NKG2D ligands (NKG2DLs) mark malignant cells for recognition by natural killer (NK) cells and cytotoxic T lymphocytes via the activating immunoreceptor NKG2D. This led to the hypothesis that NKG2DLs play a critical role in tumor immune surveillance. The human NKG2DLs MICA and MICB are expressed on tumors of epithelial origin in vivo. For the other recently described set of human NKG2DLs, the UL16-binding proteins (ULBPs), expression in vivo is as yet undefined. In this study we investigated expression and function of NKG2DLs in leukemia using a panel of newly generated NKG2DL-specific monoclonal antibodies. We report that leukemia cells from patients variously express MIC and ULBP molecules on the cell surface with MICA most frequently detected. Patient leukemia cells expressing MICA were lysed by NK cells in an NKG2D-dependent fashion. Sera of patients, but not of healthy donors, contained elevated levels of soluble MICA (sMICA). We also detected increased sMICB levels in patient sera using a newly established MICB-specific enzyme-linked immunosorbent assay. Reduction of leukemia MIC surface expression by shedding may impair NKG2D-mediated immune surveillance of leukemias. In addition, determination of sMICA and sMICB levels may be implemented as a prognostic parameter in patients with hematopoietic malignancies.

The immunoreceptor NKG2D promotes immunosurveillance of malignant cells and protects the host from tumor initiation by activating natural killer cells and costimulating CD8 T cells. NKG2D-mediated recognition of malignant cells by cytotoxic lymphocytes is enabled through the tumor-associated expression of NKG2D ligands (NKG2DL) resulting from cellular or genotoxic stress. Shedding of NKG2DL is thought to constitute a major countermechanism of tumor cells to subvert NKG2D-mediated immunosurveillance. Here, we report that the prototypical NKG2DL MICA is released by proteolytic cleavage in the stalk of the MICA ectodomain, where deletions, but not alanine substitutions, impede MICA shedding. Small compound-mediated stimulation and inhibition of MICA shedding adduced characteristics that indicated an involvement of members of the "a disintegrin and metalloproteinase" (ADAM) family. Accordingly, MICA shedding by tumor cells was inhibited by silencing of the related ADAM10 and ADAM17 proteases, which are known to promote tumor growth by releasing epidermal growth factor receptor ligands. Collectively, our data show that ADAM10 and ADAM17 are critically involved in the tumor-associated proteolytic release of soluble MICA facilitating tumor immune escape. Hence, therapeutic blockade of ADAM10 and ADAM17 seems promising for cancer treatment by targeting both growth and immune escape of tumors.

No abstract

BackgroundHuman multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.ResultsAfter transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.ConclusionPhysiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

IntroductionMultipotent human mesenchymal stromal cells (MSCs) are plasticadherent cells with triangular and fibroblastoid morphologic features, which have the capacity to differentiate into osteoblasts, adipocytes, and chondrocytes. Furthermore, they express the cell-surface markers CD73, CD90, and CD105, while being negative for CD34, CD45, and human leukocyte antigen (HLA-DR). 1 Various tissues including bone marrow, fat, umbilical cord blood, or fetal tissues have been used as sources for MSCs. 2 Not only are MSCs immunoprivileged because they are poorly lysed by T cells, but they also have strong immunosuppressive effects. [3][4][5][6] Therefore, MSCs are of clinical interest for both regenerative medicine and immune suppression. MSCs have been used successfully for treatment of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. 7 In addition, data from mouse models for rheumatoid arthritis or multiple sclerosis suggest that MSCs may be effective in the treatment of autoimmune diseases. 8 In vitro studies have analyzed the immunomodulatory effects of MSCs in detail and showed that MSCs inhibit the proliferation of T cells independent of the T-cell stimulus (ie, the suppression was observed when T cells were activated by alloantigens, mitogens, or through T-cell receptor engagement by anti-CD3 in combination with anti-CD28 antibodies). [4][5][6] Moreover, the inhibition is MHC independent. 9,10 MSCs do not only inhibit T-cell proliferation directly, but also impair dendritic cell function (ie, MSCs interfere with dentritic cell [DC] differentiation, maturation, and activation), which is shown by compromised antigen presentation and altered cytokine release. [11][12][13][14] MSCs also suppress the proliferation of NK cells and modulate the functions of B cells. 2 Most studies agree in that no cell-cell contact is required for the MSC-mediated immunosuppressive effects. 15 Instead, soluble molecules are thought to mediate this inhibition. Possible candidate molecules include transforming growth factor-␤ (TGF␤), hepatocyte growth factor, 5 indoleamine-2,3-dioxygenase (IDO), 16 prostaglandin E2 (PGE2), 11,17,18 insulin-like growth factorbinding proteins, 19 heme oxygenase-1 (HO), 20 and HLA-G5. 21 Moreover, the mechanism of the suppressive effects may be species dependent, as some molecules were described to be involved in suppressive effects of mouse MSCs such as nitric oxide (NO). 22 Another novel candidate molecule recently described to be involved in suppressive effects of mouse MSCs is the MSC-derived CC chemokine ligand CCL2; the proteolytically processed form of CCL2 modulates immunoglobulin production by plasma cells and is involved in the beneficial effects of MSCs in experimental autoimmune encephalomyelitis. 23,24 However, blocking any single one of these molecules did not completely abrogate the immuno suppressive functions of MSCs, indicating that several signaling pathways are involved and some important mediators may have not been identified yet. Several a...