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. 2017 Jun 30;8:754. doi: 10.3389/fimmu.2017.00754

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Copyright © 2017 Fransen, van Beek, Borghuis, Meijer, Hugenholtz, van der Gaast-de Jongh, Savelkoul, de Jonge, Faas, Boekschoten, Smidt, El Aidy and de Vos.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Cells from thymus, spleen, Peyer’s patches (PPs), and mesenteric lymph nodes (MLNs) were analyzed with flow cytometry. Germ-free (GF) males (M) (n = 5), GF females (F) (n = 5), conventional (conv) males (n = 8), and females (n = 10) were included as controls. Experimental groups of GF recipients of gut microbiota each contained 10 mice per group. (A) Total number of thymus cells. (B) Percentage among live thymus cells of lineage negative (CD11b, CD11c, CD19, CD45R/B220, NK1.1, and TER119) cells that were also double negative (DN) for CD4 and CD8. (C) Absolute numbers of thymus cells that were lineage negative (CD11b, CD11c, CD19, CD45R/B220, NK1.1, and TER119) and also DN for CD4 and CD8. (D) Percentage of RORyt⁺Foxp3⁺ cells among CD4⁺ T cells in PPs. (E) Percentage of RORyt⁺Foxp3⁺ cells among CD4⁺ T cells in MLN. (F) Percentage of CD25⁺Foxp3⁺ regulatory cells (Treg) among CD4⁺ T cells in MLN. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.