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. 2017 Dec 21;13(12):e1006745. doi: 10.1371/journal.ppat.1006745

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© 2017 Gurung et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 7 — Confocal microscopy of spleen sections from non-infected (Ai and Aii; two different magnifications) and MDV-infected birds (Bi and Bii; two different magnifications) are shown. CD4⁺ T cells (green) and TGF-beta⁺ cells (red) and nuclei (DAPI; Blue) are depicted. (C) 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). (D) Confocal microscopy of MDV-induced CD4⁺ T cell lymphoma cell line (265L) expressing intracellular TGF-beta (red) and nuclei (DAPI; Blue). (E) 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. (F) 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. (G) 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. (H) 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. (I)Treatment of the cells with SC-236 (PGE2 inhibitor) alone or in combination with TGF-beta blocking antibody partially restored T cell proliferation.