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Pearce et al. Neuroimmunol Neuroinflammation 2018;5:47 I http://dx.doi.org/10.20517/2347-8659.2018.46 Page 5 of 16

Studies aimed at heightening NK cell activity for immunotherapies have done so through genetic
engineering or by stimulation of NK cells directly in vivo or ex vivo [Table 2]. The tumor microenvironment
[31]
is known to be immunosuppressive which correlates with reduced NK cell activity . Because of this, few
NK cells achieve activation to carry out NK cell-mediated lysis. By stimulating NK cells, however, the
immunosuppressive tumor microenvironment may be overcome. Efforts to stimulate NK cell proliferation
and activity have focused on exposure of NK cells to cytokines including IL-2, 12, 15, 18 and 21. Once
stimulated, NK cells become lymphokine-activated killer cells. These cells have demonstrated increased
[29]
levels of cytotoxicity towards malignant tumors and proliferate at a greater rate .

Clinically, NK cell stimulation with IL-2 was approved for treatment of metastatic renal cancer by the FDA
in 1992. High-doses of IL-2 has demonstrated efficacy in treating various cancers, but increasing doses
also increases the risk of severe adverse effects. A phase III trial comparing IL-2 doses for liver and bone
metastases and primary tumors showed that the response rate for high-dose IL-2 was significantly higher
than all other groups. It was concluded high-dose IL-2 were necessary for significant clinical benefits, despite
[32]
the possible negative effects .

+
IL-21 stimulates NK cells and CD8 T-cells while also increasing the production of IFN-γ. Together with
[33]
+
IL-2 and IL-15, IL-21 enhances cytotoxic effects of NK and CD8 T-cells . A phase II clinical study treating
melanoma patients with IL-21 found that antitumor efficacy of IL-21 is comparable to that of high-dose IL-2.
[34]
The treatments were well tolerated among patients and resulted in few adverse actions .
IL-15 has been tested in phase I trials to monitor the reactions among patients after administration. The
cytokine was given as bolus intravenous infusions to patients with metastatic malignant melanoma and
renal cell cancer. The treatments caused a large swing in the distribution of lymphocytes within the blood,
suggesting its importance to the activation of NK cells and their cytotoxicity. Many adverse reactions were
[35]
recorded, however, which is thought to be the result of the method of administration .
IL-12 is another cytokine under investigation for use in immunotherapy. Studies in preclinical models using
[36]
IL-12 have shown strong antitumor effects . In one such study, the rejection of gliomas in mice was found
[37]
to be significantly enhanced in those expressing IL-12 in the CNS, as compared to those without . This
gives evidence that the expression of IL-12 cytokine can be a major factor in anti-tumor response through
stimulation of the immune system.

NK cell function is heavily dependent on cytokine support. But, even with administration of additional
cytokines, the tumor microenvironment may limit NK cell activation. To overcome dependence on
exogenous cytokines, genetically engineered NK cells have been explored for their ability to surpass
the tumor microenvironment. One study examined whether transduced expression of a nonsecretory,
membrane-bound form IL-15 (mbIL15) could sustain NK cells. The mbIL15 NK cells had enhanced survival
and viability compared to mock-transduced NK cells and NK cells that expressed non-membrane bound
IL-15. Because mbIL15 NK cells are less dependent on endogenous signaling molecules, their activity
[38]
and cytotoxicity against solid tumors is resilient to immunosuppressant effects of the tumors . Genetic
engineering of NK cells to self-activate may prove more effective than stimulation from endogenous
cytokines. Engineered NK cells avoid off targeting effects of cytokine administration to patients and may
allow for NK cell antitumor functions to be enhanced. Patients with esophageal squamous cell carcinoma,
squamous cell lung cancer, and gastric carcinoma have shown positive responses to NK treatments. Higher
survival rates were correlated to CD57 positive cells at the site of the tumor. CD57 expression is associated
with NK cells, as well as T-cells, and may serve as an additional target for enhancing NK cell effectiveness.
Clinical trials to assess the efficacy of NK therapy for gliomas have now been initiated [Table 2] with peer-
reviewed reports yet to be released.