Engagement of the OX-40 receptor in vivo enhances antitumor immunity.

The OX-40 receptor (OX-40R), a member of the TNFR family, is primarily expressed on activated CD4+ T lymphocytes. Engagement of the OX-40R, with either OX-40 ligand (OX-40L) or an Ab agonist, delivers a strong costimulatory signal to effector T cells. OX-40R+ T cells isolated from inflammatory lesions in the CNS of animals with experimental autoimmune encephalomyelitis are the cells that respond to autoantigen (myelin basic protein) in vivo. We identified OX-40R+ T cells within primary tumors and tumor-invaded lymph nodes of patients with cancer and hypothesized that they are the tumor-Ag-specific T cells. Therefore, we investigated whether engagement of the OX-40R in vivo during tumor priming would enhance a tumor-specific T cell response. Injection of OX-40L:Ig or anti-OX-40R in vivo during tumor priming resulted in a significant improvement in the percentage of tumor-free survivors (20-55%) in four different murine tumors derived from four separate tissues. This anti-OX-40R effect was dose dependent and accentuated tumor-specific T cell memory. The data suggest that engagement of the OX-40R in vivo augments tumor-specific priming by stimulating/expanding the natural repertoire of the host's tumor-specific CD4+ T cells. The identification of OX-40R+ T cells clustered around human tumor cells in vivo suggests that engagement of the OX-40R may be a practical approach for expanding tumor-reactive T cells and thereby a method to improve tumor immunotherapy in patients with cancer.

Met-HGF/SF: tumorigenesis, invasion and metastasis.

Hepatocyte growth factor/scatter factor (HGF/SF) is synthesized by mesenchymal cells and is a paracrine effector of cells, predominantly epithelial, that express the Met tyrosine kinase receptor. We have demonstrated that autocrine Met-HGF/SF expression in mouse fibroblasts results in transformation and tumorigenesis. HGF/SF-treated cells expressing Met can respond in a variety of ways: mitogenically, by scattering (motility), and by forming branching tubules in gel matrices (branching morphogenesis). HGF/SF also induces in vitro invasiveness and is angiogenic in in vivo assays. A human cell line and several mouse cell lines that we have constructed to express Met-HGF/SF in an autocrine fashion are tumorigenic, invasive and metastatic in athymic nude mice. Thus, the very complex process of invasion and metastasis can be mediated by a ligand-receptor signalling pathway, and the cell lines we have developed provide important model systems for identifying the signalling molecules that mediate these phenotypes: For example Met-HGF/SF signalling activates the urokinase plasminogen proteolysis network, thus coupling this signal transduction pathway to the proteases that mediate dissolution of the extracellular matrix. Branching morphogenesis, mediated by Met-HGF/SF signalling, is dependent on this process, as well as the formation of cell-cell junctions and interaction with the extracellular matrix. We have proposed a hypothesis for the role of Met and downstream signalling molecules in generating normal ducts and lumenal structures, as well as a model for how interruption of this signalling leads to abnormal malignant progression. Is Met involved in human cancer? Human sarcomas often inappropriately express Met, suggesting that it is an important oncogene in these cancers, and an increasing number of reports have implicated Met-HGF/SF signalling in a variety of human cancers.

Pharmacokinetics of bestatin and oral activity for treatment of experimental metastases.

Bestatin is a low molecular weight aminopeptidase inhibitor originally isolated from culture filtrates of Streptomyces olivoreticuli. The serum pharmacokinetics in mice are dependent on route of administration, with a short t1/2 (1.69 min t1/2 alpha and 12.8 min t1/2 beta), but a high initial serum level following i.v. administration. When administered via the i.p., s.c., i.m., or p.o. routes of administration, bestatin had serum t1/2 beta s of 8.56, 16.91, 19.25, or 15.4 min, respectively. The maximum area under the curve (concentration X time) occurred following i.v. and i.m. administration, with a lower level following p.o. or i.p. administration. Bestatin had therapeutic activity for experimental metastases, not only following i.v., i.p., and i.m. routes of administration but also following oral administration. Because of its brief serum t1/2, bestatin's therapeutic activity depends on aggressive (either daily or twice daily injection, especially following p.o. administration) and high-dose administration. Thus, the rate-limiting aspect of bestatin's therapeutic activity appears to be associated with its pharmacokinetics.

