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. 2018 Jan 11;8:420. doi: 10.1038/s41598-017-17981-z

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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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Exofucosylation increases expression of selectin ligands on nvTregs and their endothelial adherence. Freshly isolated nvTregs and effector DR⁺mTregs, and 14-day in vitro expanded nvTregs were tested for their ability to (A) undergo static transendothelial migration (TEM) towards SDF-1 in a Boyden Chamber assay, and to express surface (B) CXCR4, and (C) VLA-4. All T cell subsets, isolated from three donors, were stained pre- and post-expansion to determine expression of sLe^X as detected by (D) HECA452 or (E) CD15 s. (F) Pre- and post- ex vivo expanded nvTregs were examined for their relative expression of FUT7 RNA by RT-PCR. (G) Buffer- or fucosyltransferase VII-treated expanded nvTregs were stained to determine HECA452/sLe^x expression. (H) The proteins that bind mE-selectin-Ig in buffer- or FTVII-treated expanded nvTregs were identified by performing sequential immunoprecipitation on lysates with anti-CD44, anti-PSGL-1, and then anti-CD43, and detecting the proteins that bound mE-selectin-Ig by western blotting. The blot in Fig. 4H was not cropped as the samples were run in the order as shown. (I) Buffer-treated and FTVII-treated expanded nvTregs, and freshly isolated nvTregs were examined for their ability to roll and adhere to endothelium in the presence of dynamic shear forces. Data shown are mean ± SEM. Significance, determined by Student’s t test, is indicated by (*P < 0.05), (**P < 0.01), and (***P < 0.001).