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. 2017 Apr 20;7:971. doi: 10.1038/s41598-017-01065-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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In vivo treatment of donor corneas with IL-10 and TGFβ1 induces tolerogenic APCs. Two days after single subconjunctival injection of IL-10 and TGFβ1, corneas were harvested and analyzed for the expression of maturation markers on dendritic cells (A) and mRNA expression of immunoregulatory and pro-inflammatory cytokines (B). (A) Flow cytometry analysis showing decreased frequencies of MHCII⁺CD11c⁺ cells, CD40⁺CD11c⁺ cells, and CD86⁺CD11c⁺ cells in IL-10/TGFβ1-treated corneas compared to saline-treated controls (Bar graphs demonstrate mean ± SEM; N = 6 corneas/group, data shown are representative of three independent experiments). (B) Real-time PCR showing mRNA expression of IL-10 and TNFα in IL-10/TGFβ1- and saline-treated corneas 48 hours after injection before transplantation. Bar graphs demonstrate mean ± SEM; p < 0.003; N = 8 corneas/group. (C) Bone marrow-derived dendritic cells (DCs) were treated for 5 days in the presence or absence of IL-10/TGFβ1, and stimulated with LPS overnight to obtain tolerogenic DCs (tolDCs) or mature DC (mDCs), respectively. They were then co-cultured for 24 h with allogeneic splenic APCs of GFP⁺ mice to distinguish them from bone marrow-derived DCs. Frequencies of GFP⁺ APCs expressing MHCII and CD86 were quantified by flow cytometry.