Association of COX-2-induced TGF-β+ suppressor T-cells in the immunosuppression induced by Marek’s disease virus

Abstract

Marek's disease virus (MDV) is an avian alphaherpesvirus that transforms CD4+ T cells, causes a deadly lymphoma, and induces immunosuppression in the infected chickens. However, the underlying mechanisms of MDV-induced immunosuppression have not yet been established. The hypothesis for this project is that MDV infection induces the expansion of regulatory T (Treg)-cells in chickens via the activation of Cyclooxygenase-2 (COX-2)/ prostaglandin E2 (PGE2) and through the expression of the inhibitory molecule-transforming growth factor-β (TGF-β) at membrane-bound and intercellular levels. The hypothesis is that the TGF-β activation is dependent on the induction of pathways which are exclusive to the virulent virus but not the nonpathogenic strains. This PhD project aims to identify the mechanism involved in the induction of putative Treg cells in the MDV-infected chickens. As the orthologue for FoxP3 (Forkhead Box P3), a classical marker, for the identification of human and murine Tregs, had not been found in chickens, we used membrane bound TGF-β on CD4 and CD4+CD25+ T cells as a marker for identification of chicken Treg cells. An in vitro CFSE-based T cell inhibition assay was used to demonstrate the inhibitory properties of chicken TGF-β+CD4+ T cells. Similarly, data from RT-qPCR showed a high expression of immunosuppressive genes such as CTLA-4. IL-10, PD-1 and PDL-1 by chicken TGF-β+ CD4+ T cells. Using flow cytometry, the frequencies of TGF-β+ Treg-like cells, were found to be expressed at higher frequencies in higher in genetically MD-susceptible compared to that in genetically MD-resistant chicken lines. Interestingly, infection of chickens with virulent, but not the vaccine strain, induced expansion of TGF-β+ Treg-like cells at 4 days post infection in the lungs and 21 days post infection in splenocytes of the infected chickens. The results suggest that expansion of these cells is associated with MDVinduced immunosuppression. In the last chapter of this thesis, the mechanisms involved in the expansion of TGF- β+ Treg cells during MDV infection were explored. The RT-PCR, western blot and/or PGE2 ELISA assays were utilized to demonstrate the activation of COX-2/PGE2 pathway by the virulent, but not the vaccine strains of MDV. In the absence of the chemical inhibitors of the COX-2/PGE2 pathway, MDV infection upregulated ITGB8 gene, encoding integrin avb8 receptor, on chicken macrophages as determined using RT-PCR. Using gene silencing approach and the chemical inhibitors, the results showed that the macrophages, exposed to soluble factors released from MDV-infected cells, obtained the ability to induce TGF-β in an COX2/PGE2/ITGB8 dependent manner. TGF-β maturation was determined using an adopted the luciferase assay to determine the bioactivity of TGF-β. Using gene silencing techniques, the results confirmed that ITGB8-induced TGF-β maturation by the macrophages is required for induction of TGF-β+ Treg-like cells with the ability to suppress T cell proliferation in vitro and express immunosuppressive genes including CTLA4, PD1, PDL1 and IL-10. Current MDV vaccines do not provide sterilizing immunity and the vaccinated birds can shed the virus after infection. Such imperfect vaccines put the MDV viruses under high selection pressure which can drive the evolution of more virulent pathogen strains of MDV. A comprehensive understanding of host-pathogen interaction and an identification of novel targets and immune mechanisms involved in the pathogenesis of MD could prove useful in providing new insights essential for the development of more effective vaccines. Collectively, the results from this PhD project extend our understanding of the immune mechanisms involved in MDV-induced immunosuppression and could potentially be exploited for the future development of novel immune approaches against MDV.