Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin (IL)-15 and 41BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Sevenday coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56 + CD3 -NK cells from peripheral blood (range, 5.1-to 86.6-fold; n = 50), which was considerably superior to that produced by stimulation with IL-2, IL-12, IL-15, and/or IL-21 and caused no proliferation of CD3 + lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and their unstimulated or IL-2-stimulated counterparts showed marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2-stimulated NK cells against acute myeloid leukemia cells in vitro. They could be detected for >1 month when injected into immunodeficient mice and could eradicate leukemia in murine models of acute myeloid leukemia. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer. [Cancer Res 2009;69(9):4010-7]

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 anti-tumor 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 cytototoxic than unstimulated NK cells, and eradicated EWS cells engrafted in NOD/scid 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.

IntroductionThe present study was aimed to test the role of endothelin-1 (ET-1) as a possible autocrine/paracrine growth factor for cardiac fibroblasts, and to examine its interaction with cardiac natriuretic hormones. Expression of preproET-1 (ppET-1) mRNA by cultured cardiac fibroblasts from neonatal rats was demonstrated by Northern blot analysis using cDNA for rat ppET-1 as a probe. Although cardiomyocytes occupy -75% of the structural space of the heart, they constitute only one third of the total cell population (1, 2). The remaining non-myocytes consist mainly of cardiac fibroblasts in the interstitium. It has been recently shown that growth of cardiac fibroblast associated with enhanced collagen accumulation in the myocardial interstitium is involved in the remodeling process of left ventricular hypertrophy (3, 4). Several recent in vivo studies have suggested that angiotensin II (ANG I) 1 is a critical growth factor for cardiac hypertrophy. Treatment with a subdepressor dose of angiotensin-converting enzyme inhibitors (5) and angiotensin receptor antagonists (6) prevent an increase in left ventricular mass. It has been also reported that chronic infusion of a subpressor dose of ANG II into rats causes left ventricular hypertrophy(6). Since ANG II stimulates cellular proliferation (7) and several extracellular matrix gene expressions in cardiac fibroblasts (8), and induces cardiomyocyte hypertrophy (9, 10), ANG II is considered to act directly on both cardiomyocytes and fibroblasts.We (11)

Hemophagocytic lymphohistiocytosis (HLH) is caused by the hyperactivation of T cells and macrophages. The clinical characteristics associated with this disease result from overproduction of Th1 cytokines including interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α). In this study, we analyzed the production of IL-12 and IL-4, which determine Th1 and Th2 response, respectively, and IL-10, which antagonizes Th1 cytokines, in 11 patients with HLH. IL-12 was detected in plasma in all patients (mean peak value, 30.0 ± 5.0 pg/mL), while IFN-γ was massively produced in nine patients (mean peak value, 79.2 ± 112.0 U/mL). IL-4 was not detected in any of the patients. Plasma IL10 levels were elevated in all patients (mean peak value, 2,698.0 ± 3,535.0 pg/mL). There was a positive correlation between the levels of IFN-γ and IL-10 (P < .01). The plasma concentrations of these cytokines were initially high, before decreasing after the acute phase. However, the decrease in IL-10 levels was slower than that of IFN-γ. Although the concentration of IL-12 was high at the acute phase, in some patients, a peak in the level was delayed until the chronic phase. Thus, in HLH, production of cytokines that promote development of Th1 cells appears to be predominant over that for Th2 cell development. Overproduction of IL-10 was also observed indicating that a mechanism suppressing hyperactivation of Th1 cells and monocytes/macrophages functions in patients with this disease.