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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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Combined optimized treatment of A^g7/2.5 mi tetramer and IL-2:mAb complexes improves the expansion of 2.5 mi⁺ Treg cells and controls the increase of antigen-specific Foxp3⁻ T cells in NOD.Foxp3^EGFP mice. Unless otherwise indicated in the figure, analysis of spleen are shown. Seven to 14 weeks-old NOD.Foxp3^EGFP mice were injected i.p. with two doses of A^g7/2.5 mi tetramer (25 µg/dose) at day −2 and day 0 and sacrificed on day 0, 1, 2, 3 and 4 after the last injection. Spleen and pancreatic lymph nodes (PaLN) were harvested and the 2.5 mi⁺ T cell response was analyzed by tetramer staining. (A) Expansion and contraction of antigen-specific Tconv (T CD4⁺2.5 mi⁺Foxp3⁻) and Treg (T CD4⁺2.5 mi⁺Foxp3⁺) populations in the extracted organs. (B) Time-course of the CD25 expression on CD4⁺2.5 mi⁺Foxp3⁻ (Tconv) cells. Mean ± SEM of 4 independent experiments (2–3 animals per time point) is shown in graphs A and B. (C) Top, schematic representation of the combined and the combined optimized treatment with A^g7/2.5 mi tetramers and IL-2:mAb complexes. Ten to 12 weeks-old NOD.Foxp3^EGFP mice were treated as indicated in the schema. T cell responses were determined at day 8 of treatment. Bottom left, representative dot plots showing the 2.5 mi⁺ Tconv and Treg cell percentages ± SEM within total CD4⁺ T cells in the spleen. Bottom right, ratio of Tconv/Treg in Ag-specific T cells in spleen of mice treated as indicated. (D) Percentages of 2.5 mi⁺Foxp3⁺ cells within the total Ag-specific population (left) and of 2.5 mi⁺Foxp3⁻CD4⁺ T cells within CD4 T cells (right) found in spleen after immunizing mice with the combined and the combined optimized treatments. (E) Percentages of activated (CD25^hi) antigen-specific Tconv (Foxp3⁻) and Treg (Foxp3⁺) cells within the 2.5 mi⁺ T cell population in the spleen. (C,D), and (E): Error bars in graphs indicate mean ± SEM of 3 to 4 independent experiments including 2–3 animals per group of each experiment. The p values result from a Student t test analysis. **p < 0.005, ***p < 0.0005. No A^g7/GPI-specific T cell expansion was observed in naive or treated mice analyzed in this figure.