DISCUSSION
Reflective of the current study revealed several key findings that help us better understand the role of Hsp-70 and PD-1 blocker-based therapy in patients with AML. Immune checkpoints, including PD-1 and CTLA-4, are novel targets for cancer immunotherapy and made a promising tool in the path of solid cancer treatment 21,22. PD-1/PD-L1 inhibition has been demonstrated in multiple myeloma patients to improve NK cell-mediated tumor lysis 23,24. Despite the favorable outcomes of blocking such immune checkpoints, the results were not well studied in hematological cancers. meanwhile, many malignant cells over-expressed heat shock proteins, including Hsp70 and Hsp90, which indicate their crucial role in malignant progression25,26. Regarding AML, the overexpression of Hsp70 is identified 27,28, however, its role is controversial28,29. Several studies have reported that Hsp70 can induce cytotoxic activity in NK cells against tumors, including melanoma and glioblastoma 30,31. We depicted that pre-treatment of NK cells with Hsp70 and PD-1 inhibitor may stimulate their anti-tumor effects. Our results indicated that the percentage of NK cells in patients with non-M3 relapsed AML who received fludarabine and busulfan was about 12.3±4.18% of total lymphocytes similar to normal people32. However, 7.05 ± 1.14% of them expressed PD-1 in NK cell population that higher than PD-1 expression in normal NK cells (2-5%) 14,33. The main question of the present study was to evaluate the combinatory effect of Hsp70 and PD-1blocker to activate NK cells derived from patients with non-M3 relapsed AML. We found that the expression of PD-1 significantly reduced when NK cells activated with a different formula of IL-15, Hsp70, and PD-1 blocker (Fig. 3A ). However, the activatory and expanded clones in groups that received IL-15 + PD-1 blocker seemed to be more than other groups (Fig. 2B, Fig. 3B and 3C ). NKG2A, in cooperation with PD-1, exerts its inhibitory activity on NK cells through binding to classical and non-classical MHC class I molecules34. Our results exhibited that the expression of surface NKG2A in the NK cells treated in IL-15 and IL-15 + PD-1 blocker groups, considerably decreased (Fig. 3B ) and its expression was increased with adding the Hsp70 to activation media. It is worth mentioning that IL-15 is recommended to be involved in PD-1 blocking and NK cell activation through activation of the PI3K/AKT/mTOR signaling pathway 35, as also observed in our data. The over-expression of NKG2A also was reported by Fehniger et al. study, which showed induction of cytotoxicity in NK cells in parallel to increasing NKG2A when the cells were treated with IL-2/TKD (TKDNNLLGRFELSG; a 14-mer Hsp70 subunit) 36. Here our results showed a significant increase in the expression level of PIK3CB and AKT-1, the downstream molecules of NKG2D and critical regulator of NK cell activation, in the presence of IL-15 + PD-1 blocker. However, we did not detect any changes in the level of NKG2D positive NK cells, and a significant reduction was observed in NKP30 and even NKP46 post treatment of cells by Hsp70 or PD-1 blocker and both of them (Fig. 3C and D ). Meanwhile, the cytotoxic potential of NK cells enhanced in groups that received PD-1 blocker, which was concomitant with increasing in releasing of interferon gamma (IFN-γ) and upregulation of granzyme A/B and PRF116,37-40.
Although, several studies reported that a combination of Hsp70 and PD-1 blocker might over-activate NK cells in Fighting cancer cells41,42. But our results presented that the presence of Hsp70 as an activatory factor in the combination of IL-15 for increases the ambiguity of data. For example, Hsp70 in activating media caused an increase in the NKG2A positive cells, reduced IFN-γ releasing, and reduced the expression of FAS-L and TRIAL. All uncertainties may back to AML-NK cells as a source of NK cells and even the undefined role of Hsp70 these patient’s specific cells 43,44. Therefore, based on the results presented in this study, we suggested that the combination of IL-15 and PD-1 blocker can reactive AML-NK cells, increasing their killing ability against tumor cells, enhanced the key factors in NK cell function and increase releasing of IFN-γ, granzymes as well as perforin. Moreover, we clearly showed that Hsp70 acts as a disruptive factor to induce cytotoxic NK cells when combined with IL-15 and PD-1 blocker.