Elizabeth A. Carswell

Serological demonstration of H-Y (male) antigen on mouse sperm.

WE have recently overcome certain technical difficulties in applying the cytotoxicity test to mouse spermatozoa and have been able to show directly that H-2 antigens are expressed on these cells1, thus confirming less direct evidence leading to the same conclusion2. We have since applied the cytotoxicity test to sperm with antisera directed against a number of other systems of mouse alloantigens, TL3, θ4, Ly-A5 and Ly-B5, but none of these has given a positive reaction with sperm. This is not surprising because these are all “differentiation antigens”6 expressed primarily on lymphoid cells. Another antigenic system which distinguishes one mouse from another is H-Y. The H-Y antigen is carried by male cells only and is responsible for the rejection of male tissues by females of the same inbred strain7.

Immune and nonimmune effector functions of IgG3 mouse monoclonal antibody R24 detecting the disialoganglioside GD3 on the surface of melanoma cells

R24, an IgG3 mouse monoclonal antibody reactive with the disialoganglioside GD3, was found to be a potent mediator of human complement cytotoxicity and human effector cell cytotoxicity. Cytotoxicity correlated with the degree of antibody binding (GD3 cell surface expression) for each of the melanoma cell lines and melanocyte cell cultures tested. Melanoma cell lines binding low amounts of R24 (low GD3 cell surface expressors) were not lysed in R24-directed immune reactions, suggesting that a threshold number of R24 molecules bound per cell is necessary to initiate these cytotoxic mechanisms. Since both complement- and cell-mediated reactions lysed the same subpopulations of cells in each cell line, both mechanisms appeared to depend on similar threshold quantities of bound R24 molecules. However, due to the heterogeneity of R24 binding in each cell line, the numerical value for this threshold could not be determined. Only in cell lines binding greater than 10(7) R24 molecules per cell were greater than 90% of the cells lysed. Normal melanocytes in culture were not lysed by R24-directed immune mechanisms, due to their low GD3 expression, indicating that monoclonal antibodies such as R24 may show tumor specificity with regard to effector functions even though normal cells express the relevant antigen. In contrast to the potent in vitro activity of R24, treatment of nu/nu mice bearing human melanoma grafts resulted in tumor inhibition only when started within 3 days of tumor cell inoculation. No effect was seen on established tumors. Thus, this in vivo mouse model failed to predict the clinical and pathological findings observed in treatment trials of R24 in human melanoma patients--urticaria involving skin metastases, cellular infiltration of tumor tissue, and tumor regression. In addition to activating immunologic effector functions, R24 had direct effects on melanoma cells, blocking their ability to attach to surfaces and causing tumor cell aggregation. These effects were again related to the number of R24 molecules bound to the cell surface; no aggregation was seen with cell lines binding less than 4 X 10(5) molecules per cell. Both immune and nonimmune effector functions may be involved in the tumor inhibitory activity of R24 in humans.

Human Tumor Necrosis Factor (LuKII): Recent Developments

TNF(LuKII) derived from the culture media of the LuKII B cell line has been purified to a specific activity of 1.5 × 107 units/mg of protein. Fractionation of the purified TNF(LuKII) by sodium dodecyl sulfate polyacrylamide gel electrophoresis reveals the presence of seven protein bands. Peptide mapping analyses and immunoblotting studies reveal that all seven protein bands are related. Treatment of purified TNF(LuKII) with the endoglycosidase Endo F results in a reduction of the molecular weights of all of the TNF(LuKII) species, demonstrating the presence of N-linked oligosaccharides on the TNF(LuKII) molecules. The removal of the N-linked sugars does not reduce the number of molecular weight species present. 125I-labelled TNF(LuKII) binds specifically to cell surface receptors. The TNF sensitive L(M) and HeLa cell lines appear to have 200 - 300 receptors for TNF(LuKII) per cell. While the inability of a cell to bind TNF(LuKII) appears to be indicative of that cells resistance to the cytotoxic effect of TNF(LuKII), the ability of a cell to bind TNF(LukII) does not necessarily indicate sensitivity to the cytotoxic effect.