The proto-oncogene Myc drives expression of the NK cell-activating NKp30 ligand B7-H6 in tumor cells.

Natural Killer (NK) cells are innate effector cells that are able to recognize and eliminate tumor cells through engagement of their surface receptors. NKp30 is a potent activating NK cell receptor that elicits efficient NK cell-mediated target cell killing. Recently, B7-H6 was identified as tumor cell surface expressed ligand for NKp30. Enhanced B7-H6 mRNA levels are frequently detected in tumor compared to healthy tissues. To gain insight in the regulation of expression of B7-H6 in tumors, we investigated transcriptional mechanisms driving B7-H6 expression by promoter analyses. Using luciferase reporter assays and chromatin immunoprecipitation we mapped a functional binding site for Myc, a proto-oncogene overexpressed in certain tumors, in the B7-H6 promoter. Pharmacological inhibition or siRNA/shRNA-mediated knock-down of c-Myc or N-Myc significantly decreased B7-H6 expression on a variety of tumor cells including melanoma, pancreatic carcinoma and neuroblastoma cell lines. In tumor cell lines from different origin and primary tumor tissues of hepatocellular carcinoma (HCC), lymphoma and neuroblastoma, mRNA levels of c-Myc positively correlated with B7-H6 expression. Most importantly, upon inhibition or knock-down of c-Myc in tumor cells impaired NKp30-mediated degranulation of NK cells was observed. Thus, our data imply that Myc driven tumors could be targets for cancer immunotherapy exploiting the NKp30/B7-H6 axis.

Metalloprotease-mediated tumor cell shedding of B7-H6, the ligand of the natural killer cell-activating receptor NKp30.

Natural killer (NK) cells are potent immune effector cells capable of mediating antitumor responses. Thus, during immunoediting, tumor cell populations evolve strategies to escape NK-cell-mediated recognition. In this study, we report a novel mechanism of immune escape involving tumor cell shedding of B7-H6, a ligand for the activating receptor NKp30 that mediates NK-cell binding and NK-cell-mediated killing. Tumor cells from different cancer entities released B7-H6 by ectodomain shedding mediated by the cell surface proteases "a disintegrin and metalloproteases" (ADAM)-10 and ADAM-17, as demonstrated through the use of pharmacologic inhibitors or siRNA-mediated gene attenuation. Inhibiting this proteolytic shedding process increased the levels of B7-H6 expressed on the surface of tumor cells, enhancing NKp30-mediated activation of NK cells. Notably, we documented elevated levels of soluble B7-H6 levels in blood sera obtained from a subset of patients with malignant melanoma, compared with healthy control individuals, along with evidence of elevated B7-H6 expression in melanoma specimens in situ. Taken together, our results illustrated a novel mechanism of immune escape in which tumor cells impede NK-mediated recognition by metalloprotease-mediated shedding of B7-H6. One implication of our findings is that therapeutic inhibition of specific metalloproteases may help support NK-cell-based cancer therapy.

CTLA-4 is expressed by activated mouse NK cells and inhibits NK Cell IFN-γ production in response to mature dendritic cells.

NK cells express an array of activating and inhibitory receptors that determine NK cell responses upon triggering by cognate ligands. Although activating NK cell receptors recognize mainly ligands expressed by stressed, virus-infected, or transformed cells, most inhibitory receptors engage MHC class I, preventing NK cell activation in response to healthy cells. In this study, we provide insight into the regulation and function of additional receptors involved in mouse NK cell responses: CTLA-4 and CD28. CTLA-4 and CD28 engage the same ligands, B7-1 and B7-2, which are primarily expressed by APCs, such as dendritic cells. Our data demonstrate that activation of mouse NK cells with IL-2 induces the expression of CTLA-4 and upregulates CD28. CTLA-4 expression in IL-2-expanded NK cells was further up- or downregulated by IL-12 or TGF-ß, respectively. Using gene-deficient NK cells, we show that CD28 induces, and CTLA-4 inhibits, IFN-γ release by NK cells upon engagement by the recombinant ligand, B7-1, or upon coculture with mature dendritic cells. Notably, we show that mouse NK cells infiltrating solid tumors express CD28 and CTLA-4 and respond to stimulation with recombinant B7-1, suggesting that the NK cell responses mediated by the CD28/CTLA-4:B7-1/B7-2 system could be of importance during malignant disease. Accordingly, our study might have implications for immunotherapy of cancer based on blocking anti-CTLA-4 mAbs.

Downregulation of the activating NKp30 ligand B7-H6 by HDAC inhibitors impairs tumor cell recognition by NK cells.

