Association of Marek's Disease induced immunosuppression with activation of a novel regulatory T cells in chickens.

Marek’s Disease Virus (MDV) is an alphaherpesvirus that infects chickens, transforms CD4+ T cells and causes deadly lymphomas. In addition, MDV induces immunosuppression early during infection by inducing cell death of the infected lymphocytes, and potentially due to activation of regulatory T (Treg)-cells. Furthermore, immunosuppression also occurs during the transformation phase of the disease; however, it is still unknown how the disease can suppress immune response prior or after lymphoma formation. Here, we demonstrated that chicken TGF-beta+ Treg cells are found in different lymphoid tissues, with the highest levels found in the gut-associated lymphoid tissue (cecal tonsil: CT), fostering an immune-privileged microenvironment exerted by TGF-beta. Surprisingly, significantly higher frequencies of TGF-beta+ Treg cells are found in the spleens of MDV-susceptible chicken lines compared to the resistant line, suggesting an association between TGF-beta+ Treg cells and host susceptibility to lymphoma formation. Experimental infection with a virulent MDV elevated the levels of TGF-beta+ Treg cells in the lungs as early as 4 days post infection, and during the transformation phase of the disease in the spleens. In contrast to TGF-beta+ Treg cells, the levels of CD4+CD25+ T cells remained unchanged during the infection and transformation phase of the disease. Furthermore, our results demonstrate that the induction of TGF-beta+ Treg cells is associated with pathogenesis of the disease, as the vaccine strain of MDV did not induce TGF-beta+ Treg cells. Similar to human haematopoietic malignant cells, MDV-induced lymphoma cells expressed high levels of TGF-beta but very low levels of TGF-beta receptor I and II genes. The results confirm that COX-2/ PGE2 pathway is involved in immunosuppression induced by MDV-lymphoma cells. Taken together, our results revealed a novel TGF-beta+ Treg subset in chickens that is activated during MDV infection and tumour formation.Biotechnology and Biological Sciences Research Counci

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

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonMarek'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.BBSR

Infection with virulent virus induces TGF-beta⁺ Treg cells in vivo.

<p><b>(A)</b> A schematic diagram of animal trials is shown. Day old line P chicks were mock-infected or infected with the virulent virus via intra-tracheal route. Spleens were taken from 5 birds at different time points post infection. Percentages of <b>(B)</b> CD4⁺CD25⁺ T cells <b>(C)</b> CD4⁺CD25^high T cells <b>(D)</b> TGF-beta⁺CD4⁺ T cells <b>(E)</b> TGF-beta⁺CD4⁺CD25⁺ T cells are shown. <b>(F)</b> Absolute numbers of TGF-beta+ Treg cells are shown at 21 dpi. <b>(G)</b> Representative flow cytometry plots isolated from splenocytes of non-infected and MDV-infected chickens depicting the expression levels of TGF-beta within CD4⁺CD25^- or CD4⁺CD25⁺ T cell at 21 dpi. Dashed lines (Isotype control), solid line (CD4⁺CD25^- T cells), and shaded grey (CD4⁺CD25⁺ T cells). <b>(H)</b> Bar graph (mean ± SD) shows of MFIs of TGF-beta expression within CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells in non-infected and MDV infected chickens (n = 5). <b>(I)</b> The percentages of TGF-beta⁺CD4⁺ T cells within mononuclear cells isolated from the lungs at 4 dpi are depicted. * indicates a statistically significant difference (<i>P</i> < 0.05); ns, not significant.</p

MDV-induced CD4⁺ lymphoma cells express TGF-beta.

<p>Confocal microscopy of spleen sections from non-infected (<b>Ai</b> and <b>Aii</b>; two different magnifications) and MDV-infected birds (<b>Bi</b> and <b>Bii</b>; two different magnifications) are shown. CD4⁺ T cells (green) and TGF-beta⁺ cells (red) and nuclei (DAPI; Blue) are depicted. <b>(C)</b> A representative FACS histogram showing membrane bound TGF-beta on MDV-induced CD4⁺ lymphoma (265L); dotted lines (isotype control MAb) and shaded grey (anti-TGF-beta mAb). <b>(D)</b> Confocal microscopy of MDV-induced CD4⁺ T cell lymphoma cell line (265L) expressing intracellular TGF-beta (red) and nuclei (DAPI; Blue). <b>(E)</b> The variation in the absolute copy number of mRNA transcript for TGF-beta receptor I (red) and TGF-beta receptor II (blue) receptor in 265L and primary CD4⁺ T cells. The ct values for TGF-beta receptor I and II were normalized against the GAPDH house keeping gene and plotted in the standard curve. Results are mean ± S.E.M. of five independent replicates. The tissues are representative of six different infected and non-infected birds. The results from tumour cell lines are representative of 10 different experiments. <b>(F)</b> 265L lymphoma cells inhibited T cell proliferation in a transwell experiment. Splenocytes (in the lower chamber) were stimulated with Con-A, while 265L lymphoma cells were cultured in the upper chamber. T cell proliferation was analysed 3 days after the stimulation using a CFSE-based proliferation assay. <b>(G)</b> Cell culture supernatant from 265L lymphoma cells (5% of total cell culture media) inhibited T cell proliferations. Splenocytes were cultured in 100% media or 95% media and 5% supernatant and T cell proliferation was analysed after Con-A stimulation using CFSE-based proliferation assay. <b>(H)</b> 265L lymphoma cells were resistant to the effects of soluble factors. 265L lymphoma cells were cultured in 100% media or 60% supernatant plus 40% media and the proliferation was analysed using a CFSE-based proliferation assay. <b>(I)</b>Treatment of the cells with SC-236 (PGE2 inhibitor) alone or in combination with TGF-beta blocking antibody partially restored T cell proliferation.</p

