B7-H4 inhibits IL-2, IL-2 receptor α chain (CD25), and CD69 expression.

<p>Pre-activated CD3⁺ T cells were stimulated with anti-CD3 (5 µg/mL) and anti-CD28 (2 µg/mL) in the presence of B7-H4.Ig or Fc.Ig. Cells were collected for IL-2 mRNA detection (A) or IL-2 secretion (B), or stained with anti-IL-2 (C, D, E, and F) or anti-CD25 (G) or CD69 (H) at the indicated time points. Relative expression of IL-2 mRNA was detected by real-time PCR (A). Culture supernatant was collected and tested by ELISA (B). Representative dot plot of the expression of IL-2 secretion on CD3⁺, CD4⁺, CD8⁺ population and percentage of double positive IL-2 on three different subsets was shown (C). The average expression level of IL-2 on CD3⁺, CD4⁺, and CD8⁺ was shown in D, E, and F, respectively. Representative dot plot of CD25 expression and average expression level was shown (G). H. Representative histogram of the expression of CD69 was plotted and the average expression level was quantitated using Bio-Rad Quantity One program (H). 20,000 of Living cells were analyzed by flow cytometry. Data represent 3 independent experiments and are expressed as means ± SD. One star (*) indicates p<0.05, two stars (**) indicate p<0.01, and three stars (***) indicate p<0.001.