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. 2018 May 25;7(5):41. doi: 10.1038/s41389-018-0049-3

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a, b CFSE-labeled CD8⁺ T cells were activated by anti-CD3/CD28 beads, and PD1⁻, and PD1⁺ macrophages derived from GC tissue were added at 1:1 ratio. After 3 days, these cells were stained with anti-CD8 Ab, and the proliferation of CD8⁺ T cells in NO TAM (CD8⁺ T cells were cultured alone), PD1⁺ TAM (CD8⁺ T cells were cocultured with PD1⁺ macrophages), and PD1⁻ TAM (CD8⁺ T cells were cocultured with PD1^– macrophages) was analyzed. Representative flow cytometry plots are shown in (a). b Results represent the mean of three independent experiments. c, d Isolated CD8⁺T cells were activated by anti-CD3/CD28 beads, and PD1^–, and PD1⁺ macrophages derived from GC tissue were added at 1:1 ratio. After 3 days, these cells were labeled with anti-CD8 and IFN-γAbs, and the expression of IFN-γ in CD8⁺ T cells in NO TAM, PD1⁺ TAM, and PD1^– TAM was analyzed. Representative flow cytometry plots are shown in c. d Results represent the mean of three independent experiments. e, f Isolated CD8⁺T cells were activated by anti-CD3/CD28 beads, and PD1^–, and PD1⁺ macrophages derived from GC tissue were added at 1:1 ratio. After 3 days, these cells were collected and stained with anti-CD8 and perforin antibody, and the expression of perforin in CD8⁺ T cells in NO TAM, PD1⁺ TAM, and PD1^– TAM was analyzed. Representative flow cytometry plots are shown in (e). f Results represent the mean of three independent experiments. g, l Analysis of suppressive activity by PD1⁺ macrophages left untreated or incubated with 10 μg/ml anti-PD1 antibody, or polyclonal goat IgG. g CD8⁺T-cell proliferation was analyzed, and the data indicate the mean ± standard error of the mean of three independent experiments. h The expression of Perforin and IFNγ in CD8⁺T cells was analyzed, and the data indicate the mean ± standard error of the mean of three independent experiments. l the expression of IL10 in PD1⁺ macrophages was analyzed, and the data indicate the mean ± standard error of the mean of three independent experiments. m,n analysis of suppressive activity by PD1⁺ macrophages treated with NA-IL10 in the presence of 10 μg/ml anti-PD1antibody, or polyclonal goat IgG. m CD8⁺T-cell proliferation was analyzed, and the data indicate the mean ± standard error of the mean of three independent experiments. n the expression of Perforin and IFNγ inCD8⁺T cells was analyzed, and the data indicate the mean ± standard error of the mean of three independent experiments