Schematic of immunosuppression via induction of TGF-beta⁺ Treg during MDV infection.

<p>Upon infection with MDV via respiratory route, TGF-beta⁺ Treg cells, but not CD4⁺CD25⁺TGF-beta^negT cells, are expanded at 4 dpi in the lungs and at day 21 post infection in the spleens. There is an association between the induction of TGF-beta⁺ Treg cells and immunosuppression/pathogenicity of the disease. TGF-beta⁺ MDV-induced lymphoma cells express low levels of TGF-betaRI and II, while produce soluble inhibitory factors which can induce immunosuppression.</p

Induction of TGF-beta⁺CD4⁺ T cells in vitro.

<p><b>(A)</b> Representative of overlapping histogram demonstrating the expression levels of TGF-beta on gated CD4⁺ T cells isolated from 3-weeks old RIR chickens. <i>Ex vivo</i> CD4⁺ T cells (green line), CD4⁺ T cells stimulated with anti-CD3 mAb for 24 hours (red line), and 72 hrs (black line) are shown. Dotted line represents cells stained with isotype control antibody, <b>(B)</b> Compares the expression levels of TGF-beta on CD4⁺ T cells <i>ex vivo</i> (green line), or cells stimulated with anti-CD3 and rIL-2 (black line), and cells stimulated with only anti-CD3 mAb (red line) at 72 hours after stimulation. <b>(C)</b> The numbers of live cells (7AAD negative) are shown after co-cultured of the responder cells with non-Treg cells (TGF-beta^- Treg cells) (black bar) or Treg cells (TGF-beta⁺CD4⁺ T cells) (open bar) 72hrs after stimulation with Con-A. Grey bar represents the numbers of live cells in co-culture of the responder cells and TGF-beta^-CD4⁺ T cells without stimulation. A representative of three independent experiments is shown.</p

High concentrations of Treg cells in cecal tonsils is associated with immune-privileged microenvironment.

<p>Mononuclear cells were isolated from spleens or cecal tonsils of six 3-weeks old RIR birds and were stained with anti-CD4-PE, CD25-FITC, and TGF-beta-APC or isotype controls. 7AAD was used for dead cell exclusion, and the cells were analysed using FACS. Percentages of <b>(A)</b> TGF-beta⁺CD4⁺ T cells (<b>B),</b> CD4⁺CD25⁺ T cells <b>(C)</b> TGF-beta⁺CD4⁺CD25⁺ T cells in the spleens and cecal tonsils are shown. <b>(D)</b> Relative quantification of the CTLA-4, IL-10, PDL1 and PD1 molecules in cecal tonsils over spleen mononuclear cells. Fold change was calculated in non-stimulated cecal tonsils considering normalized Ct value for respective molecule from spleen as baseline. <b>(E)</b> Relative quantification of fold change in IFN-γ gene in cecal tonsils and spleens after 4 h of stimulation with PMA/Ionomycin were determined using RT-PCR. <b>(F)</b> The frequencies of IFN-γ producing cells in cecal tonsils and spleens were determined using chicken-IFN-gamma ELISPOT assay 18 hrs after PMA/Ionomycin stimulation. (<b>G)</b> CFSE histograms from one representative of T cell proliferation in cecal tonsils and (<b>H)</b> in splenocytes are shown following stimulation with 2.5 μg/ml Con-A. Empty profiles with solid line represent non-stimulated control cells and the grey shaded area represents Con A-stimulated cells. (<b>I)</b> Graphical representation of proliferation index in Con-A stimulated and non-stimulated mononuclear cells from spleens and cecal tonsils of seven birds are shown. <b>(J)</b> Relative quantification of the IL-2 cytokine gene in PMA/Ionomycin stimulated cecal tonsils and spleen mononuclear cells. The fold change in mRNA for IL-2 cytokine in stimulated cells was calculated over non-stimulated cells after normalizing against housekeeping gene. <b>(K)</b> anti-TGF-beta blocking antibody partially restored the proliferation of mononuclear cells isolated from CT using CFSE-based proliferation assay. Data are shown as means with standard deviations (error bars) of at least of three independent experiments. ns, not significant.</p

High concentrations of TGF-beta⁺CD4⁺ T cells in susceptible birds to virus-induced lymphoma.

