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.