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. 2015 Nov 19;5:16801. doi: 10.1038/srep16801

Figure 2. ATMs are closely related to Tregs to induce FOXP3+ regulatory T cells in vitro.

Figure 2

(A) FACS analysis of T cells co-cultured with ATMs or SPDCs from NC, HFD, and ob/ob mice. FOXP3+ T cells were determined as Tregs. (B) The proportion of Tregs calculated from results in (A). Data are mean ± SEM of three independent experiments. (C) Numbers of Tregs induced by ATMs or SPDCs from NC, HFD, and ob/ob mice were calculated. (D) CD25, FR4, GITR and CTLA4 expression in Tregs induced in vitro by SPDCs and ATMs. (E) CFSE labeled non Treg T cells were cocultured with SPDCs or ATMs. CFSE fluorescence of induced Tregs was analyzed on day 3. (F) The integrated proportion of proliferated Tregs among total induced Tregs is plotted. (G) The proportion of FOXP3-EGFP positive regulatory T cells was determined by confocal microscopy during in vitro Treg induction using NC ATMs. (H) Dynamics of NC ATMs (red) stained by PE-CD11b antibody and FOXP3-EGFP positive regulatory T cells (green) were visualized by confocal microscopy. (I) Box-and-whisker plots of distance between macrophages and Tregs or non-Treg T cells on day 3 (n=149 for Tregs and n=940 for non Treg T cells). (J) Box-and-whisker plots of the number of non-Treg T cells and Tregs attached to macrophages for more than 6 min (n=96 for Tregs and n=67 for non Treg T cells). In the box-and-whisker plots, lines within the boxes represent median values; the upper and lower lines of the boxes represent the 25th and 75th percentiles, respectively; and the upper and lower bars outside the boxes represent the 90th and 10th percentiles, respectively. Samples were measured in triplicate. Data pooled from two independent experiments. For (B, C, G, I, J): *P<0.05, **P<0.01, ***P<0.001.