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. 2013 Apr 8;210(4):715–728. doi: 10.1084/jem.20112061

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© 2013 Coquet et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 2. — CD27–CD70 co-stimulation promotes thymic T_reg cell development in vitro and in vivo. (A) Independent FTOCs were performed with either 1 or 5 µg/ml CD70-blocking antibody FR70 and an internal control of 1 or 5 µg/ml of control IgG2a antibody (left). Alternatively, cultures were performed in the presence of either 1 or 5 µg/ml CD27-agonist FcCD70 or control IgG1 antibody (right). After 14–16 d, T_reg cell development was assessed by disrupting the lobes and performing flow cytometric analyses for Foxp3, TCRβ, and CD4. Bar diagrams depict the percentage of Foxp3⁺ cells (T_reg cells) among TCRβ⁺CD4⁺cells. Data are from two to three separate experiments comprising four to six independent wells. Data were analyzed using the Mann–Whitney U rank test (*, P < 0.05). (B) Thymocytes were isolated from WT and Cd27^−/− neonates, stained with antibodies to CD4, CD8, CD25, and Foxp3, and analyzed by flow cytometry. Representative plots of CD25 and Foxp3 expression on gated CD4⁺CD8⁻ thymocytes are shown, and graphs depict the absolute number of Foxp3⁺ T_reg cells and Foxp3⁻CD4⁺ thymocytes. Data in histograms are mean + SEM from five WT and six Cd27^−/− mice and were analyzed by Mann–Whitney U rank test (*, P < 0.05). (C and D) Mixed BM chimeras were established by lethally irradiating B6 mice of the CD45.2 allotype and reconstituting them the next day with a 1:1 mixture of CD45.1⁺ WT and CD45.2⁺ Cd27^−/− BM cells. 8 wk later, reconstitution of recipient thymus and spleen was assessed by flow cytometry. (C) The dot plot (top left) depicts a representative analysis of the thymus and the histogram (top right) depicts the expression of CD45.2 on gated CD4⁺ cells. The histograms in the bottom panels depict the expression of Foxp3 within the gated (arrows) CD45.2⁻ WT (left) or CD45.2⁺ Cd27^−/− (right) CD4⁺ cell populations. Numbers in each plot represent percentages of the gated population. (D) The percentage of T_reg cells among total CD4⁺ cells and the absolute number of conventional CD4⁺Foxp3⁻ T cells and CD4⁺Foxp3⁺ T_reg cells in the thymus and spleen of WT and Cd27^−/− mice. Data are mean + SEM from four mice per group and are representative of three separate experiments. The Mann–Whitney U rank sum test was used to calculate significance between groups (*, P < 0.05).