Natural cell-mediated immunity in the rabbit.

Susceptibility and resistance to tumors represent the interplay of many factors. One factor felt to govern the development of tumors is natural killer and natural cytotoxic cellular activity. The constitutional resistance of rabbits to spontaneous tumor development raises questions regarding the activity of natural cell-mediated immunity in this species. We therefore examined the ability of rabbit spleen, lymph node, and peripheral blood lymphocytes to mediate natural killer cell (NK) and natural cytotoxic cell (NC) activity in vitro. Using classical approaches to the study of NK and NC activity, we found no evidence of these activities in leporine spleen, lymph node, and peripheral blood lymphocytes. Preincubation of these cells with IL-2 did not induce such activity. Antibody-dependent cell-mediated cytotoxic reactivity (ADCC), which is believed to be mediated by NK cells, was also undetectable in rabbit lymphocytes. As controls, lymphocytes from other species were capable of mediating NK, NC, and ADCC functions normally in these experiments. Finally, we were unable to identify a population of large granular lymphocytes, the cells believed to mediate NK activity in other animals. Therefore, we could not demonstrate in the rabbit either natural cell-mediated immunity or the population of cells usually associated with natural cell-mediated immunity. If such activity exists in rabbits, it is different from that seen in other animals. More likely, the basis for the natural resistance of rabbits to tumor development must be sought elsewhere.

Determinants of the ability of malignant fibroma virus to induce immune dysfunction and tumor dissemination in vivo.

The relationship of virus-induced immunological dysfunction and tumor dissemination was studied using two related tumor-causing leporipoxviruses: malignant fibroma virus (MV) and Shope fibroma virus (SFV). Recombinant viruses, produced by transferring MV's 10.7 kb BamHI C fragment to SFV, replicate in lymphocytes and suppress lymphocyte function in vitro. Those recombinants that replicate in lymphocytes and suppress lymphocyte function in vitro share about 3.5 kb from MV's C fragment. Some recombinants mimic MV in producing immune suppression and disseminated virus infection in vivo. Other recombinants, even some that are highly immunosuppressive in vitro (e.g. R71), only variably induce immune suppression in vivo, and do not cause disseminated disease. A segment of DNA from MV that transfers to Shope fibroma virus almost all of MV's virulence in vivo was identified.

Laminin receptor expression on murine tumor cells: correlation with sensitivity to natural cell-mediated cytotoxicity.

Previous studies have identified a relationship between the presence of cell surface laminin receptors on murine tumor cells and sensitivity to killing by natural killer (NK) cells. On the basis of these observations, we suggested that laminin and laminin receptors may function to facilitate the interaction of NK-sensitive murine target cells with NK cells. Our original studies were conducted with a number of genetically unrelated tumor cell lines. In order to extend these earlier observations, studies have now been conducted in which sensitivity to NK-mediated lysis and responsiveness to laminin were compared in a number of variant populations derived from the NK-sensitive cell lines Yac-1 and RL-1 and from the NK/NC-resistant line P815. All of the lines which interacted with murine NK cells as indicated by sensitivity to killing and/or by ability to "cold-target" compete with the killing of sensitive Yac-1 cells were able to bind 125I-laminin and to respond to laminin in an aggregation assay. Of 4 NK-resistant populations identified in these studies, 3 failed to respond to laminin. These studies indicate that even among genetically related tumor cell lines there is a relationship between laminin receptor expression and interaction with NK cells.

Treatment of mice with anti-asialo-GM1 antibody or poly-I:C: effects on metastasis dissociable from modulation of macrophage antitumor activity.

Treatment of C57BL/6J mice with poly-I:C or antibodies to asialo-GM1 enhances and depresses respectively natural killer (NK) cell activity while inversely altering lung metastasis, suggesting a critical role for these cells in controlling tumor formation. We assessed the effect of these treatments on antitumor activity mediated by macrophage (M phi) populations likely to be important in lung metastasis. Alveolar and lung interstitial M phi were asialo-GM1 positive (98%) and were sensitive to in vitro treatment with the antibody plus complement; however, treatment of mice with antibodies to asialo-GM1 failed to alter their tumoricidal activity in vitro. Few blood monocytes (4%) or spleen M phi (2%) were asialo-GM1 positive and their antitumor activity was similarly unaffected. In contrast, however, this same in vivo treatment resulted in a 14-fold increase in lung metastasis. Intraperitoneal injection of poly-I:C greatly reduced metastasis formation but also failed to significantly affect in vitro cytolytic activity of the M phi populations. These findings suggest that the major metastasis altering effects of these agents result from modulation of NK rather than M phi function.

Laminin production by murine melanoma cells: possible involvement in cell motility.

