Human CD274 Stable Cell Line-HEK293T

CD274 (programmed death ligand 1, also known as B7H1) is expressed in both solid tumors and hematological malignancies, and it inhibits T cell function through its receptor programmed death 1 (PD-1), which is essential for tumor cells to escape immune surveillance. Here, researchers demonstrated that the levels of both phosphorylated JNK and Cyclin D2 were significantly downregulated in CD274-deficient leukemia-initiating cells (LICs). Overexpression of Cyclin D2 completely rescued the functional loss of CD274. In addition, CD274 directly associated with JNK and enhanced downstream signaling to increase Cyclin D2 levels, promoting leukemia development. Therefore, the surface immune molecule CD274 plays a key role in LIC proliferation. The CD274/JNK/Cyclin D2 pathway promotes LIC entry into the cell cycle, which may become a novel therapeutic target for the treatment of leukemia.

The researchers examined JNK signaling in WT and CD274-deficient LICs by Western blot analysis. The phosphorylation level of JNK was significantly reduced in CD274-deficient LICs (Figure 1a). Consistently, phosphorylated JNK expression was significantly increased when CD274 was overexpressed in 293T cells (Figure 1b). Interestingly, co-immunoprecipitation experiments showed that both JNK and phosphorylated JNK were pulled down by CD274 (Figure 1c). At the same time, CD274 was also detected upon JNK immunoprecipitation (Figure 1d).

Figure 1. CD274 interacts with JNK to increase Cyclin D2 levels. a Phospho-JNK and total JNK levels in WT and CD274-deficient LICs were determined by Western blot analysis. b Phospho-JNK and total JNK levels in CD274-overexpressing (OE) 293T cells were assessed by Western blot analysis. c Strep II tagged CD274 was overexpressed in 293T cells, followed by immunoprecipitation with Strep II beads and Western blotting analysis for the levels of CD274, phospho-JNK, and JNK. d Fc-tagged JNK was overexpressed in 293T cells, followed by immunoprecipitation with protein A/G beads, and the levels of CD274 and JNK were detected by Western blot analysis. (Fang X, et al., 2016)