Background: Natural killer cells are critical immune cells for AML targeting. However, little is known about the relationship between using checkpoint inhibitors and Hsp70 as NK cell activators and later immune responses to control AML. We aimed to investigate the antitumor effects of NK cells pre-treated with ex-vivo Hsp70, human PD-1 blocker, and IL-15. Procedure: The NK cells were isolated from patients-derived MNCs using MACS and activated using the different combinations of Hsp70, PD-1 blocker, and IL-15. Then their killing potential and the expression pattern of PRF-1, PIK3CB, PD-1, AKT-1, FAS-L, TRAIL, and GER A & B were estimated. Results: Our data revealed that the PD-1 expression was significantly reduced after NK cell activation with the different formulas of NK cell activators. Also, the expression of NKG2A was reduced, particularly in the IL-15 and IL-15 + PD-1 blocker treated groups, and adding the Hsp70 increased its expression. Moreover, the cytotoxic effect of NK cells increased in all groups, especially in IL-15 + PD-1 blocker group, in parallel with increasing in IFN-γ releasing, Granzymes, and perforin expression. All changes in IL-15 + PD-1 blocker groups were associated with the up-regulation of PIK3CB and AKT-1 as key factors of NK cell activation. The presence of Hsp70 reduced IFN-γ releasing and down-regulation of PIK3CB, AKT-1, Granzymes, and perforin. Conclusions: We suggested that combining IL-15 and PD-1 blockers could enhance the killing potential of AML-NK cells. Moreover, Hsp70, in combination with IL-15 and PD-1 blocker, interferes activation of AML-NK cells through unknown mechanisms.
