The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) with the corresponding natural killer group 2, member D (NKG2D) receptor triggers cytotoxic effector activity of natural killer cells and certain T-cell subsets and provides a costimulatory signal for cytokine production. Thus, the presence of MICA=B on transformed cells contributes to tumor immunosurveillance. Consequently, the proteolytic cleavage of MICA=B is regarded as an important immune escape mechanism of various cancer cells. To investigate the molecular machinery responsible for the shedding of endogenous MICA=B, we analyzed different human tumor entities including mammary, pancreatic and prostate carcinomas. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) revealed that all tested tumor cells constitutively expressed MICA and MICB on the cell surface and also released NKG2D ligands into the supernatant. We demonstrate that the "a disintegrin and metalloproteases" (ADAMs) 10 and 17 are largely responsible for the generation of soluble MICA=B. Pharmacological inhibition of metalloproteases reduced the level of released MICA=B and increased cell surface expression. Studies using RNA interference not only revealed a prominent role of ADAM10 and ADAM17 in NKG2D ligand shedding but also a tumor cell-specific role of ADAM10 and=or ADAM17 in shedding of MICA or MICB. Moreover, we report that in the prostate carcinoma cell line PC-3, MICA was not shed at all but rather was secreted in exosomes. These data indicate that the release of NKG2D ligands from individual tumor entities is by far more complex than suggested in previously reported MICA=B transfection systems.The activating natural killer group 2, member D (NKG2D) receptor is expressed on NK cells, NKT cells, gd T cells, CD8 1 T cells and a minor immunoregulatory subset of CD4 1 T cells. 1,2 The ligation of NKG2D costimulates T cells but also directly triggers cell-mediated cytotoxicity and cytokine release in NK cells. [3][4][5][6] Thus, despite some exceptions including gastrointestinal epithelium 7 and placenta, 8 the ligands for NKG2D are not expressed on healthy tissues to avoid inadvertent damage. However, NKG2D ligand (NKG2DL) expression can be induced in response to a variety of stimuli related to cellular stress such as heat shock, viral infection, DNA damage, oxidative stress and certain proinflammatory signals. 9,10 Of note, many tumors and precancerous lesions express NKG2D ligands presumably as a bystander effect of the oncogenic process itself. 11 As NKG2DL expression renders tumors susceptible to NKG2D 1 lymphocytes, the NKG2D system plays a pivotal role in immunosurveillance. 12 On the other hand, the sustained exposure to tumor cell-bound NKG2D ligands might also result in surface modulation and functional impairment of the NKG2D receptor and induce cross-tolerance of additional NK cell activation pathways. 13,14 A hallmark of the NKG2D system is the multitude of ligands identified so far. In humans, these include the MHC class I-related chain ...

The activating natural killer group 2 member D (NKG2D) receptor is expressed on NK cells, cytotoxic T cells and additional T cell subsets. Ligands for human NKG2D comprise two groups of MHC class I-related molecules, the MHC class I chain-related proteins A and B (MICA/B) and 6 UL16-binding proteins (ULBP1-6). While NKG2D ligands are absent from most normal cells, expression is induced upon stress and malignant transformation. In fact, most solid tumours and leukaemia/lymphomas constitutively express at least one NKG2D ligand and thereby are susceptible to NKG2D-dependent immunosurveillance. However, soluble NKG2D ligands are released from tumour cells and can down-modulate NKG2D activation as a means of tumour immune escape. In some tumour entities, levels of soluble NKG2D ligands in the serum correlate with tumour progression. NKG2D ligands can be proteolytically shed from the cell surface or liberated from the membrane by phospholipase C in the case of glycosylphosphatidylinositol (GPI)-anchored molecules. Moreover, NKG2D ligands can be secreted in exosomal microvesicles together with other tumour-derived molecules. Depending on the specific tumour/immune cell setting, these various forms of soluble and/or exosome-bound NKG2D ligands can exert multiple effects on NKG2D/NKG2D ligand interactions. In this review, we focus on the role of various proteases in the shedding of human NKG2D ligands from tumour cells and discuss the not completely unanimous reported functional implications of soluble and exosome-secreted NKG2D ligands for immunosurveillance.

No abstract

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) and UL-16 binding protein (ULBP) family members expressed on tumor cells with the corresponding NKG2D receptor triggers cytotoxic effector functions in NK cells and γδ T cells. However, as a mechanism of tumor immune escape, NKG2D ligands (NKG2DLs) can be released from the cell surface. In this study, we investigated the NKG2DL system in different human glioblastoma (GBM) cell lines, the most lethal brain tumor in adults. Flow cytometric analysis and ELISA revealed that despite the expression of various NKG2DLs only ULBP2 is released as a soluble protein via the proteolytic activity of “a disintegrin and metalloproteases” (ADAM) 10 and 17. Moreover, we report that temozolomide (TMZ), a chemotherapeutic agent in clinical use for the treatment of GBM, increases the cell surface expression of NKG2DLs and sensitizes GBM cells to γδ T cell-mediated lysis. Both NKG2D and the T-cell receptor (TCR) are involved. The cytotoxic activity of γδ T cells toward GBM cells is strongly enhanced in a TCR-dependent manner by stimulation with pyrophosphate antigens. These data clearly demonstrate the complexity of mechanisms regulating NKG2DL expression in GBM cells and further show that treatment with TMZ can increase the immunogenicity of GBM. Thus, TMZ might enhance the potential of the adoptive transfer of ex vivo expanded γδ T cells for the treatment of malignant glioblastoma.

Despite aggressive treatment regimens based on surgery and radiochemotherapy, the prognosis of patients with grade IV glioblastoma multiforme (GBM) remains extremely poor, calling for alternative options such as immunotherapy. Immunological mechanisms including the Natural Killer Group 2 member D (NKG2D) receptor-ligand system play an important role in tumor immune surveillance and targeting the NKG2D system might be beneficial. However, before considering any kind of immunotherapy, a precise characterization of the immune system is important, particularly in GBM patients where conventional therapies with impact on the immune system are frequently co-administered. Here we performed an in-depth immunophenotyping of GBM patients and age-matched healthy controls and analyzed NKG2D ligand expression on primary GBM cells . We report that GBM patients have a compromised innate immune system irrespective of steroid (dexamethasone) medication. However, dexamethasone drastically reduced the number of immune cells in the blood of GBM patients. Moreover, higher counts of immune cells influenced by dexamethasone like CD45 lymphocytes and non-Vδ2 γδ T cells were associated with better overall survival. Higher levels of NKG2D ligands on primary GBM tumor cells were observed in patients who received radiochemotherapy, pointing towards increased immunogenic potential of GBM cells following standard radiochemotherapy. This study sheds light on how steroids and radiochemotherapy affect immune cell parameters of GBM patients, a pre-requisite for the development of new therapeutic strategies targeting the immune system in these patients.