Three lines of B16 melanoma cells (B16-F1, B16-F10 and B16-BL6) were examined for motility in the micropore filter assay and for synthesis in culture of the basal lamina glycoprotein laminin. All three lines synthesized laminin as judged by the incorporation of [35S]methionine into immunoreactive laminin and secreted (or shed) laminin into the culture medium as indicated by biosynthetic labeling studies and enzyme-linked immunosorbent assays. Immunoreactive laminin was also seen on the surface of the cells as indicated by immunofluorescence staining and by complement-mediated killing. Analysis of [35S]methionine-labeled laminin immunoprecipitates by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) both with and without reduction of intersubunit disulfide bonds revealed that all three cell lines produced a similar array of laminin forms, and that the Mr = 950 kD laminin molecule (but not the uncombined subunits) was secreted into the culture medium. Laminin biosynthesis appeared to be limited by the availability of the Mr = 400 kD A subunit as shown by the intracellular accumulation of excess B subunit in the form of uncombined B subunit (Mr = 200 kD) and as a disulfide-linked B dimer (Mr = 400 kD). The motility of all three cell lines was stimulated four- to five-fold by the addition of either exogenous laminin from the EHS sarcoma or culture medium from the B16 cells containing the secreted laminin. The stimulated motility was inhibited by antilaminin serum. These observations suggest that the laminin synthesized by the B16 melanoma cells themselves may facilitate their motility.

In vitro and in vivo interaction between murine fibrosarcoma cells and natural killer cells.

Murine fibrosarcoma cells were examined for sensitivity to killing by natural killer (NK) and natural cytotoxic lymphocytes from mouse spleens. These tumor cell lines were sensitive to killing by effector cells which were nonadherent to plastic or nylon wool, Thy-1 negative, asialo-GM1 negative, and present in the spleens of beige mice, nude mice, and A/J mice, as well as in the spleens of normal syngeneic and allogeneic control mice. This indicates that the cytotoxic effects were due to natural cytotoxic lymphocytes rather than to NK lymphocytes, T-cells, or macrophages. Although the fibrosarcoma cells were not killed in vitro by endogenous NK cells, these tumor cells were able to "cold target" compete for Yac-1 (an NK-sensitive target) killing and to bind to asialo-GM1-positive, nonadherent spleen lymphocytes in a target cell binding assay. This suggests that the fibrosarcoma cells were recognized by NK cells. In addition, these cell lines were killed in a 4-h NK cytotoxicity assay by polyinosinic-polycytidylic acid-activated effector lymphocytes. The interaction between NK cells and the murine fibrosarcoma cells may have in vivo significance. When syngeneic mice were treated with anti-asialo-GM1 serum to eliminate NK activity and then given i.v. injections of the fibrosarcoma cells, many more lung tumors developed than in control animals. The structural basis for the recognition of the murine fibrosarcoma cells by the NK effector cells is not known. However, laminin may be involved. When the fibrosarcoma cells, which have receptors for the laminin molecule, were preincubated with laminin, they were reduced in their ability to compete for the killing of Yac-1 cells by the NK effectors and had reduced capacity to bind to NK cells in a target cell binding assay.

Laminin inhibits the recognition of tumor target cells by murine natural killer (NK) and natural cytotoxic (NC) lymphocytes.

Tumor cells sensitive to lysis by murine natural killer (NK) or natural cytotoxic (NC) cells were shown to bind laminin. They bound 125I-labeled laminin in a time- and concentration-dependent manner, and binding of the radioactive laminin was inhibited by excess cold laminin. In the presence of laminin, cell-cell aggregation occurred. Murine tumor cells not sensitive to NK/NC-mediated killing bound much less laminin, and laminin did not induce aggregation of these cells. The addition of exogenous laminin to NK or NC cytotoxicity assays reduced target lysis in a dose-related manner. Reduction of lysis was due to an inability of NK/NC cells to bind to the targets. Target cells pretreated with laminin were reduced in their ability to cold-target compete for NK-mediated lysis of untreated target cells. These effects were unique to laminin. The control proteins (fibronectin and thyroglobulin) had no effect on NK activity. Finally, inhibition of cytolytic activity by laminin appeared to be specific for NK/NC cells. Laminin had no effect on cytolysis mediated by alloimmune cytotoxic T lymphocytes regardless of whether the targets did or did not bind laminin.

Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes and its role in NK recognition of tumor target cells.

We have identified a structure on the surface of murine NK cells that is immunochemically cross-reactive with laminin. Treatment of normal CBA/J spleen cells with monospecific anti-laminin serum plus complement completely eliminated NK cytolytic activity against YAC-1 or RL male 1 target cells. In the absence of added complement, spleen cells preincubated with anti-laminin serum were also reduced in their cytolytic activity due to a reduced capacity to bind to the target cells. Treatment with anti-asialo GM1 serum plus complement also eliminated NK activity, but pretreatment of NK cells with anti-asialo GM1 in the absence of complement did not reduce cytolytic activity. Thus, anti-laminin and anti-asialo GM1 bind to structures on the surface of NK cells that distinguish functional (laminin) from nonfunctional (asialo GM1) sites. Flow cytometric analysis revealed that approximately 15% of normal nonadherent splenic lymphocytes expressed laminin-like structures, whereas 16% expressed asialo GM1 and 19% expressed the NK alloantigen NK 2.1. Treatment of alloimmune cytotoxic T lymphocytes (CTL) with anti-laminin plus complement did not affect CTL activity. Thus, anti-laminin serum appears to detect a cell surface structure present on the NK subset of lymphocytes.