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

Abstract

Purified H-2Kk' incorporated into lipid vesicles induced a secondary allogeneic cytolytic T lymphocyte response. However, the level of the response was much less than that generated by using purified plasma membranes containing an equivalent amount of antigen. Similarly, reconstituted membranes stimulated less effectively than did intact plasma membranes. In both cases the stimulating activity of the antigen was increased by including a detergent-insoluble membrane matrix fraction during formation of the liposomes or reconstituted vesicles. Liposomes formed in the presence of the matrix were larger, were more irregular in shape, and had a higher density than those formed in its absence. Both the H-2 antigen and matrix proteins were incorporated into the same vesicles. The greater antigenicity of H-2 in vesicles containing the matrix protein might be due to either the larger size of the liposomes or interaction of the antigen with a' component(s) of the matrix.Generation ofa cytolytic T lymphocyte (CTL) response requires the recognition of cell surface antigens of the stimulator cells. The demonstration that purified membranes (1) and detergentsolubilized reconstituted membranes (2, 3) from stimulator cells are able to generate a specific in vitro secondary allogeneic CTL response has made it possible to-begin to examine the requirements for recognition at the molecular level. Purified HLA antigens (4) and partially purified'H-2 antigens (5) have been shown to stimulate a secondary CTL response when incorporated into liposomes. Similarly, liposomes containing the appropriate foreign antigens and H-2 will specifically stimulate secondary virus-specific (6-9) and tumor-specific (10,11) responses.Previous studies (2, 5) had demonstrated that reconstituted membranes could stimulate generation of an allogeneic CTL response, but the level of response was much lower than that obtained by using intact membranes. The results reported here demonstrate that activity comparable to that of intact membranes is obtained if the detergent-insoluble fraction of the membrane is not removed prior to reconstitution. Furthermore, including this isolated, detergent-insoluble fraction during incorporation of purified H-2K into liposomes results in liposomes that effectively stimulate an allogeneic response.Examination of the detergent-insoluble fraction of plasma membranes from murine tumor and lymphoid cells has shown it to consist of a relatively small number of membrane proteins which remain selectively insoluble (12). The major protein components are actin and proteins with molecular weights of70,000, 69,000, 38,000, and 36,000. '5'-Nucleotidase, a cell surface glycoprotein, also remains associated with the detergent-insoluble proteins. The proteins appear to form a matrix at the inner surface ofthe plasma membrane which is, in many respects, similar to the spectrin matrix of erythrocytes. Thin-section electron micrographs of the isolated detergent-insoluble matrix show it to be primarily in the form of closed structures si...

Direct transfer of antigen-specific cytolytic activity to noncytolytic cells upon fusion with liposomes derived from cytolytic T cell clones.

“…Cytolytic T cell clones were solubilized in the nonionic detergent NP-40 and the matrix and plasma membrane components separated from nuclear and mitochondrial constituents. Matrix proteins (detergent-insoluble fraction primarily consisting of actin) were retained to increase liposome stability (14). To this cellular protein mixture were added Sendai virus envelope proteins that had been purified from detergentsolubilized viral preparations.…”

“…Liposomes were prepared by a modification of previously published procedures (13)(14)(15). Briefly, donor cells were extensively washed in phosphatebuffered saline (PBS) to remove medium and serum contaminants, followed by a 30 min incubation at 4°C in 0.5% NP-40-Tris-HCl-saline buffer, pH 7.4.…”

H-2Kk and vesicular stomatitis virus G proteins are not extensively associated in reconstituted membranes recognized by T cells.

“…The rate oflateral diffusion of these proteins is comparable to rates observed for other membrane proteins in similar lipid mixtures (27)(28) patched with unlabeled 114.1 antibody (first-step) and rhodamine-conjugated F(ab')2 rabbit anti-mouse antibody (Cappel, Cochranville, PA) (second-step, abbreviated RFRAM henceforth); (3,4) unlabeled G patched with rabbit anti-G and rhodamine-conjugated F(ab')2 goat anti-rabbit (Cappel) (abbreviated RFGAR henceforth); (5,6) F1M H-2Kk; (7,8) FITC-G; (9,10) FITC-H-2Kk patched with alloantiserum against H-2Kk and RFRAM (fluorescein fluorescence); (11,12) FITC-G patched with rabbit anti-G and RFGAR (fluorescein fluorescence); (13,14) FITC-H-2Kk and unlabeled G; (15,16) FITC-G and unlabeled H-2Kk; (17,18) FITC-H-2Kk and unlabeled G (fluorescein fluorescence) with the G protein patched with rabbit anti-G and RFGAR; (19,20) A strong specific interaction between viral proteins and H2Kk molecules in a reconstituted membrane would provide strong support for any theory that required close proximity of these two molecules in the target membrane during specific elicitation of cytotoxic T cells. On the other hand, freely and independently diffusing viral proteins and H-2Kk molecules are also consistent with a possible close proximity of these molecules as a prerequisite for specific elicitation of CTL (32).…”

“…A similar restriction holds for the afferent immune response. Virus-specific H-2-restricted secondary elicitation of CTL has been demonstrated by using membrane fragments as well as reconstituted membranes (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). These studies show that both viral protein and H-2 antigen must be present in the same reconstituted membrane in order to elicit the cellular response.…”

A soluble divalent class I major histocompatibility complex molecule inhibits alloreactive T cells at nanomolar concentrations.

“…Soluble class I MHC-like molecules have been used to study T-cell responses (18)(19)(20)(21)(22)(23)(24). Using soluble MHC antigens which have been crosslinked, experiments have demonstrated that T-cell activation requires polyvalent MHC molecules and that a critical threshold is required for T-cell activation (18,19).…”