We have previously shown that IL-2–stimulated NK cells derived from the liver exert vigorous cytotoxicity against HCC by their binding to the TRAIL expressed on NK cells and the corresponding death receptors, and exhibit inhibitory effects on HCV replication by producing high levels of IFN-Γ. On these bases, we have successfully applied an adjuvant immunotherapy, employing IL-2-treated NK cells extracted from donor liver-allograft perfusate, to 34 liver transplant (LT) recipients having either HCC or HCV infection. The immunotherapy significantly reduced the HCC recurrence rate (p < 0.05) and transiently suppressed serum HCV-RNA load. Nevertheless, recurrent HCV infection eventually occurred in most of the HCV-infected recipients. These findings prompted us to develop a novel technique to differentiate liver-type NK cells from CD34+ hematopoietic progenitor cells for the application of an adoptive immunotherapy that can be repeated.

We incubated bone marrow-derived CD34+ cells in 5% serum-supplemented X-VIVO15 medium containing SCF, Flt-3, IL-7, and IL-15 for 28 days for the CD34+ cells to differentiate into CD56+ cells. These CD56+ cells exhibited NKp46, a specific receptor of NK cells, and were phenotypically similar to the liver-resident NK cells in that they expressed TRAIL, NKG2D, and CD94 but lacked CD158a and CD158b, which are inhibitory receptors recognizing self HLA class I (S-KIRs). Thus, the CD34+ cells effectively differentiated into unlicensed NK cells lacking S-KIRs, which have been recently shown to exhibit vigorous cytotoxicity against neoplastic cells. Additional cultivation with IL-12 and IL-18 prompted the unlicensed NK cells to actively produce IFN-Γ. An experiment using genomic HCV-replicon–containing hepatic cells indicated that the unlicensed NK cells inhibited HCV replication. The anti-HCV responses were also observed in vivo using human hepatocyte-chimeric mice, which had previously developed consistently prolonged HCV infections after inoculation with HCV genotype 1b-infected human serum; however, repeated injections of the unlicensed NK cells differentiated from the CD34+ cells resulted in a failure in the development of HCV infection.

To summarize, the unlicensed phenotype NK cells differentiated from CD34+ cells exhibited both anti-HCC and anti-HCV activities. Repeated adaptive immunotherapy with such cells may be a promising modality for preventing recurrence of HCC/HCV after LT.

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