| Natural killer (NK) cells are the first defense line against virus-infected cells and tumor cells. Dendritic cells (DCs) are at the crossroads of innate and adaptive immunity and essential mediators of immunity. The CD3-CD19-CD11cintB220+NK1.1+CD49b+Gr1-subset of cells has recently been described as IFN-y-producing killer dendritic cells (IKDC), which share phenotypic and functional properties of DC and NK cells. IKDC is shown to have TRAIL-and NKG2D-dependent NK-type cytotoxicity in vitro. When licensed by tumor cells, IKDCs up-regulates MHC class II molecules and acquired the capacity to cross-present tumor antigens to naive T cells and initiate the antitumor adaptive immune response. Thus, IKDCs might be a promising and potent agent for tumor immunotherapy. However, there are trace numbers of IKDC in lymphoid organs of naive animals (～50,000/spleen). The aim of the present study was to establish of a cell line that co-expressed of mouse membrane-bound form of IL-15and RAE-1ε for expansion and activation of IKDCs ex vivo.Part1:Construction of of BaF3-Rae-1ε-mIL-15cell.The cDNA encoding the signal peptide of CD8a, the mature peptide of IL-15and the transmembrane and cytoplasmic domain of CD8a were assembled to encode a membrane-bound form of IL-15(named mb15) using the splicing by SOE-PCR method. The RAE-le gene was amplified by PCR from pMX-pie plasmid. The purified mb15and Rae-1ε gene were subcloned into the MCS1and MCS2of the plasmid pVITR02-mcs, respectively. The resulting plasmid was named pV/mbl5/RAE-1ε. BaF3cells were transduced with pV/mb15/RAE-1ε. Cells were cloned by limiting dilution, and a single-cell clone with a high expression of RAE1ε and a surface expression of IL-15(termed BaF3/mb15/RAE) was expanded. BaF3cells expressing a membrane-bound form of IL-15(termed BaF3/mb15) or RAE1ε (termed BaF3/RAE) were produced using a similar procedure.Part2:Analysis of IKDC biology in vitro.Bone marrow-derived mature DCs were cultured with BaF3derivatives for7days, and then the percentage of IKDC in the CD11c+cell population was determined using triple staining for CD11cint, B220, and NK1.1+cells. The results showed that the percentage of IKDC increased significantly following BaF3-mb15-RAE stimulation. Furthermore, the sorted IKDCs were cultured with BaF3derivatives at a ratio of1:1, and then culture supernatants were collected for IFN-y assay. Cells were analyzed for the expression of CD40, CD80, CD86, NKG2D, FasL TRAIL and MHC class Ⅱ. The results showed that stimulation with BaF3/mbl5/RAE leads to an increased CD40, CD80, NKG2D, FasL and MHC class II production of IKDCs.In summary, we have successfully established a cell line that co-expressed of mouse membrane-bound form of IL-15and RAE-1ε (BaF3/mb15/RAE). Stimulation with BaF3-mbl5-RAE increases the percentage of IKDC and promotes CD40, CD80, MHC II and FasL expression of IKDCs, suggesting that the killing and presenting antigens capacities of IKDCs might be enhanced following BaF3/mbl5/RAE cell stimulation. This study will lay a solid experimental foundation for application of IKDC in tumor immunotherapy. |
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