Education and Cytokine Receptor Balance Play a Critical Role in Long Term Survival of the Most Differentiated Natural Killer (NK) Cells.
Authors: Felices Martin , Lenvik Todd , Ankarlo Dave , Curtsinger Julie , Tolar Jakub , Blazar Bruce R. , Anderson Stephen , Miller Jeffrey S. 
Published: October 2019

Abstract
Abstract 2148
NK cells can effectively treat advanced acute myelogenous leukemia if they survive and maintain functional competency after adoptive transfer. In the peripheral blood of healthy human donors the balance between Killer-cell immunoglobulin-like receptor positive (KIR+) NK cells, which are more differentiated and functional in terms of tumor killing and cytokine production, and KIR− NK cells is maintained by proliferation, differentiation, and survival. The mechanism that governs this process is largely unknown. Given that we and others have shown that KIR− NK cells proliferate better than KIR+ NK cells with IL-15 in vitro, we chose to study whether the balance between these two subsets could be maintained by enhanced survival of KIR+ NK cells. To explore this further, peripheral blood mononuclear cells (PBMCs) from healthy human donors were put in serum free conditions overnight and NK cell survival was assessed. A significantly larger proportion of the KIR− NK cells died (33±2.41%) compared to well characterized KIR+ NK cells: KIR2DL1/DS1 (8.9±1.22%), KIR2DL2/DL3 (6.7±0.51%), and KIR3DL1 (6.1±0.95%) (all P < 0.0001). A similar effect was seen when looking at Annexin V+ cells that still had membrane integrity. In order to further understand why KIR+ NK cells exhibited superior survival, we quantified expression of anti-apoptotic markers and found that KIR-expressing NK cells express more Bcl-xL, by Q-PCR and Western blotting. They also express more Bcl-2, by Q-PCR and flow median fluorescence intensity (MFI), particularly when the cells are treated with IL-15 overnight (P ≤ 0.0005). We next investigated expression of cell death family receptors Fas and Fas-Ligand (FasL). Under every condition tested Fas was expressed at significantly higher levels in the KIR− NK cells (P ≤ 0.03). FasL on the other hand was only expressed at higher levels in KIR− NK cells post 60 hrs in culture alone or with IL-2 but not IL-15. Although NK cells can signal through IL-7 or IL-2, only IL-15 mouse knockouts demonstrate a complete deficit of NK cells. Both IL-15 and IL-2 signal through 2 common shared components, IL-2Rb and the common g chain, and an individual cytokine specific component, IL-2Ra for IL-2 and IL-15Ra for IL-15. Interestingly, although all the components for IL-2 signaling are present on the NK cell, IL-15Ra bound to IL-15 is presented in trans from other cells or in complexes in order to achieve IL-15 signaling in NK cells, thereby discarding the necessity for IL-15Ra expression on the NK cell itself. Based on this information, the expression of all of these components was evaluated on KIR+ and KIR− NK cells. We found that IL-2Ra is expressed, by MFI, at statistically significant lower levels (P ≤ 0.0023) on KIR+ NK cells when compared to KIR− NK cells. In contrast, the common g chain is expressed, by MFI, at statistically higher levels (P < 0.0001) on KIR+ NK cells. No statistical differences were seen in IL-15Ra and IL-2Rb expression. We hypothesized that competition for common components, IL-2Rb and the common g chain, between IL-2Ra and IL-15Ra (in trans) plays a role in regulating survival through IL-15. In agreement with this premise, IL-2Ralo NK cells died significantly less than IL-2Rahi NK cells with transpresentation of IL-15 (3.5±1.5% vs. 30.8±2.6%, P = 0.0025). Transient overexpression of IL-2Ra ablates this enhanced survival. Finally we wanted to see if the educational status of the NK cell has a role in its survival. Of the KIR+ NK cells the educated NK cells, which have KIRs corresponding to self-ligands, represent the most differentiated and functional subset. We found that educated NK cells consistently had more Bcl-2 than uneducated NK cells (P ≤ 0.0021). This was associated with enhanced common g chain expression in the educated NK cell population (P = 0.0001). Previous studies have shown that CD57+ NK cells are long lived. We show here that educated NK cells had a statistically larger proportion and density of CD57 expression on a per cell basis (P < 0.0001). These data indicate that educated NK cells could survive better, through enhanced Bcl-2 upregulation, and persist longer, as determined by CD57 expression, than their uneducated counterparts. In summary we find that the mechanism for maintenance of more differentiated NK cells involves enhanced cell survival and differential responses to cytokines limited by the alpha chains of IL-2 and IL-15, which may be manipulated for therapeutic purposes.

Disclosures:
Miller: Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.