Anti-Leukemia Effects of NK Cell-Derived Exosomes

Introduction: Exosomes are 30-150 nm-sized extracellular vesicles originating from the endocytic compartment of parent cells. The exosome molecular cargo reflects the content of its cells of origin and is delivered to recipient cells in a protective glycol-lipid bilayer without degradation. Because of their small size, exosomes freely circulate within the body, can reach the bone marrow, and can cross biological barriers. 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. Importantly, NK cell activity is reduced in patients with acute myeloid leukemia (AML) relative to that in healthy donors. To overcome the decreased NK cell activity in AML, several therapeutic strategies have been evaluated for safety and efficacy, both in transplant and non-transplant settings, using autologous and allogeneic activated NK cells.

Since NK cell-derived exosomes acquire tumor-killing abilities from the parent NK cells, we hypothesize that NK cell-derived exosomes by transferring exosome content to leukemia blasts can induce the death of these target cells. In the current study, we evaluated the in vitro anti-leukemia effects of NK cell-derived exosomes.

Methods: Exosomes were isolated from the supernatants of NK cells obtained from healthy donors (n=12) using mini-size exclusion chromatography (mini-SEC). Protein levels, number and size (qNano), and exosome morphology using transmission electron microscopy were determined. The exosome cargo was studied by Western blots and on-bead 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 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. Transmission electron microscopy showed the presence of vesicles that were uniform in size (30-150nm in diameter) by NanoSight measurement. Confocal imaging of labeled NK cell-derived exosomes interacting with leukemia cells showed that they are rapidly internalized by leukemic targets. 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 were co-incubated with target cells, AML cell lines and primary leukemia cells, at different exosome:target (E:T) ratios using escalating doses of exosomes (10-70 µg). NK cell-derived exosomes mediated strong anti-leukemic activity against AML cell lines and primary leukemic blasts. Importantly, with higher doses of exosomes, higher levels of cytotoxicity were observed, suggesting that exosome-mediated lysis is concentration dependent. NK cell-derived exosomes mediated leukemia killing via different cell death pathways including apoptosis and necroptosis.

Conclusion: NK cell-derived exosomes mediating cytotoxicity against leukemic targets represents a novel therapeutic modality for patients with AML.