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. 2018 May 25;8:8106. doi: 10.1038/s41598-018-26161-6

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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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Phenotype surface markers on regulatory T cells expanded with the combined optimized treatment. Analysis of the spleen is shown. NOD.Foxp3^EGFP females received either the combined optimized treatment or A^g7/2.5 mi tetramer injections only. Spleens were isolated at day 8 of treatment and used for the determination of: (A) the MFI of ICOS, CD25 and GITR, on the surface of 2.5 mi⁺ (left) and polyclonal (right) Foxp3⁺CD4⁺ T cells from treated animals; (B) the percentages of CXCR3⁺ cells within antigen-specific (right) or polyclonal (left) Foxp3⁺CD4⁺ T populations. 2.5 mi⁺ cells were excluded from polyclonal T cells for all the analysis. Data shown in (A) is a representative example from 3 independent experiments; n = 2-3 animals/experimental group. Data in (B) is a compilation of 3 independent experiments with 2-3 animals per treatment group. No A^g7/GPI-specific T cell expansion was observed in any experiment.