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.