Cord Blood IL-15 Activated NK Cells Increase Cord Blood CD34⁺CD133⁺CD45^lo Cell Function through IFN-y Production and Direct Cell Contact

Delayed engraftment following cord blood (CB) transplantation remains a significant challenge. While cell dose is clearly limiting, CB derived hematopoietic stem cells (HSCs) also exhibit a deficit in homing and engraftment. IL-15 activated natural killer (NK) cells have been shown as potential promoters of homing and engraftment of CB HSCs. However, the role of NK cells and their underlying mechanisms in promoting CB HSCs requires further study. Here, we explore the effect of IL-15 activated CB NK cells on the functional properties of CB CD34⁺CD133⁺CD45^lo HSCs. In addition, we define the mechanistic interaction between NK cells and HSCs that may increase CB-HSC engraftment and improve patient outcome post-HSC transplantation.

We first determined whether IL-15 activated NK cells could improve HSC function in vitro. Purified CD56⁺CD3^- NK cells from CB were stimulated overnight with IL-15 and cultured at a 1:5 cell ratio with autologous purified CD34⁺CD133⁺CD45^lo cells (CB HSCs). IL-15 activated NK cells increased CXCR4 expression on CB HSCs when compared to cultures with resting NK cells or CB HSCS alone. As HSCs must migrate to the bone marrow in order to engraft and facilitate long-term immune reconstitution, we next assessed whether NK cells also impact on HSC migration, clonogenicity and proliferation. We found that elevated levels of CXCR4 on CB HSCs cultured with activated NK cells also translated into enhanced chemotaxis towards SDF-1α in vitro. IL-15 activated CB NK cells also increased CB HSC clonogenicity as evaluated by short-term in vitro cultures. The effect of activated NK cells on the clonogenic capacity of CB HSCs was cell dose dependent with the highest effect observed at a ratio of 1:10. To study CB HSC proliferation, CFSE stained CD34⁺CD133⁺CD45^lo cells were cultured either alone, with resting NK cells or IL-15 activated NK cells. Cultures with IL-15 activated NK cells significantly increased CB HSC proliferation when compared to cultures with resting NK cells or CB HSCs alone [median percentage of proliferating CB-HSCs; 38.4% (34%-44.6%) vs. 46.7% (36%-53.4%) vs. 69% (59.6%-78.5%)]. Moreover, following the ability of IL-15 activated NK cells to upregulate CB HSC proliferation, we investigated whether CB HSCs still retained their long-term engraftment potential. We found that proliferating CB HSCs still recalled both their short-term and long term clonogenic capacity as evaluated by CFU assays and cobblestone cultures followed by long-term culture (LTC-IC) respectively. Finally, we demonstrated that IL-15 activated NK cells also possessed the ability to activate pAkt/pErk and pStat3. As pAkt/pErk and pStat3 are key mediators of cell survival proliferation, our findings identify that NK cells may promote the survival and proliferation of CB HSCs through activating pAkt/pErk and pStat3. These data suggest that IL-15 activated NK cells from CB are endowed with properties to promote the functional profile of CB HSCs that contribute to improved engraftment.

To further understand the underlying mechanisms through which IL-15 activated NK cells exert their ability to upregulate the functional profile of CB HSCs, we used antibody blockade experiments. We showed that the ability of NK cells to increase CXCR4 expression on CB HSCs was mediated via the provision of IFN-γ, but not TNF-α or TNF-β. Whereas, the effect of NK cells on CB HSC function studied through clonogenicity, proliferation and signalling studies was only partially dependent on IFN-γ production by IL-15 activated NK cells. Using transwell experiments, we further determined that the ability of activated NK cells to upregulate CB HSC function is also partly dependent on direct NK cell/HSC cell contact. Subsequently, we found that the addition of blocking antibody against 2B4 in cultures containing IL-15 activated NK cells and CB HSC partially reversed the ability of NK cells to increase the clonogenic capacity, proliferation and Akt/Erk and Stat3 signalling of CB HSCs. Thus, the ability of IL-15 activated NK cells to increase the functional profile of CB HSCs depends on IFN-γ production and cell-cell contact involving 2B4.

Our combined studies demonstrate a novel effect of IL-15 activated CB NK cells and their key factors as potential mediators of stem cell homing and engraftment, which could be utilized to develop strategies that will benefit all patients with haematological malignancies and improve CB transplantation.