Panoply™ Human IL2RA Knockdown Stable Cell Line

IL2 receptor signaling is crucial for human NK cell activation and the gain of effector functions. The molecular mechanisms underlying the termination of IL2 activation in human NK cells remain unclear. Previous reports have shown that PR/SET domain 1 reduces cell growth and increases apoptosis in malignant NK cell lines in an IL2-dependent manner, suggesting that IL2 signaling pathway genes may be downregulated through direct transcriptional repression. Using ChIP-Seq, researchers identified a PRDM1 binding site located within the first intron of CD25 (IL2RA), encoding an IL2 receptor subunit that modulates IL2 signaling sensitivity, in genetically engineered K562 cells or IL2-activated primary NK cells. Ectopic expression of PRDM1 in two NK cell lines that lack PRDM1 downregulated CD25 expression at both the transcript and protein levels. In both malignant NK cell lines, shRNA-mediated knockdown of CD25 resulted in progressive NK cell depletion in response to low IL2 concentrations. In contrast, ectopic expression of CD25 in primary human NK cells resulted in a gradual increase in the number of CD25-transduced cells in response to low IL2 concentrations. Together, these results reveal that PRDM1 plays a critical role in suppressing IL2-induced NK cell expansion by directly inhibiting CD25 in activated human NK cells.

Here, researchers used retroviral expression vectors expressing shRNAs within a mir30 backbone to transduce two different CD25 shRNAs into KHYG1 cells, resulting in stable knockdown of CD25 expression (Figure 1A). GFP+ cells were then quantitatively tracked by FACS at serial time points after transduction, and a GFP competition assay was performed (Figure 1B). At a relatively high IL2 concentration (50 IU), CD25 knockdown KHYG1 cells were slightly reduced (Figure 1C). However, after 11 days of culture at a lower IL2 concentration (25 IU), CD25 knockdown KHYG1 cells gradually and significantly decreased, as reflected by a decrease in GFP+ cells in the unsorted population (Figure 5D). Next, the researchers transduced another PRDM1-deficient NK cell line, NK9218, with empty vector or one of two CD25 shRNAs and observed a gradual decrease in the GFP+ population in CD25 knockdown cells, but not in empty vector-transduced cells, after 10 days of culture with 25 IU (Figure 1E) or 12.5 IU (Figure 1F) of IL2.

Figure 1. Stable knockdown of CD25 results in decreased cell growth in low IL2 concentrations in malignant NK cell lines. (Akman B, et al., 2021)