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. 2017 Sep 6;7:10740. doi: 10.1038/s41598-017-11354-2

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© The Author(s) 2017

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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Salp15 does not induce increased anergic or Treg numbers but augments the levels of CD73 in FoxP3⁺ cells. (A) Heatmap representing genes associated with anergy in CD4 T cells according to the RNAseq analysis in the presence of Salp15 (S) or Salp15ΔP11 (C) at 2 and 4 days of activation. (B) Percentage of FoxP3⁺ and anergic CD4 T cells upon their exposure to 25 μg/ml of Salp15 or Salp15ΔP11 for 2 days. The average ± SE of triplicates is indicated. The p values (Student´s t test) correspond to the comparison between the Salp15ΔP11 and Salp15 groups. The data presented is representative of 2 independent experiments with similar results. (C) Heatmap corresponding to genes associated with regulatory T cells according to the RNAseq analysis in the presence of Salp15 (S) or Salp15ΔP11 (C) at 2 and 4 days of activation. (D) Percentage of FoxP3-positive CD4 T cells upon exposure to 25 μg/ml of Salp15 or Salp15ΔP11 in vitro for 2 days during activation. The values correspond to the average ± SE of 5 mice per group. No significant differences were detected between the Salp15ΔP11 and Salp15 groups. The data is representative of 2 independent experiments. (E) Normalized reads corresponding to the expression levels of Nt5e at 2 and 4 days of activation in vitro in the presence of Salp15 (S) or Salp15ΔP11 (C). (F) Surface expression levels of CD73 on CD4⁺FoxP3⁺ T cells (top) activated for 2 days in the presence of 25 μg/ml of Salp15 (red histogram) or Salp15ΔP11 (black histogram). The shaded histogram represents an unstained control. The table shows the average mean fluorescence intensity (MFI) ± standard deviation (SD) of 5 mice per group. Differences of the means were analyzed by the Student´s T-test. (G) Average adenosine levels in the supernatants of CD4 T cells activated in the presence of 25 μg/ml of Salp15 or Salp15ΔP11 for 2 days. The results represent 3 independent mice. (H) Increased percentage of CD4⁺FoxP3⁺CD73^high cells in the blood of CB6F1 mice transplanted with B6 splenocytes at day 50 post induction. The histogram on the right represents the average ± SE of 5 control (Salp15ΔP11) and 5 Salp15-treated mice. *p < 0.05.