Natural killer (NK) cells are central effector cells during innate immune responses against cancer. Natural cytotoxicity receptors expressed by NK cells such as NKp30 are involved in the recognition of transformed cells. Recently, the novel B7 family member B7-H6, which is expressed on the cell surface of various tumor cells including hematological malignancies, was identified as an activating ligand for NKp30. To investigate expression and regulation of B7-H6, we generated monoclonal antibodies. Our study reveals that B7-H6 surface protein and messenger RNA (mRNA) expression in various tumor cell lines was downregulated upon treatment with pan- or class I histone deacetylase inhibitors (HDACi) as well as after small interfering RNA-mediated knockdown of the class I histone deacetylases (HDAC) 2 or 3. B7-H6 downregulation was associated with decreased B7-H6 reporter activity and reduced histone acetylation at the B7-H6 promoter. In certain primary lymphoma and hepatocellular carcinoma samples, B7-H6 mRNA levels were elevated and correlated with HDAC3 expression. Finally, downregulation of B7-H6 on tumor cells by HDACi reduced NKp30-dependent effector functions of NK cells. Thus, we identified a novel mechanism that governs B7-H6 expression in tumor cells that has implications for potential cancer treatments combining immunotherapy with HDACi.

Organ-specific cellular requirements for in vivo dendritic cell generation.

Bone marrow-derived dendritic cell (DC) precursors seed peripheral organs, where they encounter diverse cellular environments during their final differentiation into DCs. Flt3 ligand (Flt3-L) is critical for instructing DC generation throughout different organs. However, it remains unknown which cells produce Flt3-L and, importantly, which cellular source drives DC development in such a variety of organs. Using a novel BAC transgenic Flt3-L reporter mouse strain coexpressing enhanced GFP and luciferase, we show ubiquitous Flt3-L expression in organs and cell types. These results were further confirmed at the protein level. Although Flt3-L was produced by immune and nonimmune cells, the source required for development of the DC compartment clearly differed among organs. In lymphoid organs such as the spleen and bone marrow, Flt3-L production by hemopoietic cells was critical for generation of normal DC numbers. This was unexpected for the spleen because both immune and nonimmune cells equally contributed to the Flt3-L content in that organ. Thus, localized production rather than the total tissue content of Flt3-L in spleen dictated normal splenic DC development. No differences were observed in the number of DC precursors, suggesting that the immune source of Flt3-L promoted pre-cDC differentiation in spleen. In contrast, DC generation in the lung, kidney, and pancreas was mostly driven by nonhematopoietic cells producing Flt3-L, with little contribution by immune cells. These findings demonstrate a high degree of flexibility in Flt3-L-dependent DC generation to adapt this process to organ-specific cellular environments encountered by DC precursors during their final differentiation.

Human NK cells are alerted to induction of p53 in cancer cells by upregulation of the NKG2D ligands ULBP1 and ULBP2.

Natural killer (NK) cells are immune cells sensing and eliminating foreign, stressed, transformed, and senescent cells through specialized surface receptors, such as NKG2D, that interacts with several virus- or stress-inducible ligands, including ULBP1 and -2, which are expressed on target cell surfaces. For example, induction of DNA damage or cellular senescence pathways in tumor cells led to upregulation of NKG2D ligands that activate NK cells. Although, both pathways activate p53, the relationship of p53 activation to upregulation of NKG2D ligands has not been addressed. In this study, we report that induction of wild-type p53, but not mutant p53, strongly upregulated mRNA and cell surface expression of ULBP1 and -2, whereas expression of other NK cell ligands was not affected. We defined intronic p53-responsive elements in these two novel p53 target genes. Coculture of wild-type p53-induced human tumor cells with primary human NK cells enhanced NKG2D-dependent degranulation and IFN-γ production by NK cells. Accordingly, treatment of certain wild-type p53-expressing tumor cell lines with the p53-reactivating small molecular compound RITA resulted in upregulation of ULBP2 mRNA and cell surface protein expression. Taken together, our findings define the involvement of p53 in the regulation of specific NKG2D ligands that enhance NK cell-mediated target recognition. One implication of our work is that activating p53 after adoptive transfer of NK cells might constitute an effective combinatorial strategy of NK cell-based immunochemotherapy in cancers in which wild-type p53 function is preserved.

Mimitin - a novel cytokine-regulated mitochondrial protein.

BACKGROUND: The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin - a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners. RESULTS: By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18-24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway. CONCLUSION: Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S), and some changes of mimitin gene expression modulate activity of apoptotic caspases 3/7, suggesting that this protein may indirectly participate in apoptosis.