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. 2018 Mar 13;9:1056. doi: 10.1038/s41467-018-03348-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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Fig. 5 — CAF-S1 stimulate CD25⁺FOXP3⁺ T lymphocytes. a Percentage of CD4⁺CD25⁺ or CD4⁺CD25⁻ lymphocytes migrating towards CAF-S1. Data are shown as mean ± SEM. n = 4 independent experiments. P values from Student’s t-test. b Percentage of alive CD4⁺CD25⁺ or CD4⁺CD25⁻ T lymphocytes in absence (Control) or presence of CAF-S1. Data are shown as mean ± SEM. n = 4. P values from Student’s t-test. c Density curves showing CXCR4 expression in CD4⁺CD25⁻ (green), untreated CD4⁺CD25⁺ (orange) or after culture with CAF-S1 (red), compared to control isotype (blue). Cell count is normalized, as percentage of maximal number of cells (% of max). d CXCR4 protein levels in CD4⁺CD25⁻ or CD4⁺CD25⁺ lymphocytes without (Control) or with CAF-S1. Specific MFI are shown as mean ± SEM. n = 5. P values from paired ttest. e CXCL12α and CXCL12β mRNA levels after silencing of CXCL12α- or/and CXCL12β in CAF-S1 cells. Data are shown as mean ± SEM of fold change to control. n = 5. P values from one sample ttest. f Percentage of migrating CD4⁺CD25⁺ lymphocytes after CXCL12α/β silencing in CAF-S1 cells. Data are shown as mean ± SEM. n = 5. P values from paired ttest. g Same as in (f) after CXCL12α/β or CCL2 silencing. n = 3. h Flow cytometry plots showing CD4⁺CD25⁺ and FOXP3⁺ cells in absence (Control) or presence of CAF-S1 transfected with siCtrl or silenced for both CXCL12α and β (siCXCL12α/β). i, j, k Percentages of CD25⁺FOXP3⁺ among CD4⁺ cells (i), of alive CD25⁺FOXP3⁺ lymphocytes (j) and of CD25⁺FOXP3⁺ lymphocytes relative to alive cells (k). Data are shown as mean ± SEM. n = 4. P values from Student’s t-test. l CFSE fluorescence intensities quantifying CD4⁺ effector T cells (Teff) proliferation. Teff were incubated alone (black curve), with CD3⁺CD25⁺ beads (grey), or in presence of CD4⁺CD25^HighCD127^lowCD45RA^low (+Treg) either pre-incubated with CAF-S1 fibroblasts (red) or not (blue). Scatter plot shows percentage of suppression (see Methods). m Flow cytometry plots showing CD4⁺CD25⁺FOXP3^+/− cells without (Control) or with CAF-S1 transfected with siCtrl or silenced for B7H3, CD73 or IL-6. n Percentages of alive FOXP3^High lymphocytes as in (m). P values from paired ttest. n ≥ 6