Abstract
We have recently established a continuous natural killer (NK) cell line, KIL (for Killer Lymphocyte), from a coculture of normal murine whole bone marrow (WBM) and the OP-9 stromal cell line expressing the Notch ligand, Jagged2, in the presence of stem cell factor and IL-7 (
Dehart et al. Blood 2005;105:3521
). A clonal prototype, KIL C.2, was derived from a C57BL/6 mouse and has the CD3−NK1.1+CD122+ phenotype, expresses the NKG2D activating receptor, and proliferates extensively upon in vitro stimulation with IL-2 (20ng/ml). We now show that KIL C.2 stimulated with IL-2 for 48 hours produce large quantities of interferon-gamma (10,300 ± 1850 pg/ml) and modest amounts of tumor necrosis factor-alpha (200 ± 60 pg/ml). IL-4 and IL-10 secretion was below background levels. We then evaluated the cytolytic potential of KIL C.2 using a FACS-based flourolysis assay using GFP-labeled target cells. We observed that KIL C.2 potently lyse allogeneic A20 (H2d) lymphoma tumor targets without prior activation. In addition, KIL C.2 is moderately lytic against two syngeneic (H2b) targets: a myeloma cell line, 5T33MM, and BLL C.2, a tumorigenic leukemia/lymphoma cell line that was established from a WBM culture and has the CD19+CD25+CD43+sIg− pre-B-cell phenotype (S.T. and T.M.C., unpublished data). To study the mechanism of cytolysis we performed killing assays in the presence of anti-NKG2D (MI-6) or anti-NK1.1 (PK136) blocking antibodies. Blockade of either NK1.1 or NKG2D reduced cytotoxicity against BLL C.2 targets by 50%. BLL C.2 has the C57BL/6 background, thus does not express H60 but expresses the other NKG2D ligands Rae1 at high levels and Mult1 at moderate levels as determined by quantitative RT-PCR. In contrast, blocking NK1.1 and NKG2D had no effect on cytolysis of A20 suggesting the presence of alternative activation pathways such as those mediated by MHC class I alloantigen receptors. To examine the in vivo tumoricidal potential of KIL C.2, we used a C57BL/6 → BALB/c T-cell depleted (TCD)-WBM transplantation and A20-luciferase tumor model and monitored tumor progression using the Xenogen bioluminescence imaging system. BALB/c mice lethally irradiated and rescued with 2.5 × 107 TCD-WBM along with A20-luciferase inoculation uniformly died from tumor. Mice given TCD-WBM and 1 × 106 donor splenocytes did not develop tumor but died from lethal graft-versus-host disease (GVHD). In contrast, mice given TCD-WBM and two injections of KIL C.2 at doses of 1 × 107 per mouse were protected from tumor progression without developing signs of GVHD. These results show that KIL C.2 is a highly functional NK cell line with the capacity for proliferation, cytokine secretion, and cytotoxicity against syngeneic as well as allogeneic tumors. As such, KIL C.2 provides a useful cell line model for studies of NK cell biology and suggests a potentially effective strategy for NK cell-based immunotherapy.Disclosure: No relevant conflicts of interest to declare.
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2006, The American Society of Hematology
2006
