Membrane Fas ligand activates innate immunity and terminates ocular immune privilege.

It has been proposed that the constitutive expression of Fas ligand (FasL) in the eye maintains immune privilege, in part through inducing apoptosis of infiltrating Fas(+) T cells. However, the role of FasL in immune privilege remains controversial due to studies that indicate FasL is both pro- and anti-inflammatory. To elucidate the mechanism(s) by which FasL regulates immune privilege, we used an ocular tumor model and examined the individual roles of the membrane-bound and soluble form of FasL in regulating ocular inflammation. Following injection into the privileged eye, tumors expressing only soluble FasL failed to trigger inflammation and grew progressively. By contrast, tumors expressing only membrane FasL 1) initiated vigorous neutrophil-mediated inflammation, 2) terminated immune privilege, and 3) were completely rejected. Moreover, the rejection coincided with activation of both innate and adaptive immunity. Interestingly, a higher threshold level of membrane FasL on tumors is required to initiate inflammation within the immune privileged eye, as compared with nonprivileged sites. The higher threshold is due to the suppressive microenvironment found within aqueous humor that blocks membrane FasL activation of neutrophils. However, aqueous humor is unable to completely block the proinflammatory effects of tumor cells that express high levels of membrane FasL. In conclusion, our data indicate that the function of FasL on intraocular tumors is determined by the microenvironment in conjunction with the form and level of FasL expressed.

Pharmacological purging of syngeneic bone marrow ex vivo: effect of treatment with doxorubicin and lonidamine on normal and leukaemic cells of DBA/2 mice.

The in vivo effect of in vitro treatment with doxorubicin plus lonidamine on normal and leukaemic cells was investigated in a mouse model of syngeneic bone marrow transplantation. Different numbers of L5178Y tumour cells or normal bone marrow cells alone, or mixtures of bone marrow and leukaemic cells were incubated with doxorubicin (0.25, 0.5, 0.75, 1 microgram/ml) and/or lonidamine (50 micrograms/ml) and reinfused in DBA/2 mice. Lonidamine potentiated the cytotoxic effect of doxorubicin dependent on doxorubicin dosage and tumour cell concentration. Survival after injection of 10(4) in vitro-treated tumour cells was 42% for doxorubicin 0.75 micrograms/ml alone versus 100% for the combination with lonidamine and 50% for doxorubicin 1 microgram/ml alone versus 100% combination. Reinfusion of normal bone marrow incubated with doxorubicin alone or in combination with lonidamine in lethally irradiated mice did not occur in 12-14% of mice injected, indicating that the repopulating ability of stem cells was spared. These data suggest the potential usefulness of lonidamine in ex vivo purging of bone marrow before autologous bone marrow transplants in haemopoietic malignancies.

Cyclophosphamide (Cy)-facilitated adoptive immunotherapy of a Cy-resistant tumour. Evidence that Cy permits the expression of adoptive T-cell mediated immunity by removing suppressor T cells rather than by reducing tumour burden.

A cyclophosphamide (Cy)-resistant immunogenic tumour, the L5178Y lymphoma, was used to demonstrate that Cy-treatment of a host bearing this tumour enables passively transferred tumour-sensitized T cells to cause complete tumour regression without any need for Cy to cause a reduction in tumour burden. It was shown that whereas infusion of tumour-sensitized T cells from immune donors had very little effect on growth of the tumour, and whereas treatment with 150 mg/kg of Cy caused appreciable enhancement of tumour growth, combination therapy with Cy plus immune T cells caused complete tumour regression and resulted in long-term survival. Evidence that Cy treatment facilitated the expression of adoptive immunity against the L5178Y lymphoma by eliminating tumour-induced suppressor T cells consisted of the demonstration that tumour regression caused by combination treatment with Cy and immune T cells could be inhibited by infusing the recipient with Cy-sensitive, L3T4+ T cells from tumour-bearing but not from normal donors.

Changes in tumor cell adhesiveness affecting speed of dissemination and mode of metastatic growth.

Adhesion variants can be isolated from suspension growing highly metastatic murine ESb tumor cells under reproducible conditions from uncloned as well as from cloned ESb tumor cells. One such variant, ESb-MP, has been analyzed in detail. In vitro it had similar growth properties and high invasive capacity as the parental ESb cells. In vivo, ESb-MP cells showed a reduced growth capacity as compared to ESb cells. This was seen at the site of tumor cell transplantation (increased latency period) as well as at the site of secondary tumor growth in internal organs. ESb-MP tumor cells disseminated much later than ESb cells from the primary tumor into the blood stream. Both tumor lines metastasized to the liver but they affected liver functions in a different way: ESb cells infiltrated the liver diffusely and exerted toxic effects on liver parenchyma very quickly. This resulted in early increase of liver enzyme activity in the blood. In contrast, liver infiltrated by ESb-MP cells showed a more focal type of colonization and the organs seemed to be functioning for much longer periods. In fact, animals inoculated with ESb-MP cells subcutaneously or intravenously had an increased life expectancy compared to ESb-tumor-bearing animals of about 300%. The organotropism of both tumor lines remained similar although there were kinetic and quantitative differences, especially with regard to the kidney. In late stages of tumor growth, ESb-MP-tumor-bearing animals developed a high percentage of metastases in the kidney and around and within the spinal cord, thereby causing a syndrome of hind-leg paralysis. This syndrome was remarkable in its reproducibility, especially after intravenous tumor cell inoculation. The changed adhesiveness thus seemed to have affected the tumor latency period, the speed of dissemination into blood and internal organs, the mode of organ infiltration (focal vs. diffuse) and of metastatic growth, parameters which all might contribute to the greatly reduced overall malignancy.