Role of Src-PI3K signaling activation in regulation of STAT5b-deficient NK cell function in response to IL-2

Abstract

BackgroundMutations in the Signal Transducer and Activator of Transcription (STAT)5b, are associated with low numbers of NK cells and poor cytolytic function. IL-2 enhances cytotoxicity and requires STAT5b as a downstream effector. It can also induce Src protein activation to promote lytic granule convergence, an essential step in mobilizing lytic granules to the immunologic synapse for cytotoxicity. Subsequently, after lytic granule convergence and conjugation of the NK cell to the target cell activation of PI3K signaling pathway activation leads to target lysis. We have previously shown that IL-2 can partially rescue NK cell cytotoxicity in STAT5b-deficient cells. However, how IL-2 stimulation induces PI3K activity outside of IL-2/STAT5b signaling is not entirely clear. Using 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 Src, MEK, ERK, and pS6 activation. MethodsWe 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. Src, MEK, ERK1/2, and pS6 activationwere evaluated in primary NK cells and STAT5-KD NK cell lines by WB and FC in presence or absence of IL-2. ResultsThe STAT5b-KD NK cell lines mimics 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 PI3 K activation (MEK, ERK, and pS6) after IL-2 and observed increase levels of pAKT and pERK compared to HD andWT with lower levels of pS6. The activation of these protein correlated with lytic granule convergence and restored cytotoxicity suggesting that IL-2 through Src activates PI3 K by way of pAKT and pERK. ConclusionsOur results suggest restored granule convergence and partial improvement in NK cell killing happens through the non-canonical IL-2 pathway by Src-PI3K-MEK-ERK activation.