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

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.

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.

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 Pirbright 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.