Figure 1.

MDM2 is crucial for human Treg cell function. (A) The expression level of MDM2 was detected in Treg cells (CD4⁺FOXP3⁺) and Tconv cells (CD4⁺FOXP3⁻) of CD4⁺ T cells isolated from healthy donor PBMC, as indicated by MFI. (B) Naïve CD4⁺ T cells (CD4⁺CD25^lowCD127^highCD45RA⁺) from healthy donors were differentiated into iTreg cells in vitro in the presence of anti-CD3/CD28 beads, IL-2 (100 U/ml), and TGF-β (5 ng/ml), and the differentiation efficiency was measured after 7 days, as indicated by the percentage of FOXP3⁺. Human iTreg cells were used for a series of experiments 7 days post-differentiation. (C) MDM2 knockdown assay was performed in human iTreg cells using MDM2 shRNA-bearing lentiviruses, and the expression levels of MDM2 and FOXP3 were examined by Western blot. (D) MDM2 knockdown assay was performed in human iTreg cells, followed by the analysis of CTLA-4 and CD25 expression. (E) IL-2 and IFN-γ production from human iTreg cells were assessed after knockdown of MDM2 by lentiviruses carrying MDM2 shRNA. (F) The percentages of IL-2 and IFN-γ production from human iTreg cells after MDM2 knockdown were analyzed as demonstrated in (E). (G) In vitro suppression assay was performed in human iTreg cells infected by shCK or MDM2 shRNA-bearing lentiviruses. (H) The proportions of Teff cell (CD4⁺CD127^highCD25^low) proliferation were analyzed as demonstrated in (G) (n = 3). Error bars represent mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001.