Functional Characterization Of Interleukin-15 Gene Transduction Into The Human Natural Killer Cell Line NKL

ObjectiveNatural killer (NK) cells are a key component of the innate immune system that is involved in the early defense against virus-infected or transformed cells by non-major histocompatibility complex (MHC)-, non-T-cell receptor (TCR)-restricted mechanisms. NK cells not only mediate spontaneous anti-rumor effector functions but also regulate innate and adaptive immune responses. Adoptive transfer of activated or allogeneic NK cells is effective in the treatment of certain types of leukemia and solid tumors. Given the importance of NK cells to immunity, a variety of approaches have been taken to try and selectively augment NK cell responses to tumors, including genetic modification.The cytokine interleukin- 15 (IL-15) is present in a wide variety of tissues and cell types, and shares many biologic activities with IL-2 and can directly induce CD34+ cells to differentiate into NK cells in the absence of IL-2. This cytokine is also a potent regulator of NK cell proliferation, survival and cytolytic activity. In previous work, we have reported that IL-15 gene-modified NK-92 cells exerted more efficiency against cancer but extensive utilization of this gene-modified human NK-92 cell line is limited by its tumor spectrum.Compared with primary NK-92 cells, NKL cells appear to have a different anti-tumor spectrum. NKL is one of six malignant NK cell lines that have currently been established and are sufficiently well characterized. The NKL cell line was established from the peripheral blood of a patient with CD3-CD16+ CD56+ large granular lymphocyte (LGL) leukemia and grows in the presence of IL- 2 at a concentration of 100 U/mL. Among all the NK cell lines, NKL is probably the one that has retained the most original features of NK cells and could therefore potentially be used as effector cells in adoptive immunotherapy.In this report, the human (h) IL-15 gene was stably transfected into NKL cells. We analyzed the characterization of hIL-15 gene-modified NKL cells and investigated the molecular mechanisms for IL-15 gene modification in improving the proliferation, anti-apoptosis activity of NKL cells and enhancing the natural cytotoxicity against hepatocarcinoma cells.Methods1. Electroporation: electroporation was used to transfer hIL-15 gene into NKL cells.2. CTLL-2 proliferation assay: CTLL-2 proliferation was used to detect biologic activity of IL-15 in the culture supernatants.3. ELIAS: ELISA was performed to detect the concentration of IL-15 in the culture supernatants.4. RT-PCR assay: RT-PCR was used to analysis the expression of apoptosis-associated genes and cytotoxicity-associated genes in NK cells.5. CCK: CCK was used to determine the proliferation of NK cells in different concentration of IL-2 or IL-15.6. FACS: FACS was used analysis the cell apoptosis, cell cycle, the expression of NKG2D and NKG2A.7. MTT: MTT was used to determine the cytotoxity of NK cells against hepotocarcinomar cells.Results1. hIL-15 gene-modified NKL cell line was successfully established.2. The proliferation of hIL-15 gene-modified NKL cells was increased with low levels of IL-2 or IL-15.3. hIL-15 gene increased the anti-apoptotic effect of NKL cells.4. Anti-apoptosis gene expression was up-regulated in hIL-15 gene-modified NKL cells.5. No detectable change of cell cycle was shown in hIL-15 gene-modified NKL cells.6. The cytotoxicity of hIL-15 gene-modified NKL cells against hepatocarcinoma cells was significantly enhanced in vitro.7. The cytotoxicity-associated gene expression in hIL-15 gene-modified NKL cells was up-regulated.8. The cytotoxicity against hepatocarcinoma cells by hIL-15 gene-modified NKL cells was augmented in vivo.ConclusionNK cells play a crucial role as a first line of defense against virus infection and tumor cells, and have been used as a strategic weapon in cancer cell therapy and hematopoietic stem cell transplantation, through activation of endogenous NK cells and adoptive transfer of in vitro-activated autologous NK cells or permanent NK cell lines among which the NK-92 cell line has already finished clinical trials.To create more effective NK cells against human tumors, we have established hIL-15 gene-modified NKL cells with many original features of NK cells. Investigations have demonstrated that the hIL-15 gene modification has improved the NK cell proliferation potential at low concentrations of IL-2 and IL-15, and NKL-IL15 cells proliferate even in the absence of IL-2, with less apopototic cells by serum starvation. Furthermore, with the improvement of antiapoptosis in IL-15 gene-modified NKL cells, the antiapoptosis gene Bcl-2, Bcl-xl and Mcl-1 expression was significantly increased and accompanied apoptosis gene down-regulation (Fas, Bim and Noxa). Interestingly, natural cytotoxicity against hepatocarcinoma cells was significantly augmented in the IL-15 gene-transferred NKL cells. NKL-IL15 cells could inhibit the proliferation of hepatocarcinoma cells in vitro and in vivo. The mechanisms were associated with higher expression of IFN-γ, FasL and perform in IL-15 gene-modified NKL cells than that in parental cells. These results indicate that IL-15 gene modification of NKL cells enhances their suitability for large-scale production in vitro and the IL-15 gene-modified NKL cells maybe more suitable for clinical application.