Natural Killer Cell Derived Exosomes As a Novel Therapeutic for Acute Myeloid Leukemia

Introduction: Natural killer (NK) cells play a critical role in the innate immune response through their capacity to lyse malignant cells without prior antigen-specific priming. NK cells have been evaluated for safety and efficacy in acute myeloid leukemia (AML), both in the transplant and non-transplant settings. Exosomes, small, 30-150 nm-sized extracellular vesicles originating from the endocytic compartment of parent cells, have recently emerged as a universal intercellular communication system. Exosomes are released by virtually all cells and carry proteins, lipids, and nucleic acids from the parent to recipient cells at short and long distances. The exosome molecular cargo reflects the content of the parent cell and is delivered to recipient cells in a membrane-protected vesicle. We hypothesize that exosomes produced by human activated NK cells carry the machinery necessary for the killing of leukemic cells.

Methods: Venous blood (20-50 mL) was obtained from healthy donors (n=10). NK cells were isolated using Ab-based immunomagnetic selection from the recovered peripheral blood mononuclear cells. NK cells were cultured in the presence of interleukin-2 and interleukin-15, and NK-cell supernatants were used for exosome isolation by size exclusion chromatography. Protein levels, numbers and size (qNano), and exosome morphology (transmission electron microscopy) were determined. Exosome cargos were studied by Western blots and/or flow cytometry for NK cell activating and inhibitory receptors, immune inhibitory molecules and for perforin and granzyme B. Cytotoxicity of the NK cell-derived exosomes for K562 targets, AML cell lines (Kasumi, MLL-1) and primary leukemia blasts was measured using flow cytometry-based assays.

Results: Activated human NK cells produced large quantities of exosomes. PKH-26-labeled NK cell-derived exosomes were avidly taken up by leukemic blasts. NK cell derived exosomes carried activating NK cell receptor NKG2D, natural cytotoxicity receptors, perforin, granzyme B, transforming growth factor beta (TGF-β), killer-cell immunoglobulin-like receptors and PD-1. NK cell derived exosomes mediated anti-leukemia activity against K562 targets, AML cell lines and primary leukemia blasts. Lysis of leukemic blasts by NK cell-derived exosomes was exosome concentration dependent.

Conclusion: We report that NK cell derived exosomes have anti leukemia activity in vitro. These data provide a foundation for the future development of new therapeutic strategies using NK cell-derived exosomes for the elimination of leukemia.