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Functional Characterization of a Continuous Natural Killer Cell Line, KIL, In Vitro and in Adoptive Immunotherapy.

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. Blood2005;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 ( H2 d ) lymphoma tumor targets without prior activation. In addition, KIL C.2 is moderately lytic against two syngeneic ( H2 b ) 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that was established from a WBM culture and has the CD19 + CD25 + CD43 + sIg − pre-B-cell phenotype (S.T. and T.M.C., unpublished data).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 × 10 7 TCD-WBM along with A20- luciferase inoculation uniformly died from tumor. Mice given TCD-WBM and 1 × 10 6 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 × 10 7 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.