The increased IL-17-producing γδT cells promote tumor cell proliferation and migration in neuroblastoma

Abstract

Neuroblastoma (NB) is the most common solid extracranial malignancy in children with a considerable chance of metastatic progression. Prevalent evidence supports the anti-tumor role of γδT cells and these cells have been testing in clinical trials for constraining tumor growth. A small subpopulation of γδT cells releasing IL-17, however, were demonstrated to exert tumor-promoting effects in many aspects. In this study, we found an augment of IL-17⁺ γδT cells both in in vitro PAM-stimulated γδT-cell expanding culture and circulating γδT cells in NB patients. These patient-origin cells expanded in vitro by PAM in the presence of IL-17 polarizing condition were shown to promote the proliferation and migration of NB cells. Furthermore, an intrinsic preference for IL-17 polarization in NB γδT cells was revealed by mRNA microarray and Western Blot, which pointed to an up-regulated expression of multiple Th17-development related genes in addition to an increased phosphorylation level of STAT3.

Introduction

Neuroblastoma (NB) is the most common extra-cranial solid pediatric tumor [1]. Surgical removal and chemotherapy are main therapies for NB. However, high recurrence/metastasis rates discourage the treatment efficacy [2]. Currently, multiple immunotherapy strategies, including adoptive immune cell transfer, as well as GD2 monoclonal antibody, have been testing for NB treatment [3,4]. Increasing evidence suggests that γδT-cell, a distinct and conserved population of lymphocytes with strong tumoricidal activity, could be a possible candidate for NB immunotherapy [5].

γδT cells have been illustrated to shape immune responses in inflammatory diseases and cancers [6]. Producing multiple cytokines (including IFN-γ and IL-17) upon pathogenic stimulation, γδT cells are able to exert direct cytotoxicity as innate cells and to activate adaptive immunity. It was demonstrated that γδT cells could induce cytotoxic responses against various tumors in vitro, including NB [[7], [8], [9]]. Clinical application of γδT cells for tumors treatment were also explored in several studies. In human peripheral blood, Vγ9Vδ2T cells are characterized with Th1 signature and 50–80% of them produce IFN-γ (Tγδ1 cells) responsible for their anti-infection and anti-tumor immunity. γδT cells exhibit functional plasticity and heterogeneity with regard to the cytokine production and < 5% of peripheral Vγ9Vδ2T cells producing IL-17 (IL-17-producing γδT cells or Tγδ17 cells) [10]. Tγδ17 cells have been revealed to be important in the pathogenesis of autoimmune diseases, allergy, and cancers [11,12]. Particularly, Tγδ17 cells were demonstrated to promote multiple tumor development and progression [13]. In human colorectal carcinoma, Tγδ17 cells were positively associated with tumor-stage advancing, tumor size, tumor invasion and metastasis [14]. It was evidenced that tumor-infiltrating Tγδ17 cells were the main producers of IL-17 in various tissues and closely implicated in cancer progression and metastasis [[15], [16], [17], [18]]. The pro-tumor effect conferred by Tγδ17 cells was likewise found to be true in animal models [[19], [20], [21]].

Our previous study has shown an elevated percentage of circulating γδT cells in NB patients [22] and these γδT cells exhibited a reduced IFN-γ production and cytolytic ability against NB cell lines, probably due to the reduction of DNAM-1 expression [23]. In the present study, the ratio of IL-17-producing γδT cells was similarly found to increase in the peripheral blood of NB patients. In light of this finding, we were interested in knowing the characteristics of these cells in NB. In an in vitro culture system with IL-17 polarizing condition and pamidronate (PAM) stimulation, we successfully expanded the γδT cells and induced IL-17-producing γδT-cells. Furthermore, the condition medium from IL-17-producing γδT-cell culture was identified to exert significant promotive effects on proliferation and migration of SH-SY5Y and SK-N-BE2 cells. The tumor-promoting effects of condition medium were partially due to IL-17A secreted by these γδT cells. We also verified the increased expression in genes and promoted signaling pathway related to IL-17 production using microarray analysis and Western Blot. Results from our study indicate that IL-17-producing γδT cells are likely to produce some specific factors, mainly IL-17A, thus exerting tumor-promoting effects on NB.