NKG2D is an activating receptor for NK, NKT, CD8+, and γδ+ T cells, whose aberrant loss in cancer is a key mechanism of immune evasion. Soluble NKG2D ligands and growth factors, such as TGFβ1 emanating from tumors, are mechanisms for down-regulating NKG2D expression. Cancers thereby impair the capacity of lymphocytes to recognize and destroy them. In this study, we show that exosomes derived from cancer cells express ligands for NKG2D and express TGFβ1, and we investigate the impact of such exosomes on CD8+ T and NK cell NKG2D expression and on NKG2D-dependent functions. Exosomes produced by various cancer cell lines in vitro, or isolated from pleural effusions of mesothelioma patients triggered down-regulation of surface NKG2D expression by NK cells and CD8+ T cells. This decrease was rapid, sustained, and resulted from direct interactions between exosomes and NK cells or CD8+ T cells. Other markers (CD4, CD8, CD56, CD16, CD94, or CD69) remained unchanged, indicating the selectivity and nonactivatory nature of the response. Exosomal NKG2D ligands were partially responsible for this effect, as down-modulation of NKG2D was slightly attenuated in the presence of MICA-specific Ab. In contrast, TGFβ1-neutralizing Ab strongly abrogated NKG2D down-modulation, suggesting exosomally expressed TGFβ as the principal mechanism. Lymphocyte effector function was impaired by pretreatment with tumor exosomes, as these cells exhibited poor NKG2D-dependent production of IFN-γ and poor NKG2D-dependent killing function. This hyporesponsiveness was evident even in the presence of IL-15, a strong inducer of NKG2D. Our data show that NKG2D is a likely physiological target for exosome-mediated immune evasion in cancer.

Exosomes are nanometer-sized vesicles, secreted by normal and neoplastic cells. The outcome following interaction between the cellular immune system and cancer-derived exosomes is not well understood. Interleukin-2 (IL-2) is a key factor supporting expansion and differentiation of CTL and natural killer (NK) cells but can also support regulatory T cells and their suppressive functions. Our study examined whether tumor-derived exosomes could modify lymphocyte IL-2 responses. Proliferation of healthy donor peripheral blood lymphocytes in response to IL-2 was inhibited by tumor exosomes. In unfractionated lymphocytes, this effect was seen in all cell subsets. Separating CD4 + T cells, CD8 + T cells, and NK cells revealed that CD8 + T-cell proliferation was not inhibited in the absence of CD4 + T cells and that NK cell proliferation was only slightly impaired. Other exosome effects included selective impairment of IL-2-mediated CD25 up-regulation, affecting all but the CD3 + CD8 À T-cell subset. IL-2-induced Foxp3 expression by CD4 + CD25 + cells was not inhibited by tumor exosomes, and the suppressive function of CD4 + CD25 + T cells was enhanced by exosomes. In contrast, exosomes directly inhibited NK cell killing function in a T-cell-independent manner. Analysis of tumor exosomes revealed membrane-associated transforming growth factor B 1 (TGFB 1 ), which contributed to the antiproliferative effects, shown by using neutralizing TGFB 1 -specific antibody. The data show an exosome-mediated mechanism of skewing IL-2 responsiveness in favor of regulatory T cells and away from cytotoxic cells. This coordinated ''double hit'' to cellular immunity strongly implicates the role of exosomes in tumor immune evasion. [Cancer Res 2007;67(15):7458-66]

No abstract