Fig. 4

Defective in vitro differentiation and function of M2r and M1 macrophage in absence of WASP. BMDM from WT and Was^−/− mice were cultured in presence of IL-4, IL-10 and TGF-β to differentiate them into M2r macrophages. a qPCR analysis of M2 specific gens expression (WT n = 5; Was^−/− n = 5). Data are representative of three independent experiments. b qPCR analysis of proinflammatory gene expression in WT (n = 5) and Was^−/− (n = 5) M2r-macrophage after restimulation with LPS for 4 h. Data are representative of three independent experiments. c CFSE (carboxyfluorescein succinimidyl ester)-labelled naive CD4⁺CD25⁻ T cells were cultured in presence of plate bound anti-CD3 (2μg/ml) and either WT or Was^−/− M2r macrophages for four days. T-cell proliferation was determined by flow cytometer. Data are representative of three independent experiments. d Naive CD4⁺CD25⁻ T cells were cultured in the presence of plate-bound anti-CD3 (2 μg/ml), TGF-β (2 ng/ml) and either WT or Was^−/− M2r macrophages for 5 days. FoxP3 expression were analysed by flow cytometry and quantified. Data are representative of three independent experiments. BMDM from WT and Was^−/− mice were cultured in presence of LPS and INFγ to differentiate them into M1 macrophages. e qPCR analysis of M1 specific gens expression (WT n = 5; Was^−/− n = 5). Data are representative of three independent experiments. f CFSE-labelled naive CD4⁺CD25^– T cells was cultured in the presence of plate-bound anti-CD3 (2μg/ml) and WT or Was^−/− M1 macrophage for 3 days. T-cell proliferation was determined by flow cytometer. Data are representative of three independent experiments. Data shown in a–f are mean ± SEM and P-value was obtained by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001