Differences between liver and peripheral blood NK cells have not been extensively investigated in human because of the limited availability of appropriate human samples. We have recently determined the phenotypic and functional properties of NK cells extracted from donor and recipient liver perfusate in clinical living donor liver transplantation [10]. Donor liver NK cells exhibited the most vigorous cytotoxicity against a hepatocellular carcinoma cell line after in vitro IL-2 stimulation, compared to the activities of donor and recipient peripheral blood NK cells and recipient liver NK cells (Fig. 4.3). IL-2 stimulation increased TRAIL expression, which has been shown to be critical for NK cell-mediated antitumor cell killing without affecting normal cells, on liver NK cells (Fig. 4.4) [31, 32]. In addition, we have confirmed that death-inducing TRAIL receptors, TRAIL-R1/death receptor (DR) 4, and TRAIL-R2/DR5, which contain cytoplasmic death domains and signal apoptosis, are expressed in HCC [33, 34]. These findings suggest a novel concept to prevent recurrence of HCC after liver transplantation, the adoptive transfer of IL-2-stimulated NK cells extracted from donor liver graft into recipients.

In order to apply this NK cell immunotherapy to cases of deceased donor liver transplantation, we demonstrated for the first time the phenotypical and functional properties of liver NK cells that were extracted from a deceased donor liver graft perfusate under current good manufacturing practice conditions (cGMP) [35]. First, a large number of NK and NKT cells were verified among liver mononuclear cells, with both subsets possessing characteristics different from those of peripheral blood mononuclear cells. Second, in vitro stimulation with IL-2 induced liver NK cells to strongly upregulate activation markers, cytotoxicity, and cytokine production while maintaining the expression of inhibitory receptors. These results were compatible with those for a living donor liver graft perfusate [10]. Finally, we confirmed that the final product met the lot release criteria and contained a low T-cell number, thereby reducing the possibility of graft versus host disease (GVHD) in a recipient.

On the basis of these results, we started a clinical trial of adjuvant immunotherapy with activated donor liver NK cells for preventing the recurrence of HCC after living donor liver transplantation in 2006. The lymphocytes in the peripheral blood of liver transplant recipients who received immunotherapy in the early postoperative period showed significantly enhanced cytotoxicity against an HCC cell line (HepG2), compared with those in the peripheral blood of liver transplant recipients who did not receive the therapy in the same period (Fig. 4.6a). Correspondingly, the proportions of TRAIL⁺ NK cells in the peripheral blood of liver transplant recipient who received immunotherapy also significantly increased (Fig. 4.6b). Although the long-term benefits of this approach with regard to HCC recurrence after liver transplantation remain to be elucidated, there were no severe adverse events including GVHD, thus far. With the collaboration of Professors A. Tzakis and S. Nishida at the University of Miami, FL, we successfully performed a phase I study of adoptive immunotherapy using liver NK cells for deceased donor liver transplant recipients with HCC (ClinicalTrials.gov identifier: NCT01147380) beginning in 2009. Both clinical studies are under evaluation and these results will be published soon.