IL-2/IL-18 prevent the down-modulation of NKG2D by TGF-β in NK cells via the c-Jun N-terminal kinase (JNK) pathway

Abstract

TGF-β is known to play a major role for the reduced NKG2D expression seen in cancer patients. However, the mechanisms for reduced TGF-β-induced down-regulation of NKG2D are unclear. In this study, we observed that IL-2/IL-18 increased the NKG2D expression in the TGF-β treated NK cell line in a dose-dependent manner. Incubation with the JNK inhibitor SP600125 inhibited the NKG2D expression induced by IL-2/IL-18 in the TGF-β treated human NK cell line. Moreover, the NK cytotoxicity assay showed that the reduced NK cytotoxicity by TGF-β was recovered by IL-2/IL-18 treatment. The results indicate that IL-2/IL-18 strongly prevented the TGF-β-induced NKG2D down-regulation in NK cells via the JNK pathway. Taken together, the protected expression of NKG2D by IL-2/IL-18 provides insight into the mechanism of NKG2D regulation and it also supplied useful information for creating a novel therapeutic approach to treat TGF-β-secreting cancer cells.

Introduction

Natural killer (NK)² cells are populations of lymphocytes that can eliminate virus-infected and transformed cells [1], [2], [3], [4], [5], [6]. This elimination activity against tumors and virally infected cells is a process that utilizes a combination of signals from activating and inhibitory receptors. One of these activating receptors has recently been found to be NKG2D, which is expressed on most NK cells and CD8 T cells [7], [8]. NKG2D is a C-type lectin-like homodimeric surface receptor, and it can be signal-transduced through the transmembrane adapter protein DAP10 that contains an YXXM motif in the cytoplasmic domain, which ultimately transmits an activation signal [7]. Furthermore, in NK cells, NKG2D-mediated activation can overcome the inhibitory signal from self recognition [9], [10]. Studies have demonstrated that NKG2D receptor play a critical role in immune surveillance. However, despite the pivotal role of NKG2D receptor for immune cells, tumors still grow despite that NKG2D ligands are expressed on the tumor cell surface in many malignant tumors. This suggests that tumor cells have a mechanism to escape the NKG2D/NKG2D ligand system. Indeed, ligands of NKG2D receptor such as MHC class I chain-related molecules A (MICA) and MICB in humans and the Rae1 and H60 families in mice [11], [12], [13] are not constitutively expressed, but their expression is induced by stress or tumor formation [14], [15]. This ligand’s characteristic up-regulated in tumor cells suggest that the NKG2D/NKG2D ligand system may play a pivotal role in anti-tumor activity.

TGF-β inhibits the anti-tumor immune responses through an impairment of the functions of dendritic cells and by inhibiting the activation of immune cells. TGF-β may act directly as a tumor progression factor. Recently, TGF-β was reported to be a key molecule for regulating NKG2D-mediated immune escape of human tumor cells by down-modulating the NKG2D expression in immune effector cells [16], [17], [18]. The knockdown of TGF-β by siRNA technology blocked the malignancy and tumorigenicity of glioma cells [17]. However, the molecular mechanism of NKG2D down-regulation by TGF-β is unclear.

Cytokine combination treatment has played a pivotal role in the recent protocols of cancer immunotherapy. In particular, IL-18, which is an IFN-γ-inducing factor, enhances the T and NK cell immunoregulatory functions (cytokine production and cell proliferation) [19], [20], [21] and the expression of cytolytic weapons such as Fas ligand (FasL) and perforin [22], [23], [24]. The systemic administration of IL-18 has demonstrated considerable therapeutic activity in several murine tumor models [23], [25]. In addition, the IL-2/IL-18 combination has been reported to mediate the anti-tumor activities in mice by expanding the NK cell population [26]. Cytokine-activated NK cells have recently been shown to activate APCs (antigen presenting cells, especially DC (Dendritic cells)) and to induce their maturation and cytokine production. These activated DCs have been known to support the immune surveillance activity of NK cells. While cross-talk between APC and NK cells has been observed by many studies, the molecular mechanisms of this interaction are not fully understood [27], [28].

In this study, we investigated the ability of a cytokine treatment to regulate the NKG2D expression in NK cells. We show that IL-2/IL-18 can protect the TGF-β induced NKG2D down-modulation in NK cells via the JNK pathway.