NK cells Activation by IL-15, Hsp70 and PD-1 blocker
To assess the effect of IL-15, Hsp70, and PD-1 blocker as activation mediators for NK cell therapy, CD56+ cells were isolated from nine patients with non-M3 relapsed AML using manual magnetic cell sorting (MACS). Totally, 80.22± 2.54 % of purified cells were NK (CD56+/16+CD3-) cells, 16 ± 1.85 % were NKT (CD56+/16+CD3+) cells and lower than 1.82 ± 0.25 % of them were T cells (CD56-/16-CD3+) (Fig. 2A ).
In the next step, purified cells were incubated with different components, including IL-15, PD-1 blocker, and Hsp70, which were reported to be essential for their expansion and activation(Fig. 2B) . The results displayed that the number of PD-1 expressing NK cells and the mRNA level of PD-1 was significantly reduced in all groups treated with IL-15 in combination with Hsp70 and PD-1 blocker, compared to inactive and IL-15 treated group (Fig. 3A) . Meanwhile, the expression of NKG2A, as an inhibitory receptor, was reduced in those groups that received in PD-1 blockers. The results indicated that Hsp70 in combination with IL-15 can promote the expression of NKG2A (Fig. 3B) . Looking at NKP30 and NKP46 as activator receptors signified that their level reduced in those groups that activated with PD-1 blocker, Hsp70, and their combinations compared to inactive NK cells and IL-15 treated group (Fig. 3C and 3D).
The different activator combinations’ effect in NK cell-mediated cytotoxicity
As mentioned earlier, the combination of IL-15, Hsp70, and PD-1 blocker reduced the expression pattern of both activatory and inhibitory receptors of the patient’s NK cells. Nevertheless, the main question is how these components affect the cytotoxic potential of NK cells. Our results indicated that although cytotoxicity of treated NK cells on the KG1 cell line, as target cells, enhanced in all treated groups, it was dominant in groups with PD-1 blocker in their formulation (Fig. 4A and 4B). Meanwhile, the rate of LDH released from KG-1 cells co-cultured with NK cells only exhibited elevation in IL-15 + Hsp70 + PD-1 blocker group that was not significant (Fig. 4C). As the lactate dehydrogenase (LDH) assay is used in NK cell cytotoxicity assessment against tumor cells, its release is detected at 4 hours post activation 13. For the LDH release assay, we removed condition media after 24 hours, but our result did not show a significant difference between groups that may be associated with the NK and KG1 cells co-culture time which incubated over 4 hours. Interferon gamma (IFN-γ) is another factor that its released enhanced post activation of NK cells. IFN-γ level significantly increased in IL-15 + PD-1 blocker treated group compared with inactive NK cells (p<0.03, Fig. 3D ). Also, we found that in those groups that Hsp70 was in their formulation, the secretion of IFN-γ significantly reduced (Fig. 3D) . The reason for this variation may back to use of fludarabine in these patients. Actually it has been reported that fludarabine increases the secretion of interferon, and therefore, after using the protein, its secretion no longer shows an increase 14.