Recombinant interferon-gamma (IFN-gamma) induced the expression of HLA-DR when added to the culture medium of HLA-DR- melanoma cell lines. In addition, IFN-gamma induced the expression of another class II antigen, HLA-DC, on a HLA-DR+ and -DC-melanoma cell line and to a lower level on a -DR- and -DC-melanoma line. IFN-gamma also enhanced the expression of HLA-ABC and beta 2-microglobulin, as well as HLA-DR on DR+ melanoma cells. In contrast, IFN-alpha gave no induction of expression of HLA-DR and DC on two DR- melanoma lines, while it did enhance the expression of HLA-ABC and of beta 2-microglobulin. The expression of 3 out of 6 melanoma-associated differentiation antigens was enhanced by IFN-gamma treatment. The modulation of antigens by IFN-gamma was both dose and time dependent. A minimum incubation time of 48 h was necessary for the appearance of HLA-DR on the two HLA-DR- melanoma lines, whereas HLA-ABC and beta 2-microglobulin were already increased after 24 h. A dose of 20 U/ml IFN-gamma started to induce the expression of HLA-DR and DC on melanoma cells GLL-19 and Me-43 and a plateau of maximum antigen expression was reached with 100 U/ml. Analyses of IFN-gamma-treated cells by flow microfluorometry showed a homogeneous distribution of increased staining intensity rather than the appearance of two cell populations. Immunoprecipitation experiments using detergent-solubilized 125I-labeled membrane proteins of IFN-gamma-treated melanoma cells and a monoclonal anti-HLA-DR antibody confirmed the presence of HLA-DR antigens. When IFN-gamma-treated cells were cultured without IFN the induced or enhanced expression of HLA antigens was reversible. Eight days after removal of IFN, the HLA-DR level was reduced by more than 90% and the level of HLA-ABC and beta 2-microglobulin by more than 50%. The demonstration of the ability of HLA-DR- melanoma cells to express HLA-DR after IFN-gamma treatment was extended to cells from other types of tumor such as gliomas, colon carcinomas and one cervical carcinoma cell line.

Temperature-jump experiments were performed with di-, tetra-, and hexasaccharides derived from type III pneumococcal polysaccharide using a homogeneous corresponding antibody IgG 45-394. A decrease in stability of the oligosaccharide-antibody complexes with decreasing chain length was observed and entirely reflected in the decrease of the association rate constants which were 1.7 X 10(4) M-1 s-1 for the di-, 3.7 X 10(5) M-1 s-1 for the tetra-, and 1.1 X 10(6) M-1 s-1 for the hexasaccharide at 23 degrees C. The dissociation rate constants for all oligomers were about 12 s-1. This marked chain-length dependence of the association rate constants as well as their low values are unexpected for a single binding step. A mechanism is proposed which consists of a fast formation of a labile oligosaccharide-antibody precomplex followed by a slow isomerization step which is induced by the oligosaccharide ligands but which is chain-length independent.

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Thirty monoclonal antibodies from eight laboratories exchanged after the First Workshop on Monoclonal Antibodies to Human Melanoma held in March 1981 at NIH were tested in an antibody-binding radioimmunoassay using a panel of 28 different cell lines. This panel included 12 melanomas, three neuroblastomas, four gliomas, one retinoblastoma, four colon carcinomas, one lung carcinoma, one cervical carcinoma, one endometrial carcinoma, and one breast carcinoma. The reactivity pattern of the 30 monoclonal antibodies tested showed that none of them were directed against antigens strictly restricted to melanoma, but that several of them recognize antigenic structures preferentially expressed on melanoma cells. A large number of antibodies were found to crossreact with gliomas and neuroblastomas. Thus, they seem to recognize neuroectoderm associated differentiation antigens. Four monoclonal antibodies produced in our laboratory were further studied for the immunohistological localization of melanoma associated antigens on fresh tumor material. In a three-layer biotin-avidin-peroxidase system each antibody showed a different staining pattern with the tumor cells, suggesting that they were directed against different antigens.