Panoply™ Human CD40 Over-expressing Stable Cell Line

More than 250 million people worldwide are chronically infected with hepatitis B virus (HBV), which can lead to serious complications. Host genetic susceptibility is a key factor in chronic hepatitis B (CHB), and previous genome-wide association studies have found a single nucleotide polymorphism (SNP) rs1883832 in the 5′ untranslated region of CD40 that predisposes to chronic HBV infection, but the underlying mechanism remains unclear. Here, researchers aimed to investigate whether rs1883832 is a true functional SNP (fSNP) of CD40 and how it regulates HBV clearance in hepatocytes. They found that rs1883832 is a true fSNP of CD40 and identified ANXA2 as a negative regulator that preferentially binds to the risk allele T of rs1883832, thereby reducing CD40 expression. In addition, CD40 inhibits HBV replication and transcription in hepatocytes by activating the JAK-STAT pathway. BST2 was identified as a key interferon-stimulated gene regulated by CD40 after JAK-STAT pathway activation. Inhibition of the JAK/STAT/BST2 axis attenuated the antiviral effect induced by CD40. In summary, the functional variant of CD40 regulates HBV clearance by regulating the ANXA2/CD40/BST2 axis, which may provide new ideas for personalized treatment of HBV.

Knockdown of CD40 reduced the levels of p-STAT1, p-STAT3, and IRF9 in HBV-infected HepG2-NTCP cells, HBV-transfected HepG2 cells, and HepG2.2.15 cells, while overexpression of CD40 increased the expression of p-STAT1, p-STAT3, and IRF9 in HBV-transfected Huh7 cells and HepG2.2.15 cells (Figure 1C). In addition, the researchers also explored whether the subcellular distribution of IRF9 was also affected by CD40. The results showed that after knockdown of CD40, the expression of IRF9 in the nucleus was reduced, while after overexpression of CD40, the expression of IRF9 in the nucleus was increased (Figure 1D). The subcellular localization of IRF9 was observed by immunofluorescence. The results showed that after knockdown of CD40, the translocation of IRF9 to the nucleus was reduced, while overexpression of CD40 produced the opposite effect (Figure 1E). The researchers then tried to detect the activity of the interferon-stimulated response element (ISRE). The luciferase activity level of CD40-silenced cells transfected with a luciferase plasmid carrying an ISRE motif in the promoter region was significantly lower than that of control cells transfected with an empty plasmid. However, higher luciferase activity was observed in CD40-overexpressing cells (Figure 1F). These results suggest that CD40 may play an anti-HBV role in hepatocytes by activating the JAK-STAT pathway.

Figure 1. CD40 activates JAK-STAT signaling pathway. (Chen J, et al., 2023)