oa In Vitro Mimicking of IL-15 Delivery through Trans-Presentation Is a Potent Driver of NK Cell Expansion and Effector Functions

Background

Natural Killer (NK) cells are critical mediators of tumor immunosurveillance. In humans, clinically relevant NK-dependent anti-leukemia effects have been originally demonstrated in the context of hematopoietic stem cell transplantation from HLA-haploidentical related donors. More recently, adoptive transfer of allogeneic NK cells has been recognized as a potentially successful immunotherapeutic strategy allowing for leukemia allorecognition without transplantation-related mortality. Several sources of allogeneic NK cells for adoptive immunotherapy have been described. Among these, hematopoietic progenitor cells from cord blood (HPC-CB) represent an emerging “off-the-shelf” source of NK cells potentially capable of leukemia allorecognition. Various methods for in vitro differentiation of HPC-CB into NK cells have been reported. However, such methods have been generally unable to propagate large numbers of mature NK cells with enhanced alloreactivity, a critical requirement for NK-based immunotherapy. IL-15, a critical factor for survival, proliferation and activation of NK cells, is physiologically delivered in combination with a high affinity IL-15 binding protein (IL-15 receptor alpha or IL-15Ra), a mechanism known as IL-15 trans-presentation. To mimic this mechanism, pre–B-lymphocyte BaF/3 cells have been double-transfected with IL-15Ra and IL-15 (BaF/3 IL-15Ra/IL-15). When used as feeders, these cells can trans-present IL-15, thereby inducing extensive NK cell propagation independent of exogenous soluble cytokines. We have recently set up the BaF/3 co-culture system with the purpose of obtaining large-scale generation of HPC-CB-derived NK cells with potent leukemia alloreactivity.

Methods

BaF/3 IL-15Ra/IL-15 were maintained in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin. One day prior use in co-culture, BaF/3 IL-15Ra/IL-15 were incubated with 10 ug/ml mitomycin C to inhibit proliferation. NK cells were cultured in 24-well plates containing SCGM medium supplemented by 10% heat-inactivated human serum. BaF/3 IL-15Ra/IL-15 transfectants at 1:1 or 1:2 effector: feeder (E:F) ratio (5 × 10⁵ or 1 × 10⁶) were added at D1 and every five days thereafter until D15. On D19, cultured cells were screened by flow cytometry to determine NK number, viability, purity and receptor expression. On D20, NK cytotoxicity to the K562 and U937 myeloid leukemia lines was determined by a non-radioactive calcein AM release assay.

Results

Nineteen days after co-culture start, we obtained 4.35 × 10⁶ from 5 × 10⁵ starting NK cells (8.7-fold increase) in the presence of a 1:2 E:F ratio. A 1:1 E:F ratio allowed for less extensive NK cell propagation. Regardless of the E:F ratio, harvested NK cells were >94% pure and expressed high levels (58–90%) of activating natural cyctotoxicity receptors (NCR) (NKp46, NKp44, NKp30) and NKG2D. Expression of inhibitory killer immunoglobulin-like receptors (KIR) was comparatively lower (0–30%). Consistent with this data, NK cells displayed potent and similarly high cytotoxicity to K562 and U937 AML lines, namely 69–82% at 20:1 effector:target ratio.

Conclusions

We show here that IL-15 trans-presentation may support efficient propagation of polyclonal NK cell with enhanced anti-leukemia functional capabilities. These results provide strong mechanistic rationale for studies exploring the effects of IL-15 trans-presentation delivery to HPC-CB-derived NK cells. Our next objective is to induce differentiation of NK cells (CBNK) from CBSC, and then enhance the cytotoxicity of differentiated NK cell using a secondary culture system based on IL-15 trans-presentation with the scope of generating high numbers of NK cells with anti-leukemia reactivity.