60P - Adaptive NK cells as a therapeutic option for childhood leukaemia

Background

Natural killer (NK) cells are promising tools for immunotherapy of different malignancies including childhood leukaemia and are shown to exhibit memory-like features. Epigenetic alterations including DNA methylation contribute to the generation of memory in these cells. Studying the DNA methylation profile of activated NK cells helps identify candidate genes of activation biomarkers and new approaches to enhance NK cytotoxicity by targeting intrinsic epigenetic regulators.

Methods

We investigated the DNA methylation profile in NK cells upon priming with activating cytokines. Basically, two types of peripheral blood NK cells were used in this study: Freshly antibody-bead isolated NK cells and expanded NK cells, generated by a 7-day coculture with irradiated K562 cells expressing membrane bound IL-21. Memory-like features were achieved by 16-h stimulation of NK cells with IL-12, IL-15, and IL-18, as previously published. The activation of NK cells was confirmed by higher production of Interferon-γ in preactivated cells. DNA was extracted from the FACS sorted NK cells, based on NK cells markers, in both experimental groups and were analysed by Illumina methylation bead array.

Results

Downstream bioinformatic analyses revealed differential methylation patterns with hypomethylated CpG sites predominantly in preactivated NK cells and in expanded NK cells, independent of cytokine pre-activation.

Conclusions

Our data suggest that the in vitro expansion of NK cells in coculture with K562 cells at least partially recapitulates the memory formation by cytokine-preactivation due to the encounter with K562 cell surface targets prior to being stimulated with cytokines resulting in DNA hypomethylation at a variety of loci in some genes encoding immune system ligands, receptors and regulators such as IL-5, C-C Motif Chemokine Receptor 5 (CCR5) and Programmed Cell Death Ligand 1 (PDL1). Targeted therapy of JAK-class Ph-like ALL with ruxolitinib might interfere with interleukin-signaling pathways in NK cells disrupting memory-formation and cytotoxic activities as shown by live cell imaging and multiparametric full spectrum flow cytometry.

Legal entity responsible for the study

The authors.

Funding

Research institute Children’s Cancer Center and Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Disclosure

All authors have declared no conflicts of interest.