IL-4–exposed macrophages suppress T-cell proliferation in a cell-cell contact–dependent manner. (A) CFSE-labeled wild-type (WT) splenocytes were left untreated (black line) or were stimulated with plate-bound anti-TCR/anti-CD28 (filled) and were either cultured alone (T cells only) or were cocultured with control or IL-4–treated (10 ng/mL) BMDM from WT or Stat6^−/− mice at ratio of 1:3 (BMDM:splenocytes). Cells were collected after 4 days, stained for CD4 and CD8 and analyzed by flow cytometry. Histograms show the CFSE profile of gated CD4 and CD8 T cells. (B) CFSE-labeled WT splenocytes were cultured with untreated BMDM (black bars) or IL-4–treated BMDM (gray bars) from WT or Stat6^−/− mice at ratios of 1:3, 1:6, and 1:10 (BMDM:splenocytes). Bars show the mean ± SD from 3 separate cultures per condition (**P < .001; *P < .05). (C) T-cell suppression is dependent on cell-cell contact. Supernatants of untreated or IL-4–treated WT and Stat6^−/− BMDM were collected after 24 hours of stimulation and added to CFSE-labeled WT splenocytes that were left untreated (black line) or were stimulated (filled) as described in panel A. “T cells only” indicates cultures without addition of supernatant. The data are representative of 3 independent experiments.