphocyte Functions without Inducing CTL Unresponsiveness. PLoS One. 2016 Feb 24;11(2):e0150134 2. Bahram Shojadoost, Shahriar Behboudi, Villanueva AI, Jennifer Brisbin, Ali Ashkar, Shayan Sharif. Vitamin D3 modulates the function of chicken macrophages. Res. Vet. Sci. 2015 June, 100: 45-51
Start Year 2019
 
Description Ash Manor school challenge week science workshops 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Provide information on research activity at The Pirbright Institute and informing on pathogens infecting farmed animals.
Year(s) Of Engagement Activity 2019
 
Description Big Bang Fair 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The Big Bang Near Me is a programme of regional and local Big Bang Fairs that take place all across the UK. Together with The Big Bang UK Young Scientists & Engineers Fair and The Big Bang Competition, it forms part of the wider Big Bang programme, bringing science, technology, engineering and maths (STEM) to life for young people
Year(s) Of Engagement Activity 2019
URL https://nearme.thebigbangfair.co.uk/about/
 
Description CABI_Research activity of Avian Immunology Group at The Pirbright Institute 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Release of research activity within Avian Immunology group at The Pirbright Institute
Year(s) Of Engagement Activity 2018
 
Description Diamond open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Over 300 people visited our stand at the open day, in which we described the importance of our work for food security and public health. Children from all ages were involved and learned about viruses and played with the huge virus particle that we had produced. One of the major impact was that we described to several people why we work on some viruses that we do not have them in the UK, and the importance of the work for the UK and UK economy and public health was explained.
Year(s) Of Engagement Activity 2017
URL http://www.diamond.ac.uk/Public/VisitUs.html
 
Description Food security and the control of infectious disease in livestock 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A workshop was organized by myself at St Catherine's College Oxford for the duration of three days. Representative from industry, funding agencies and media (publishers) attended this workshop. Over 20 delegates from Egypt and 20 delegates from the UK attended this meeting. The purpose of this workshop/ meeting was to promote international collaborations, provide training for undergraduate students, promote the importance of managing and controlling infectious disease in livestock and public health.
Year(s) Of Engagement Activity 2016
 
Description Participation in student day at the Pirbright Institute 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Student open day was participated by postgraduate students registered at several Universities, and scientists from Pirbright institute. Students were given a chance to discuss their research activities and future plans with senior scientists. Due to high interest from the students, the institute has decided to organize another open day.
Year(s) Of Engagement Activity 2016
 
Description Surrey Open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Described the research activity of the Pirbright Institute for students from school, graduate and postgraduate students, and provided information on research facilities in the institute.
Year(s) Of Engagement Activity 2017
URL https://www.surrey.ac.uk/opendays/vet
 
Description University of Surrey School of Veterinary Medicine Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Presented research activity from the Pirbright Institute
Year(s) Of Engagement Activity 2018
 
Description Vet Times 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Information on research activity of Avian Immunology group at the Pirbright Institute
Year(s) Of Engagement Activity 2018