Exosomes bearing HLA-G are released by melanoma cells

Abstract

Tumor cells release membrane vesicles, named exosomes, capable of specific cytotoxic T-lymphocyte activation by transferring tumor antigens to dendritic cells. By contrast, the nonclassical human leucocyte antigen (HLA)-G class I molecule displays immunotolerant properties and can be ectopically expressed by tumor cells, thereby allowing their escape from immunosurveillance. We describe here that a melanoma cell line, named Fon, established from an HLA-G–positive melanoma biopsy, spontaneously expressed high levels of the HLA-G1 membrane-bound isoform. Exosomes released by Fon cells were purified and analyzed both for their density on sucrose gradient and their protein composition by Western blotting and flow cytometry. Besides the expression of well-described proteins such as Lamp-2, notably, these melanoma-derived exosomes bore HLA-G1. In addition, exosomes harboring HLA-G1 were secreted by the HLA-G–negative M8 melanoma cells transfected with the HLA-G1 cDNA. Thus, the presence of tolerogenic HLA-G molecules on melanoma-derived exosomes may provide a novel way for tumors to modulate host's immune response.

Introduction

Exosomes are small membrane vesicles secreted by a multitude of cell types, including reticulocytes 1, 2, 3, platelets [4], mast cells [5], B and T lymphocytes 6, 7, 8, 9, and dendritic cells [10]. Tumor cells were also recently described as releasing exosomes 11, 12. These 60- to 90-nm vesicles originate from fusion of late multivesicular endosomes/lysosomes with the plasma membrane. Of particular interest is the recent finding that exosomes released by tumor cells contain and transfer tumor antigens to dendritic cells allowing cytotoxic T lymphocyte (CTL) cross-priming 11, 12. Thus, tumor-derived exosomes could be useful for antitumoral treatment.

Tumor cells have developed a number of strategies that enable them to escape from immune detection. One of them is the expression of inhibitory ligands such as the nonclassical human leucocyte antigen (HLA) class I molecule HLA-G that mediates a negative signal through interaction with inhibitory receptors present on immunocompetent cells [13]. HLA-G is normally absent on healthy tissues except for trophoblast [14] and thymus [15]. Interestingly, both its transcription and protein expression are upregulated on some tumors cells, as demonstrated in biopsies from patients with melanoma 16, 17, 18, breast cancer [19], renal carcinoma 20, 21, primary cutaneous lymphoma [22], lung cancer [23], glioma [24], epithelial cutaneous malignant lesions [25], and colorectal cancer [26]. In contrast to the data obtained with surgically removed tumor lesions, the detection of HLA-G on in vitro established tumor cell lines has been mainly unsuccessful 27, 28, 29, 30. This is probably because of the loss of HLA-G expression during long-term cell culture. Consequently, up to now, one renal carcinoma cell line [20] and four glioma cell lines [24] have been found to display low HLA-G–membrane-bound expression. Recently, short-term ovarian carcinoma cell lines were described as exhibiting constitutive HLA-G surface expression that was gradually lost upon long-term in vitro propagation [31].

HLA-G can be expressed as seven HLA-G protein isoforms, four of which are membrane-bound (HLA-G1, -G2, -G3, and -G4) and three being soluble (HLA-G5, -G6 and -G7) [32]. These HLA-G isoforms exhibit immunomodulatory properties such as inhibition of natural killer (NK) cytolysis and CTL responses 33, 34, 35, 36, 37. The full-length HLA-G1 isoform, which exhibits a structure similar to that of classical HLA class I molecules, interacts with at least three inhibitory receptors, immunoglobulin (Ig)-like transcript-2 (ILT-2 or CD85j) present on NK cells, T cells and antigen-presenting cells (APC) 38, 39, ILT-4 (CD85d) on APC [40], and p49/KIR2DL4 (CD158d) on NK cells 41, 42.

In the present report, we investigated whether HLA-G was present in exosomes of HLA-G–positive tumor cells, thus representing a potential additional pathway for these tumors to escape from immunosurveillance. We studied a melanoma cell line, named Fon, which was derived in vitro from an HLA-G–positive tumor lesion [43]. To our knowledge, this is the first description of a melanoma cell line expressing HLA-G1 at its cell surface and at a high level. Interestingly, this melanoma cell line secreted exosomes that also contained HLA-G1. The expression of HLA-G on both tumor cells and tumor-derived exosomes may have important insight in tumor progression and metastasis.