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. 2019 Sep 10;9:12962. doi: 10.1038/s41598-019-49344-1

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© The Author(s) 2019

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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Identification of apoptotic CTLA-4⁺ cells by TUNEL staining of the spleen of dogs naturally infected by L. infantum presenting organized (A–C) or disorganized (D–F) splenic white pulp. Scale bar: 25 μm. Red arrows indicate isolated CTLA-4 expression. Green arrows indicate cells undergoing apoptosis without expressing CTLA-4. Yellow arrows indicate double labelling (CTLA-4⁺ + apoptosis). (G) Quantitative analysis of CTLA-4⁺ cells and apoptotic cells/mm² according to the disorganization of the splenic white pulp and the parasite load. ANOVA: p = 0.0263. Mann-Whitney test (*): (B) p = 0.0303.