Correlation between H-ras p21TLeu61 protein content and tumorigenicity of NIH3T3 cells.

We have prepared a number of NIH3T3 clonal cell lines that contain an H-ras transforming gene with an A----T transversion at the 61st codon. The clonal lines contain 1 to 3 cell equivalents of the transforming oncogene and some lines look more morphologically transformed than others. Using Y13-238, a rat monoclonal antibody that recognizes H-ras p21 but not Ki- or N-ras in rodent cells, we found that the degree of morphological change is correlated with the relative amount of transforming protein in the selected clonal lines. Nude mice were injected with cells from lines containing different amounts of the transforming protein, ranging from approximately 1 to 10 times the level of normal H-ras protein present in NIH3T3 cells. Tumors arose in all mice that received cells containing the transforming protein. Their time of appearance (tumor latency) was correlated with the number of cells injected and the amount of transforming protein present in each clonal line; however, the subsequent rate of growth and ultimate size of the tumors were similar. Thus, it appears that the transforming protein has a significant effect on some early step in tumor development. Our results also show that relatively low amounts of transforming ras protein are sufficient to cause tumorigenicity in NIH3T3 cells and that higher amounts of the transforming protein cause proportionately faster responses.

Halothane anesthesia decreases human monocyte hydrogen peroxide generation. Protection of monocytes by activation with gamma interferon.

In an effort to determine the impact of halothane anesthesia on certain human cell-mediated immune functions, normal, purified human monocytes and lymphocytes were exposed to halothane in vitro at varying concentrations for up to 8 hours. Subsequently, these human effector cells were analyzed for their ability to function in several cell-mediated immunologic assays. Natural killer cell activity against K-562 was unaffected by halothane in most of the donors tested. Similarly, the ability of purified monocytes to inhibit MBL-2 tumor cell growth was unchanged. Halothane appeared to decrease the proliferative response of lymphocytes to phytohemagglutinin (PHA) in approximately 50% of the normal donors tested. In contrast, the ability of monocytes to lyse antibody-coated red cell targets (ADCC) was unaffected by even maximal exposure to halothane. Of interest was the finding that human monocytes exposed to as low as 2% halothane anesthesia for 4 hours displayed a dramatic down-regulation of hydrogen peroxide (H2O2) release. Since it is known that hydrogen peroxide and other incompletely reduced forms of oxygen secreted by monocytes can play a major role in the antimicrobial, antitumor, and inflammatory functions of these cells, this finding may help explain the enhanced susceptibility of post-operative patients to infections.

The effect of anesthetic agents on human immune system function. I. Design of a system to deliver inhalational anesthetic agents to leukocyte cultures in vitro.

A novel system for exposing purified human mononuclear leukocyte subsets or any cultured cell to inhalational anesthetic agents has been devised. Monocytes and lymphocytes are purified by counter-current centrifugal elutriation and put into culture vessels with and without appropriate functional activators. The culture vessels are placed into one of four anesthetic agent exposure chambers, each containing a different concentration of the anesthetic agent to be tested. The system that delivers the anesthetic agent to the culture vessels is essentially the same as the one used in the operating room; the actual levels of anesthetic agent delivered are monitored. In this report, we present evidence that halothane can be successfully tested using the above-cited experimental design; this agent substantially inhibits the secretion of interferon by human mononuclear cells. The ability of monocytes to secrete alpha interferon in response to polyinosinic: polycytidylic acid (poly I:C) was significantly depressed following 4 h in vitro exposure to increasing concentrations of halothane; the secretion of gamma interferon by lymphocytes in response to phytohemagglutinin (PHA) was also depressed, although to a lesser degree. The system presented should allow for the in vitro exploration of the effects of inhalational agents on purified human leukocyte subset functions as well as for the analysis of the effect of these agents on monocyte/lymphocyte interactions.