Panoply™ Human CD19 Knockdown Stable Cell Line

PI3K activity determines positive and negative selection of B cells, a key process for immune tolerance and B cell maturation. PI3K activation is balanced by phosphatase and tensin homolog (Pten), the major antagonistic phosphatase of PI3K. However, the extent of feedback regulation between PI3K activity and Pten expression during B cell development is unclear. Here, researchers show that PI3K control of this process is achieved post-transcriptionally through an axis composed of a transcription factor (c-Myc), a microRNA (miR17-92), and Pten. Enhancing the activation of this axis by overexpressing miR17-92 reconstitutes impaired PI3K activity in CD19-deficient B cells for positive selection and restores most of the B cell developmental impairments seen in CD19-deficient mice. Using a genetic approach of deletion and complementation, researchers show that the c-Myc/miR17-92/Pten axis tightly controls PI3K activity and the sensitivity of immature B cells to negative selection imposed by activation-induced cell death.

Here, researchers found that immature B cells lacking CD19 had increased sensitivity to BCR-induced AICD relative to control cells (approximately 1.5-fold; Figure 1A). Based on these results, researchers hypothesized that the c-Myc/miR17-92/Pten axis may also play a role in regulating the AICD threshold of immature B cells. To mechanistically validate this, they used the immature B lymphoma cell line WEHI-231, which was previously shown to undergo AICD after BCR ligation and is used as a model for negative selection of B cells. Knockdown (kd) of CD19 in WEHI-231 cells effectively inhibited the PI3K pathway, as evidenced by altered phosphorylation of Akt and GSK3, as well as reduced expression of c-Myc after BCR ligation. Inhibition of PI3K in CD19 knockdown cells reduced the activity of the c-Myc/miR17-92/Pten axis, as evidenced by decreased miR19b expression in CD19 knockdown cells compared with CD19 WT cells (Figure 1B) and increased Pten mRNA and protein expression (Figures 1B and 1C). Furthermore, impaired PI3K activity in CD19 knockdown cells resulted in enhanced sensitivity to AICD, as evidenced by a 2-fold increase in apoptosis compared with CD19wt cells (Figure 1D). In support of a role for the c-Myc/miR17-92/Pten axis in mediating increased AICD in CD19kd cells, the researchers showed that GSK3 inhibitors effectively rescued both CD19kd and CD19wt WEHI cells from AICD, as evidenced by a 50% reduction in apoptosis (Figure 1D). Furthermore, Pten expression was suppressed after infection with a vector encoding a Pten-specific short hairpin RNA (shRNA) (shPten) that rescued from AICD, whereas overexpression of Pten after infection with a vector encoding human Pten (Pten-OE) enhanced AICD in WEHI cells after anti-IgM treatment (Figures 1E).

Figure 1. Modification of CD19 Expression Alters c-Myc/miR17-92/Pten Axis Activity and the Consequential Sensitivity to AICD. (Benhamou D, et al., 2016)