KIR Incompatible NK Cells Effectively Lyse Osteosarcoma Cells.

Recurrent or metastatic pediatric solid malignancies, including osteosarcoma, carry a dismal prognosis despite modern multi-modality treatment approaches. Although the success of KIR (Killer Immunoglobulin-Like Receptor) incompatible, haploidentical stem cell transplantation has been documented in hematological malignancies in adults and children, this approach has not been thoroughly examined in pediatric solid tumors. In this study, we evaluated the potential for KIR-incompatible lysis of osteosarcoma cells, in vitro. We hypothesized that the killing of osteosarcoma cell targets could be predicted by the degree of inhibitory KIR receptor/ligand mismatch with NK cell effectors. To test this hypothesis, healthy donor NK cells were isolated by magnetic bead sorting and their KIR phenotype determined by flow cytometry. Consistent with previous studies, donor NK cells exhibited a high prevalence of all three relevant inhibitory KIRs (KIR2DL1, KIR2DL2/KIR2DL3, KIR3DL1). Conversely, examination of three established osteosarcoma cell lines (HOS, SaOS, and U2OS) demonstrated significant variability in cell surface HLA class I expression, by flow cytometry. QRTPCR analysis of these cell lines revealed variable expression of the three known inhibitory KIR ligands (HLA-C groups 1 and 2, HLA-Bw4). This variable KIR-ligand expression allowed evaluation of NK-mediated lysis of targets with varying KIR receptor-ligand incompatibility. Following a 12-hour incubation of donor NK cells in IL-2, lysis of osteosarcoma targets was measured in an annexin V flow cytometric assay. Osteosarcoma cell lines that expressed fewer KIR ligands consistently showed greater susceptibility to NK-mediated cytotoxicity. These findings were consistent using NK effectors from different donors. Additionally, we observed that at high passage number (>20), SaOS cells demonstrated down-regulation of KIR ligand expression. These changes correlated with increased lysis by the same donor NK cells. Our findings suggest:

1. Variable expression of KIR ligands in osteosarcoma cell lines allows potential susceptibility to KIR-incompatible, NK cell-mediated lysis;

2. The killing of osteosarcoma cells by NK cells can be predicted by the degree of receptor/ligand mismatch; and

3. During expansion, osteosarcoma cells may alter expression of KIR ligands, resulting in increased or decreased susceptibility to lysis by KIR-incompatible NK cells.

Further studies are needed to explore the utility of KIR-incompatible, haploidentical stem cell transplantation for patients with high-risk osteosarcoma.