Role of PI3K-MEK-ERK1/2 pathway activation in regulation of STAT5b-deficient NK cell function in response to IL-2.

Background: Mutations in the Signal Transducer and Activator of Transcription (STAT)5b, are associated with low numbers of NK cells and poor cytolytic function. IL-2 induce Src protein activation to promote lytic granule convergence and cytotoxicity. In addition, the PI3K-MEK-ERK1/2 pathway is activated during conjugation of the NK cell to the target cell, mediating NK cell function. We have previously shown that IL-2 can partially rescue NK cell cytotoxicity in primary STAT5b-deficient NK cells. However, how IL-2 stimulation induces PI3K activity outside of IL-2/STAT5b signaling is not entirely clear. Using primary STAT5b-deficient NK cells (p.Q41X) and STAT5b-KD NK cell lines as a human model, we elucidate the role of the non-canonical IL-2 signaling pathway through PI3K-MEK-ERK1/2 pathway activation.
Methods: We used multiparametric flow cytometry, functional NK cell assays (standard Cr51 release assay), and microscopy to elucidate the effect of non-canonical IL-2 signaling in human STAT5-deficient NK cells (p.Q41X) and conditional doxycycline STAT5-KD YTS and NK92 NK cell lines. STAT5b expression was evaluated by Western blot. PI3K-MEK-ERK1/2 pathway activation was evaluated in primary NK cells and STAT5-KD NK cell lines by WB and FC in presence or absence of IL-2. Results: STAT5b-KD NK cell lines mimic the abnormalities observed in primary STAT5b-deficient NK cells (p.Q41X). Mutant primary and cell lines showed abnormal NK cell maturation, decreased convergence of lytic granules to the microtubule-organizing center (MTOC) leading to impaired NK cell cytotoxic capacity partially restored after IL-2 stimulation. We measure downstream mediators of PI3K activation (AKT, MEK, ERK1/2) after IL-2 and observed increase levels of pAKT, pMEK, and pERK1/2, compared to HD and WT. These results revealed that PI3K controls NK cell lysis through regulation of MEK and ERK1/2 activation. Conclusions: Our results suggest restored granule convergence and partial improvement in NK cell killing happens through the non-canonical IL-2 pathway by PI3K-MEK-ERK1/2 activation.

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