Allogeneic cytolysis of reconstituted membrane vesicles.

Abstract

The successful use of lipid bilayer model membranes as targets for cytotoxic lymphocytes is described.Lipid vesicles were made from a mixture of dipalmitoyl lecithin, dimyristoyl lecithin, and cholesterol. Membrane proteins of LSTRA or EL4 tumor cells (as source of H-2 antigens), human eye muscle membrane proteins (as supporting proteins), and 51Cr marker were inserted into the lipid vesicles. Incubation of the reconstituted vesicles with lymphocytes sensitized in mixed lymphocyte cultures against allogeneic cells resulted in the specific release of intravesicular 5tCr. Vesicle damage was mediated by thymus-derived lymphocytes. H-2 antigens could be incorporated into vesicles without eye muscle proteins. However, immune damage of the vesicles could not be demonstrated when vesicles inserted with H-2 antigens in the absence of eye muscle proteins were used as targets. Thymus-derived (T) lymphocyte-mediated cytotoxic reactions to alloantigens, virus antigens, minor histocompatibility antigens, and haptens all involve direct or associated recognition of gene products of the major histocompatibility complex (MHC) (1-4). Thus, T cell receptors recognize on target cells both MHC and other antigenic ligands. The mechanism whereby T cell reactivity is restricted by target cell MHC phenotype is not yet understood. Model membranes should be useful for the study of allogeneic T cell cytotoxicity as well as the MHC restriction phenomenon, because they involve welldefined systems in which lipid vesicles may be reconstituted with purified MHC gene products in the presence or absence of other cell membrane proteins or exogenous antigens. Although murine MHC components have been incorporated into synthetic liposomes (5), and reconstituted vesicles have been utilized in the in vitro restimulation of cytotoxic T cells involved in MHC-restricted Sendai virus immunity (6), such reconstituted vesicles have not yet been reported as targets for cytotoxic T cells.In a different system designed to study the mechanism of eye muscle injury in human Graves ophthalmopathy, lipid vesicles inserted with human eye muscle membrane proteins (EMP) were damaged when incubated with lymphocytes from patients with Graves ophthalmopathy in the presence of thyroglobulin (Tg) or thyroglobulin-anti thyroglobulin complexes (TgA), whereas lymphocytes from control subjects damaged such vesicles only in the presence of TgA (7). Lipid vesicles lacking EMP or containing other tissue proteins in equivalent amounts were not lysed even if TgA complexes were present, indicating that EMP have some characteristic that renders lipid vesicles susceptible to lysis by immune cells.The first step in our approach of using model membrane systems as targets in cell-mediated cytolytic reactions was therefore the use of lipid vesicles into which both murine cellThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact...

Internal Disintegration Model of Cytotoxic Lymphocyte‐Induced Target Damage

“…Thus, simply stating it as a possible mechanism does little to elucidate the mechanism itself. Our experiments do, however, help us to redirect our thinking on the lytic process away from the plasma membrane and help to explain why fifteen years of attempts to use subceliular fragments as targets for the CTL have been unsuccessful or unconfirmed (Hollander et al 1979).…”

Secondary cytolytic T lymphocyte stimulation by purified H-2Kk in liposomes.

“…Hollander et al (27) have demonstrated that liposomes can be specifically lysed by CTL. The liposomes used in that study were made by including a protein extract of human eye muscle during their formation.…”

Novel fluorescence method to visualize antibody-dependent hydrogen peroxide-associated "killing" of liposomes by phagocytes

“…Model membranes containing antigens or lipid haptens can participate in both afferent and efferent immune reactions in vitro and in vivo (1)(2)(3)(4)(5)(6)(7)(8)(9). Considerable interest has been focused on the use of liposomes or lipid vesicles as targets of immune effector cell function (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Studies employing T cells (6)(7)(8)(9), neutrophils (10, 1 1), and macrophages (1 1-20) have been reported.…”