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. 2017 Aug 8;7:7498. doi: 10.1038/s41598-017-07685-9

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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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Gr1⁻ but not Gr1^± cells exhibit enhanced immunosuppressive capacity after oleate treatment. Bone marrow cells were polarized in the presence of 40 ng/ml GM-CSF and treated with the indicated compounds for 7 days. Gr⁺ and Gr1⁻ populations were purified via beads and co-cultured with purified CD4⁺ T cell in the ratios indicated for functional assay (A). NO production from the co-culture supernatant was quantified by Griess reaction (B). CTLs were polarized and coincubated with purified Gr1− cells for 18 hours. The suspension cells were harvested and cocultured with CFSE labelled E.G7 OVA cell line for 90 mins. The proportion of Annexin V⁺ cells gated from CFSE⁺ population was used to calculate the cytotoxicity of CTLs (Formula in the Methods section) (C). Surface marker panel of Gr1⁻ cells from either control or oleate group was performed via flow cytometry (D). Purified Gr1⁻ cells were stimulated by 1 µg/ml LPS in 24 hours and then supernatants were collected for cytokine analysis (E). Shown is the mean ± SD from two to four independent experiments. *p < 0.05; **p ≤ 0.01;***p ≤ 0.001.

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