Functional NK Cell Repertoires Are Determined By Survival Mechanisms Through IL-2Ra and FasL
Authors: Felices Martin , Lenvik Todd , Ankarlo Dave , Foley Bree , Curtsinger Julie , Blazar Bruce R , Anderson Stephen K , Miller Jeffrey S. 
Published: October 2019

Abstract
NK cell based immunotherapy can be used to treat advanced acute myelogenous leukemia and data shows that success depends on the function of NK cells and how long the cells are maintained in the recipient. An important determinant of NK cell function is expression of Killer-cell immunoglobulin-like receptors (KIRs), which are involved in the maintenance of self tolerance and acquisition of NK cell functional competence, a process termed education. Little is known about how NK cell education influences NK cell survival. Utilizing a serum starvation assay we found that KIR+ and educated NK cells survived better when compared to those that did not have a KIR or KIR matching cognate HLA-ligand respectively (Figure 1A and 1B). Under basal conditions both KIR+ and educated NK cells had more anti-apoptotic proteins (Bcl-2 and Bcl-xL) and expressed less Fas. When NK cells were incubated with serum and IL-15 (10 ng/ml) followed by IL-15 withdrawal, a substantial increase of pro-apoptotic Bim was found on uneducated NK cells (1.4 fold increase; P = 0.01). Under this same condition educated NK cells expressed more FasL (1.5 fold increase; P = 0.0003), indicating that they could be driving cell death on neighboring NK cells when cytokine is limiting. Since both NK cell survival and homeostasis are mediated by cytokine signaling, we studied expression of IL-15 and IL-2 signaling components. The most significant change was IL-2Ra, which was expressed at higher levels on uneducated NK cells (3 fold increase; P = 0.0004) when the cells were stimulated with serum and IL-15 (1 ng/ml) for 72 hrs. Higher IL-2Ra expression correlated well with cell death and Bim expression so we decided to test if modulating its expression would alter NK cell survival in an IL-15 withdrawal setting. Compared to the control, transient overexpression of IL-2Ra lead to a 1.55 fold decrease in NK cell numbers (P = 0.01) while siRNA knockdown of IL-2Ra lead to a 1.5 fold increase in NK cell numbers (P = 0.07). Importantly, at the time of harvest there was a 1.4 fold decrease in cell death in the IL-2Ra knockdown condition when compared to the control (P = 0.0007). Given that no IL-2 or crosslinking signals were present, the mechanism must differ from the well-described role of IL-2 on activation induced cell death. Since FasL is upregulated on educated NK cells when IL-15 is limiting and IL-2Ra renders NK cells more sensitive to cell death, we tested if educated cells could drive cell death of IL-2Rahi NK cells when survival signals are scarce using a co-culturing assay and FasL blocking antibodies. IL-2Rahi NK cells were more sensitive to apoptosis when co-cultured with KIR+ NK cells (presumably enriched for educated subsets) than with KIR- NK cells from the same donor that were subjected to IL-15 stimulation and withdrawal (34±4.8% vs. 20.6±4.7%; P = 0.002). The effect was reduced when FasL was blocked (P = 0.003), and no differences in killing were seen on the IL-2Ralo NK cells regardless of the treatment. Taken together these findings indicate that educated NK cells outlive their counterparts through two mechanisms: decreased expression of proteins involved in cell death and by killing competing NK cells when IL-15 is limiting. Finally, since we have previously reported on expansion of educated NK cells on transplant patients post CMV reactivation and since CMV reactivation can be associated with decreased relapse, we wanted to investigate if CMV reactivation could alter NK cell survival. There was increased survival on the NK cells from adult donor HCT (2 fold increase at 6 months; P = 0.03) and umbilical cord blood (3 fold increase at 100 days; P = 0.01) allogeneic transplant patients that had undergone CMV reactivation supporting the physiologic role of NK cell survival in vivo from a pathologic challenge. Taken together, these findings show that NK cell functional repertoires are determined by class I interactions, infection, and NK-NK interactions through IL-2Ra, Bim, and FasL to mediate clonal dominance that might be exploited in order to enhance NK cell survival and function after adoptive transfer of allogeneic NK cells for therapeutic use in cancer.

Disclosures:
No relevant conflicts of interest to declare.