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. 2018 Sep 10;8:13543. doi: 10.1038/s41598-018-31757-z

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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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Expression of the immune checkpoints PD-1, LAG-3, BTLA and CTLA-4 by CD4⁺ T cells responding to common allergen sources. (A) Unstimulated PBMCs of study donors were analyzed for expression of indicated immune checkpoints using the gating strategy depicted in the upper panels. Blue histograms show the expression of indicated immune checkpoints of a representative allergic donor and numbers indicate percent receptor positive cells. Scatter plots on the right side show reactivity of antibodies to PD-1, LAG-3, BTLA and CTLA-4 and isotype control of 6 donors (3 NA, 3A). (B) CFSE-labeled PBMCs of four to six donors (2 NA, 4A) were left unstimulated or stimulated with allergenic extracts for 7 days. 7-AAD negative CFSE^low CD4⁺ T lymphocytes were analyzed for the expression of the indicated immune checkpoints as shown in the blue histograms for one representative donor (left) and summarized in cumulative scatter plots (right). For expression analysis in unstimulated CD4⁺ T cells the gate was set to CFSE^high cells. Open histograms represent isotype control stainings. gMFI; geometric mean fluorescence intensity. Statistical analysis was performed using the Friedman test (**P ≤ 0.01 and ***P ≤ 0.001).