Liposomes as a tool to study the role of membrane presentation in the immunogenicity of a MuLV-related tumor antigen.

Abstract

Abstract The immunogenicity of a tumor cell surface-associated antigen is closely related to its presentation; usually the antigen that is presented on the cell membrane is a better immunogen than the antigen in soluble form. We studied the immunogenicity of the Gross virus cell surface antigen GCSAa in a syngeneic rat lymphoma model. Two injections of irradiated Gross virus-induced (C58NT)D lymphoma cells into W/Fu rats induced a high antibody response against GCSAa. Crude plasma membranes prepared from (C58NT)D cells could also induce a good anti-GCSAa antibody response when mixed with complete Freund adjuvant (CFA) and injected, whereas soluble GCSAa from the cytosol was a poor immunogen. To investigate the requirement for GCSAa to be associated with cell membranes to elicit an effective antibody response, soluble GCSAa prepared from the cytosol of (C58NT)D cells was incorporated into multilamellar liposomes made of dipalmitoyl phosphatidylcholine, cholesterol, and dicetylphosphate in 7:2:1 molar ratio, and was used as immunogen. High antibody responses specific to GCSAa were obtained, but emulsion of the liposome preparation in CFA was also required. Because CFA could be replaced by incomplete Freund adjuvant but not by live BCG microorganisms, CFA was tentatively replaced by the addition to the liposome preparation of either a powerful chemotactic tripeptide, f-Met-Leu-Phe, or lipid A, or muramyldipeptide. In most of the experiments, however, the liposome preparation failed to show a higher immunogenicity than that of the soluble antigen. Alternatively, when the liposome preparation was incubated in vitro with peritoneal exudate cells before its injection into syngenic rats without any adjuvant, high antibody responses were observed in the animals. No significant antibody response was obtained in rats that also received either peritoneal exudate cells that were previously incubated with soluble antigen, or spleen cells that were depleted in adherent cells and previously incubated with the antigen preparations. The role of liposome presentation of GCSAa in the expression of its immunogenicity was also studied. The requirement for a constitutive association of GCSAa with liposomes was confirmed because injection of the soluble antigen mixed with preformed liposomes and emulsified in CFA did not induce a significant antibody response. The lipidic lamellae were separated from the aqueous phase after mechanical disruption of the liposome-GCSa preparation, and were used as immunogen; GCSAa associated with the lipidic lamellae was found to be as immunogenic as the intact liposome-GCSAa preparation. These results strongly suggest the increase in GCSAa immunogenicity by liposomes results from a rapid in vivo recognition of unaltered liposomes by macrophages and is related to the presentation of the antigen in a membrane-like structure.