The immunoreceptor NKG2D activates natural killer cells and costimulates CD8 T cells. The MHC class I-related MICA molecules are ligands of NKG2D and are expressed on malignant, but not on normal, cells. As NKG2D plays an important role in the immunosurveillance of tumors, studies suggest that release of MICA from cancer cells constitutes an immune escape mechanism that systemically impairs antitumor immunity. Here, we investigated the potential of soluble MICA (sMICA) as a marker in cancer. Analysis of sMICA in sera of 512 individuals revealed significantly ( p < 0.0001) higher levels in patients with various malignancies (n 5 296, median 161 pg/ml) than in healthy individuals (n 5 62, median <30 pg/ml). Patients with benign diseases (n 5 154, median 84 pg/ml) exhibited intermediate sMICA levels. In cancer patients, elevated sMICA levels correlated significantly with cancer stage and metastasis ( p 5 0.015 and p 5 0.007, respectively). While release of MICA is thought to impair tumor immunity, determination of sMICA levels may provide useful additional information in the diagnosis and staging of cancer. ' 2005 Wiley-Liss, Inc.

Glucocorticoid-induced TNF-related protein (GITR) has been shown to stimulate T cell-mediated antitumor immunity in mice. However, the functional relevance of GITR and its ligand (GITRL) for non-T cells has yet to be fully explored. In addition, recent evidence suggests that GITR plays different roles in mice and humans. We studied the role of GITR-GITRL interaction in human tumor immunology and report for the first time that primary gastrointestinal cancers and tumor cell lines of different histological origin express substantial levels of GITRL. Signaling through GITRL down-regulated the expression of the immunostimulatory molecules CD40 and CD54 and the adhesion molecule EpCAM, and induced production of the immunosuppressive cytokine TGF-beta by tumor cells. On NK cells, GITR is constitutively expressed and up-regulated following activation. Blocking GITR-GITRL interaction in cocultures of tumor cells and NK cells substantially increased cytotoxicity and IFN-gamma production of NK cells demonstrating that constitutive expression of GITRL by tumor cells diminishes NK cell antitumor immunity. GITRL-Ig fusion protein or cell surface-expressed GITRL did not induce apoptosis in NK cells, but diminished nuclear localized c-Rel and RelB, indicating that GITR might negatively modulate NK cell NF-kappaB activity. Taken together, our data indicate that tumor-expressed GITRL mediates immunosubversion in humans.

Expression of ligands of the immunoreceptor NKG2D such as MICA and MICB has been proposed to play an important role in the immunosurveillance of tumors. Proteolytic shedding of NKG2D ligands from cancer cells therefore constitutes an immune escape mechanism impairing anti-tumor reactivity by NKG2D-bearing cytotoxic lymphocytes. Serum levels of sMICA have been shown to be of diagnostic significance in malignant diseases of various origins. Here, we investigated the potential of soluble MICB, the sister molecule of MICA, as a marker in cancer and its correlation with soluble MICA. Analysis of MICB in sera of 512 individuals revealed slightly higher MICB levels in patients with various malignancies (N = 296; 95th percentile 216 pg/ml; P = 0.069) than in healthy individuals (N = 62; 95th percentile 51 pg/ml). Patients with benign diseases (N = 154; 95th percentile 198 pg/ml) exhibited intermediate MICB levels. In cancer patients, elevated MICB levels correlated significantly with cancer stage and metastasis (P = 0.007 and 0.007, respectively). Between MICB and MICA levels, only a weak correlation was found (r = 0.24). Combination of both markers resulted only in a slightly higher diagnostic power in the high specificity range. The reduction of MICA and MICB surface expression on cells by shedding and the effects of sMICA and sMICB in serum on host lymphocyte NKG2D expression might play a role in late stages of tumor progression by overcoming the confining effect of NK cells and CD8 T cells. While MICB levels are not suited for the diagnosis of cancer in early stages, they may provide additional information for the staging of cancer disease.

The human NKG2D ligands (NKG2DL) MICA and MICB have been shown to be expressed on tumors of epithelial and hematopoietic origin in vivo. Recently we reported that MICA is shed from the cell surface of tumor cells and is present in sera of tumor patients (J Immunol169:4098 (2002), Blood102:1389 (2003)). Since the strength of an anti-tumor response by NK cells and CD8 T cells is critically depending on NKG2DL expression levels, down-regulation of MICA-expression on tumor cells represents an immune escape mechanism that diminishes anti-tumor reactivity of NKG2D-bearing lymphocytes. However, no data are yet available regarding the correlation of soluble MICA (sMICA) levels with specific tumor entities, aggressiveness of the disease, and hence the potential implementation of sMICA as novel marker in differential diagnosis and prognosis of cancer. In this study, we determined sMICA levels in sera of 512 individuals including 296 patients with various cancers, 154 patients with benign disorders and 62 healthy individuals. Healthy individuals revealed significantly lower sMICA values (median<30pg/mL) than patients with benign diseases (84pg/mL; p=0.005) and cancer patients (161pg/mL; p<0.0001). In addition, sMICA levels differed significantly between cancer patients and patients with benign disorders (p<0.0001) that represent the most relevant control group for differential diagnosis. In cancer patients, while there was no association between sMICA levels and tumor size (p=0.456), cell differentiation (p=0.271), or lymph node involvement (p=0.674), sMICA correlated significantly with cancer stage and metastasis (p=0.015 and p=0.007, respectively). Our data indicate that release of MICA might play a role in late stages of tumor progression by overcoming the confining effect of NK cells and CD8 T cells. Thus, determination of sMICA levels provides valuable information for cancer staging, and sMICA in serum seems to be an indicator for systemic manifestation of malignancy rather than for local tumor extent.

Members of the TNF superfamily mediate multiple cellular functions, including cellular proliferation, differentiation and cell death. Many members of this protein family are shed from the cell surface as soluble forms, which affects cell-cell interactions by reduction of ligand densities and distally modulates effector cells bearing the respective receptor. The TNF family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and NFkappaB and abrogates suppression of murine but not human regulatory T cells. Its cognate ligand GITRL has been found in various healthy tissues. Recently we reported that NK cells express GITR, while tumor cells express GITR ligand (GITRL), and GITR/GITRL interaction downregulates NK cell-mediated anti-tumor effector mechanisms like cytotoxicity and IFN-gamma production. Here we report that human tumor cells spontaneously release a soluble form of GITRL (sGITRL) detectable in culture supernatants by ELISA. Furthermore, we found elevated levels of sGITRL in sera of patients with various malignancies compared to healthy controls. We demonstrate that the release of GITRL from tumor cells can be blocked by inhibition of metalloproteinases, concomitantly causing accumulation of GITRL on the tumor cell surface as determined by FACS analysis. Upregulated GITRL surface expression further increased inhibition of NK cell anti-tumor effector mechanisms, while, in contrast, presence of sGITRL in cocultures of GITRL-expressing tumor cells and GITR-positive NK cells enhanced NK cell cytotoxicity and IFN-gamma production. Thus, in line with the results obtained with other TNF family members, conversion of membrane bound GITRL to its soluble form is associated with downregulation of its function, potentially due to blocking its cognate receptor. Thus, release of sGITRL substantially influences the interaction of tumor cells with NK cells. In addition, determination of sGITRL levels may be implemented as a diagnostic marker in patients with malignancies. Further prospective studies are currently being conducted addressing the value of GITRL as a tumor marker in different tumor entities.