**11. Conclusions**

chemotherapy, infused donor NK cells could successfully expand in the patients of AML [164]. Remarkably, among 10 patients, three patients were observed to have detectable NK cells up to 1 month after infusion and have a disease-free survival rate of 100% at a median of 2 years. This promising outcome may due to the higher cell dose of transferred donor NK cells used in this study; thus, higher doses of infused NK cells may be important for

To produce sufficient number of donor NK cells and achieve the optimal expansion of NK cells *ex vivo*, numbers of studies were performed with variable approaches, such as cytokine stimulation and utilizing engineered feeder cells. Up to now, clinical NK cell doses can be

affect the effector function of expanded NK cells. A crucial factor among these complicated production protocol is how to activate NK cells *ex vivo*. Addition of cytokines such as IL-2 or IL-15 was used to promote survival and expansion of NK cells. Although IL-2 stimulation may result in activation-induced cell death (AICD) of NK cells *in vivo*, IL-15 stimulation does not have this side effect [166, 167]. Subsequently, human-derived feeder cells were used to develop approaches in expanding NK cells. Fujisaki et al. first reported the use of genetically modified APCs that could expand NK cells from peripheral blood 500- to 1000 fold [13]. These artificial APCs can be further modified with costimulatory molecules and membrane-bound cytokines [168, 169]. The expanded NK cells express high level of activating receptors and CD16 molecule and are proved to be potent mediators of cytotoxicity. However, these activated NK cells often become "exhausted" and cannot maintain their effector function and expansion *in vivo* [170]. Denman CJ et al. used APCs modified with membrane-bound form of IL-21 to break this barrier [168]. However, after cytokine withdrawal, these *ex vivo* expanded NK cells have been shown to have shorter survival *in vivo*

Another alternative approach is to generate "memory-like" NK cells *ex vivo*. Cooper et al. reported that IL-12/15/18-preactivated NK cells obtained the ability to produce increased IFN-γ upon restimulation for up to 4 months after adoptive transfer [24]. Therefore, cytokine preactivation before infusion may amplify and sustain the beneficial effects of NK cells during allo-HSCT. The enhanced antitumor activity of murine IL-12/15/18-preactivated NK cells was first demonstrated in a murine tumor model [172]. Similar to murine NK cells, human IL-12/15/18-preactivated NK cells have been recently shown to have memorylike properties, including increased IFN-γ production, enhanced proliferation, and high expression of IL-2 receptor [26]. Recently, a phase I study of adoptively transferred cytokine-induced memory-like NK cells has demonstrated sustained anti-leukemia responses in patients with relapsed or refractory AML [173]. Therefore, cytokine-induced memory-like NK cells may possess long-lasting effector function *in vivo*, which can be harnessed in the treatment of leukemia patients. However, the safety of such therapy could be complicated by the induction of aGvHD after allo-HSCT. Adoptively transfer of donor-derived IL-15/4- 1BBL-activated donor NK cells contributed to aGvHD [158], likely through upregulation of activating receptor expression and inflammatory cytokine production. Therefore, the effect of IL-12/15/18-preactivated NK cell infusion on GvL and GvHD after allo-HSCT needs fur-

NK cells/kg [165]. However, there are numerous factors that may

better HSCT outcome.

142 Natural Killer Cells

reached even up to 10<sup>8</sup>

ther investigation.

compared with freshly activated NK cells [171].

A number of recent studies demonstrated that NK cells played a critical role in diseaserelapse prevention. Based on the progress made in the field of NK cell therapy, the criteria for choosing HSCT donor have been significantly changed and shown to be associated with better outcomes. However, the role of NK cells in HSCT is not fully understood. The GvL effect of NK alloreactivity may be affected by the complexity of HSCT including the degree of T-cell depletion, donor sources, degree of HLA mismatch, different intensity of preparative regimens, and the origin of leukemic blasts. Furthermore, the haplotype of donor KIR genes and the intensity of interaction between KIR and HLA may also influence the GvL effect of NK cells. Donor KIR B haplotype may be favorably associated with better overall survival. The education of NK cells is also critical for their beneficial effect. The stronger KIR/HLA affinity may contribute to the generation of NK cells with enhanced GvL effect. Intriguingly, "unlicensed" NK cells may also have chance to undergo "re-education" and acquire potent effector function after HSCT. Several studies reported the favorable association between CMV reactivation and better HSCT outcome. This beneficial effect of CMV reactivation was mainly due to the expansion of NKG2C<sup>+</sup> NK cells. Moreover, these NKG2C<sup>+</sup> NK cells display memory-like properties and enhanced cytotoxicity toward leukemic blasts. NK alloreactivity has been demonstrated to play potential role in suppression of aGvHD. However, some groups found KIR mismatch or alloreactive donor NK cell infusion also worsen aGvHD. Therefore, the role of NK alloreactivity in GVHD still needs to be carefully studied.

Since the GvL effect of NK alloreactiviy is affected in various aspects. Some critical questions are still remained to answer so as to ensure NK cells play the most desirable role during HSCT. The fate of NK cells is still unpredictable after transfusion. The optimal approach to expand most powerful NK cells which can maintain their proliferative potential and effector function *in vivo* is still not confirmed. The risk of the clinical usage of these activated NK cells is still needed for evaluation. The ligands of several activating receptors are undefined and the interactions between NK cells and other immune cells need further investigation. Further studies are also required to explore the mechanism underlying the process of NK cell "education" and "memory". Recently, monoclonal antibodies and immunomodulatory drugs are utilized to modulate NK cell function and proved to have benefit effect on the outcome of HSCT, whereas it is still needed to confirm the suitable hematopoietic malignancies for these treatments. The answers to these questions and continuous progress in understanding NK cells biology will optimize donor NK cells-based therapy and benefit the outcomes of HSCT.
