Legends:
Figure 1 The PD-1 expression on T, NKT and NK cells derived from MNCs of patients with non-M3 relapsed AML. A.  Schematic presentation of different cell types and characterization of PD-1 expressing cell types  in patients’ MNCs. B.  Quantification demonstration of different cells in the patients’ MNCs. C . PD-1 expressing cell types graph illustrate  11.6% of total lymphocyte cells express PD-1 receptor and 9.8% of CD56+ and CD16+ cells, known as NK cells, express PD-1 receptor. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical analysis performed using unpaired two-way Anova. (n=7, *p<0.05, **p<0.01, and ***p<0.001). CD: cluster of differentiation; MNC: mononuclear cells; PD-1: Programmed cell death protein.
Figure 2 NK cell isolation and activation in different component. A. Purification of NK cells before isolation showed about 17% NK cells which showed more than 80% NK cells after isolation with MACS, less than 2% T cells, and 16% NKT cells. B.Morphologically, NK cells in different components, including IL-15, Hsp70, and PD-1 blocker, displayed round clones with the ability of expansion and single activated NK cells, (n=7). Images were obtained with a 20x objective, the magnification used for morphological assessment (scale = 100 µm).
Figure 3 The expression of activatory and inhibitory receptors in different activation NK cells . Purified NK cells were activated in presence of IL-15, Hsp70 and PD-1 blocker for 24 hours A. The expression of PD-1 was assessed at mRNA and protein level. The results indicated of higher reduction of PD-1 in those group that received Hsp70, PD-1 blocker and their combination than IL-15 or in active NK cell, (n=9, p<0.02). B. The expression of NKG2A was significantly reduced in all groups (n=9, p<0.04) except combination of IL-15 and Hsp70 (n=9, p>0.05). C.The expression of NKP30 and D. NKP46 as activator receptors reduced in all combinatorial groups post activation (n=9, p<0.006), which means that their expression was higher in inactivated NK cells. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical analysis performed using unpaired two-way Anova. (*p<0.05, **p<0.01, and ***p<0.001).
Figure 4 The effect of different activator combinations on NK cell-mediated cytotoxicity. A. Morphological illustration and flowcytometry results of different activator combinations’ effect in NK cell-mediated cytotoxicity on KG-1 cell line. Images were obtained with a 40x objective, the magnification used for morphological assessment (scale =50 µm). B. NK cell cytotoxicity potential increased in all groups compared to the inactive group. However, it was significant in those groups that had PD-1 blocker in their formulation, (n=9, p<0.02). C. The LDH released from KG-1 cells co-cultured with NK cells only increased in IL-15 + Hsp70 + PD-1 blocker group that was not significant (n=7, p>0.05). D.IFN-γ level in the group that received IL-15 + PD-1 blocker significantly increased compared to other groups (p<0.03). Although, it was not significant in comparison to IL-15 treated group (n=7, p>0.05). E . GZMA and F.GZMB expression level was increased in IL-15 + Hsp70 + PD-1 blocker group compared to other groups (n=7, p<0.01).G. PRF-1 was higher in IL-15 + Hsp70 + PD-1 blocker treated group (n=7, p>0.02). Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical analysis performed using unpaired two-way Anova. (*p<0.05, **p<0.01, and ***p<0.001).
Figure 5 NK cell gene regulation after activation in patients with AML. A. Fas ligand expression upregulated in activated groups. However, it was over-activated when the NK-cells were treated with IL-15 + Hsp70 (n=7, p<0.0001). B. The expression of TRAIL gene was upregulated in IL-15+Hsp70 treated NK cells which is just significant compared to IL-15+ Hsp70 + PD-1 blocker (n=7, p<0.008). C. AKT-1 expression level showed a significant increment in the IL-15 + PD-1 blocker treated NK cells compared to other groups (n=7, p<0.01). D. The expression level of PIK3CB enhanced in the IL-15+PD-1 blocker treated NK cells but as shown in the plot there is no significance with IL-15 + Hsp70 + PD-1 blocker group (n=7, p<0.01). Statistical analysis performed using unpaired two-way Anova. (*p<0.05, **p<0.01, and ***p<0.001).