Cytotoxicity of activated natural killer cells against pediatric solid tumors

Abstract

Purpose: To develop new therapies for children with solid tumors, we tested the cytotoxicity of natural killer (NK) cells expanded by coculture with K562-mb15-41BBL cells. We sought to identify the most sensitive tumor subtypes, clarify the molecular interactions regulating cytotoxicity, and determine NK antitumor potential in vivo.

Experimental design: We tested in vitro cytotoxicity of expanded NK cells against cell lines representative of Ewing sarcoma (EWS; n = 5), rhabdomyosarcoma (n = 4), neuroblastoma (n = 3), and osteosarcoma (n = 3), and correlated the results with expression of inhibitory and activating NK receptor ligands. We also compared expanded and primary NK cells, determined the effects of activating receptor ligation and of chemotherapeutic drugs, and assessed the therapeutic effect of NK cell infusions in xenografts.

Results: In 45 experiments, EWS and rhabdomyosarcoma cell lines were remarkably sensitive to expanded NK cells, with median cytotoxicities at 1:1 effector/target ratio of 87.2% and 79.1%, respectively. Cytotoxicity was not related to levels of expression of NK receptor ligands, nor was it affected by pretreatment of target cells with daunorubicin or vincristine, but was markedly inhibited by preincubation of NK cells with a combination of antibodies against the NK-activating receptors NKGD2 and DNAM-1. Expanded NK cells were considerably more cytotoxic than unstimulated NK cells, and eradicated EWS cells engrafted in nonobese diabetic/severe combined immunodeficient Il2rgnull mice.

Conclusions: Among pediatric solid tumors, EWS and rhabdomyosarcoma are exquisitely sensitive to expanded NK cells. The NK expansion method described here has been adapted to large-scale conditions and supports a phase I clinical study including patients with these malignancies.

Figure 1

Susceptibility of cell lines derived from pediatric solid tumors to cytotoxicity by expanded NK cells. Shown are results of 4-hour cytotoxicity assays performed at 1:1 E:T ratio (A) and at 1:2 E:T ratio (B). Including in the tests were cell lines derived from Ewing sarcoma family of tumors (EWS) (TC71, SK-N-MC, ES8, EW8 and A673), rhabdomyosarcoma (RMS) (RH30, RH36, RH41 and TE32), neuroblastoma (NB) (NB1691, JF and SK-N-SH) and osteosarcoma (OS) U-2 OS, HOS and MG-63. Each symbol represents the mean of triplicate measurements relative to control cultures with no NK cells. Horizontal bars correspond to median values in each group.

Figure 2

Relation between susceptibility to expanded NK cell cytotoxicity and expression of ligands for NK cell inhibitory or activating receptors in pediatric solid tumor cell lines. For each surface molecule, the mean fluorescence intensity (MFI) (mean of triplicate measurements) measured in each cell line is plotted on the Y axis, with the percent cytotoxicity measured in 4-hour assays at 1:1 E:T ratio plotted on the × axis. Lines in each plot correspond to linear regression analyses. R² values were 0.05 for HLA-Class I, 0.004 for MIC A/B, 0.19 for ULBP1, 0.19 for ULBP2, 0.03 for ULBP3, 0.06 for CD112, and 0.18for CD155.

Figure 3

Cytotoxicity of expanded NK cells against EWS cells. (A) Cytotoxicity of expanded NK cells (squares) compared to that of primary NK cells (circles) against the EWS cell lines ES8 TC71 and EW8. Results obtained from 3 different donors at the E:T ratios indicated are shown. Each symbol is the mean of triplicate measurements relative to control cultures with no NK cells. (B) Live cell confocal photography of the EWS cell line (labeled with green fluorescent protein) cultured with expanded NK cells at 2:1 E:T ratio. The time of culture is shown in the lower left corner of each photograph. Microscopy was performed with a Nikon TE2000E2 microscope equipped with a Nikon C1Si confocal using 488nm and 561nm DPSS lasers for excitation. Temperature was maintained at ~37°C and 5% CO2 using an environmental control chamber. Images were acquired with a Nikon 40× 1.3 NA DIC objective every 20s for 2hr using Nikon EZC1 software. Full movie is in Supplementary Material.

Figure 4

Cytotoxicity of expanded NK cells against EWS cell lines at low E:T ratios. Shown are percentage of cytotoxicity (relative to cultures with no NK cells) in co-cultures lasting 4 hours and 24 hours at different E:T ratios. Symbols are mean of triplicate measurements; each NK donor is indicated by a different symbol. In the case of the 4-hour measurements, standard deviations for all cell lines and all donors were <4% at 1:1, <5% at 1:10, <7% at 1:20 and at 1:50; for 24-hour measurements, they were <1% at 1:1, <5% at 1:10, <6% at 1:20 and <10% at 1:50.

Figure 5

Effect of ligating NK-activating receptors on the cytotoxicity of expanded NK cells against EWS cell lines. Co-cultures of EWS with NK cells were performed after incubating NK cells for 20 minutes with anti-NKG2D and anti-DNAM-1 antibodies, alone or in combination. Each bar corresponds to mean percent cytotoxicity (± SD) of triplicate measurements (relative to control cultures with no NK cells). * = P <0.01; **= P<0.0001 by t test.

Figure 6

Anti-tumor capacity of expanded NK cells in vivo. (A) NOD/scid IL2RGnull were injected with 2 × 10⁵ ES8 i.p. Seven days later mice were either irradiated with 3.25 Gy, treated with 1 × 10⁷ expanded NK cells and 20000 IU IL-2 i.p. daily injections for 5 days with or without prior irradiation, or left untreated. Kaplan-Meier curves indicate the survival of each group of mice; P values in comparisons between groups by log rank test are shown. (B) Xenogen imaging of ES8-luciferase tumors in 4 groups of 3 mice each. Mice received the treatment described in A or no treatment.