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. 2018 Nov 8;8:16544. doi: 10.1038/s41598-018-35037-8

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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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Diphtheria toxin treatment of DEREG mice in ongoing PCM reduces the number of CD4⁺Foxp3⁺ Treg cells and the expression of Treg cells markers. The phenotypic analysis of CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ T cells of lung infiltrating leukocytes from DT-treated and control (PBS treated) DEREG mice was performed by flow cytometry at weeks 6 and 10 week after P.brasiliensis infection. The lung cells were obtained as described in Material and Methods and labeled with antibodies conjugated to different fluorochromes. The lung infiltrating leukocytes were gated by FSC/SSC analysis. The cells were gated for CD4 and then for Foxp3 expression (A). Treg cells markers (CTLA-4, ICOS and GITR) were then assessed in CD4⁺Foxp3⁺ T cells (B). One hundred thousand cells were acquired on FACS CANTO II and subsequently analyzed by FlowJo software. Data are expressed as means ± SEM of three independent experiments using 5 mice per group (**p < 0.005 and ***p < 0.001).