Fig. 3

CD147-dependent Foxp3 stabilization requires a trans-acting ligand. A, B YFP + cells and naïve CD4 + cells were sorted from the LNs and spleens of Foxp3-YFP mice. A YFP + cells were seeded in V-shaped 96-well plates or flat 24-well plates and incubated with anti-CD3/anti-CD28-coated microbeads and 50 IU/ml IL-2. Flow cytometry analysis the YFP + cells. B Naïve CD4 + cells were seeded in V-shaped 96-well plates or flat 24-well plates and incubated with anti-CD3/28-coated microbeads, 25 IU/ml IL-2, 10 µg/ml anti-IL6 and 10 ng/ml TGF-β. Flow cytometry analysis the YFP + cells. C Naïve CD4 + cells were seeded in V-shaped 96-well plates or flat 24-well plates and incubated with anti-CD3/28-coated microbeads, 25 IU/ml IL-2, 10 µg/ml anti-IL6 and 10 ng/ml TGF-β. Five days later, the same number of cells in the two wells were collected for RNA-seq analysis. A heatmap of differentially expressed genes. The columns represent samples, and the rows represent genes. Gene expression levels in the heat maps are z score. D CD4 + CD25 + cells were seeded V-shaped 96-well plates or flat 24-well plates and incubated with anti-CD3/anti-CD28-coated microbeads and 50 IU/ml IL-2. Flow cytometry analysis of Foxp3+ cells. E Naïve CD4 + cells were seeded in V-shaped 96-well plates or flat 24-well plates and incubated with anti-CD3/28-coated microbeads, 25 IU/ml IL-2, 10 µg/ml anti-IL6 and 10 ng/10 ng/ml TGF-β. Flow cytometry analysis of Foxp3+ cells. F Naïve CD4 + T cells were sorted from human peripheral blood and then seeded in V-shaped 96-well plates or flat 24-well plates and incubated with anti-CD3/28-coated microbeads, 25 IU/ml IL-2, 10 µg/ml anti-IL6 and 10 ng/ml TGF-β. For all experiments, 2 × 10⁵ cells were cultured in V-shaped 96-well plates to allow sufficient contact, 5 × 10⁴ cells were cultured in flat 24-well plates to allow limited contact, and Foxp3 levels were detected by flow cytometry