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. 2017 Apr 21;7:1005. doi: 10.1038/s41598-017-00691-x

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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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NOD1 silencing promotes a tolerogenic DC phenotype. (a) To obtain tolerogenic DCs (tol. DC), iDCs were differentiated in the presence of IL-10 (30 ng/ml) for 7 days. Fully differentiated tolerogenic DCs and conventional iDCs (differentiated in the absence of IL-10) were silenced with control or NOD1 siRNA for 72 hours. ILT2, ILT3, ILT4, HLA-DR, CD86, PD-L1 and PD-L2 were analysed by flow cytometry. IL-12 secretion was monitored by ELISA. Data represent mean and SD of at least three independent experiments. (b) iDCs or tolerogenic DCs were co-cultured (co-cu) with naïve, allogeneic CD4⁺ T-cells. After 12 days of co-culture, only T-cells were re-stimulated with anti-CD3/PMA for 48 hours, and CD25 and FOXP3 expression was analysed by flow cytometry, and IL-2, IFN-γ and IL-13 secretion was measured by multiplex assay. Lymphocytes were gated on live, CD3⁺, CD4⁺ cells after doublet exclusion. Data represent mean and SD of three independent experiments. For statistical analysis a one-way ANOVA with Tukey’s post hoc test was performed.