<p><b>A)</b> Gating strategy for detection of TGF-beta+ Treg cells are shown. Mononuclear cells were isolated from spleens of 3 weeks-old naïve chicken lines, and were surface stained with anti-CD4-PE, anti-CD25-FITC and anti-TGF-beta-APC mAbs or isotype controls. The 7AAD staining was used for dead cell exclusion. The percentages and mean fluorescent intensity (MFI) of CD4⁺ cells expressing TGF-beta are shown in FACS density plots. The percentages of <b>(B)</b> TGF-beta⁺CD4⁺ T cells, <b>(C)</b> TGF-beta⁺CD4⁺CD25⁺ T cells and <b>(D)</b> CD4⁺CD25⁺ T cells in different chicken lines are shown. The values are representative from five different birds for each group. The mean ± SD value are shown. * indicates a statistically significant difference (<i>P</i> < 0.05); ns, not significant.</p

Identification and characterization of chicken TGF-beta⁺ Treg cells.

<p>A) Mononuclear cells isolated from the spleens of 3 weeks old RIR birds were independently cultured in the presence of media only, rIL-2, PHA or Con-A at 40°C. After 72 hrs, the cells were stained with anti-CD4, and anti-CD25; 7AAD staining was used for dead cell exclusion. Data (mean ± SD) represents the percentages of CD4⁺CD25⁺ T cells in different cell culture conditions. The results are representative of three independent experiments with three biological replicates. (B) Representative FACS profiles and percentages TGF-beta⁺CD4⁺ T cells within CD4⁺ T cell population are shown. Mononuclear cells, isolated from spleens of 3 weeks-old naïve RIR chickens, were stained with anti-CD4-PE, anti-TGF-beta-APC mAbs and/ or isotype controls. The 7AAD was added for exclusion of dead cells. C) Representative of FACS profile and percentages TGF-beta⁺CD4⁺ T cells within three subpopulations: CD25^high (R1); CD25^low (R2); CD25^-ve(R3) CD4⁺ T cells are shown. D) The percentages of CD25⁺ cells within TGF-beta⁺CD4⁺ T cells are shown. E) TGF-beta expressions on gated CD4⁺ T cells isolated from lungs, thymus, cecal tonsil, and blood of 3-weeks old RIR chickens were analysed using flow cytometry. The number in each quadrant represents percentages of the cells. The results are representative of data obtained from one out of six chickens. F) The intracellular staining of TGF-beta (Red) and cell surface CD4 (green) are shown using confocal microscopy. Mononuclear cells from spleen were stained with primary mouse anti-chicken CD4 (IgG2b), and mouse anti-TGF-beta (IgG1) monoclonal antibodies followed by staining with anti-mouse IgG1-Alexa Flur 488 or IgG2b-Alexa Flur 568 secondary antibodies, respectively. DAPI was used to visualize the nucleus. G) <i>In vitro</i> inhibitory function of TGF-beta⁺CD4⁺ T cells using a CFSE based proliferative assay 72 hrs after co-culture. CFSE-labelled responder cells were co-cultured with or without TGF-beta⁺CD4⁺ T cells in the presence or absence of 2.5 μg/mL Con-A. Filled grey profiles represent non-stimulated controls without co-culturing with TGF-beta⁺CD4⁺ T cells. Empty profiles represent stimulation with 2.5 μg/mL Con-A in co-culture with TGF-beta⁺ CD4⁺ T cells (green lines), or without co-culturing with TGF-β⁺CD4⁺ T cells (red lines). H) <i>Ex vivo</i> splenocytes isolated from RIR chickens were stimulated with PMA and stained for CD4 and TGF-beta. The levels of Phospho-AKT were analysed using Phosflow by gating on CD4⁺TGF-beta⁺ (R1) or CD4⁺TGF-beta^- (R2) T cells. Filled grey profiles represent isotype control, thin lines represent non-activated cells, and thicker lines represent the expression of Phospho-AKT in PMA-activated cells. The percentages of cells expressing Phospho-AKT for activated cells are shown for the two sub-populations. The results represent of at least three independent experiments.</p