FIG. 7.

TGF-β is important for M2 macrophages to suppress T-cell proliferation and the induction of Tregs in vitro. A: Suppression of T-cell proliferation (CPM) by stimulating 25 × 10³ macrophages for 24 h and further coculture with splenocytes and αCD3 for 72 h. B: Macrophages (25 × 10³) were cultured and stimulated for 24 h in a transwell upper chamber. Splenocytes and αCD3 were added to the lower chamber, and cells were coincubated for 72 h and assessed for proliferation (CPM). C: Cell contact–dependent suppression of proliferation (CPM) evaluated by stimulating 25 × 10³ macrophages for 24 h followed by fixation and coculture with T cells, splenocytes, and αCD3 for another 72 h. D: CFSE-stained splenocytes cocultured with 25 × 10³ prestimulated macrophages, as described above, for 96 h. Cells were either gated for CD4⁺ or CD8⁺ (left), and CFSE dilution was analyzed (right). E: Macrophages (25 × 10³) were cultured and stimulated for 24 h before coculture with splenocytes from NOD-FoxP3-GFP mice for 96 h. Cells were gated for CD4⁺ cells, and the percentage of GFP (FoxP3)-expressing cells was analyzed. F: Macrophages (25 × 10³) were cultured with CD25⁺-depleted splenocytes from NOD-FoxP3-GFP mice for 96 h. Cells were gated for CD4⁺ cells, and the percentage of CD25⁺GFP⁺-expressing cells was analyzed. G: Macrophages (25 × 10³) were cultured with CD4⁺CD62L⁺CD25⁻ T cells from NOD mice for 96 h. Cells were gated for CD4⁺ cells, and the percentage of CD25⁺FoxP3⁺-expressing cells was analyzed. The results are representative of three independent (A–F) experiments and one experiment (G). Statistical comparison was conducted against untreated macrophage control (black bars; n = 4). Control represents splenocytes without macrophages (white bars). Error bars are presented in SEM. *P < 0.05.