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

Abstract

It is shown that liposomes containing (i) a fluorescein-labeled murine histocompatibility antigen and the G protein of vesicular stomatitis virus or (ii) H-2Kk and fluorescein-labeled viral protein (FITC-G) can elicit H-2-restricted syngeneic antiviral cytotoxic T 'cells as assayed by 5tCr release from appropriate virus-infected target cells. Fluorescence recovery after photobleaching was -used to measure the diffusion coefficients of these reconstituted proteins in four different sam-ples: (i) FITC-H-2Kk; jiU) FITC-H-2Kk and G; (iii) FITC-G; and (iv) FITC-G and H-2K . The same rate of lateral diffusion (D' = 1 X 10-8 cm2/sec at 37C in 25% cholesterol/75% dimyristoylphosphatidylcholine) was obtained in every case. Both proteins, fluorescent as well as nonfluorescent, could be patched by using specific antibodies. When G was patched with antibody, FITC-H2Kk did not copatch. When H-2Kk was patched with antibody, FITC-G did not copatch. These diffusion and patching measurements rule out the possibility that these proteins have either extensive oligomeric associations or strong specific pairwise associations.There is now much evidence that virus-specific cytotoxic T lymphocytes (CTL) are dually specific for.virus and for selfcell surface antigens encoded by the major histocompatibility complex (1). Effector lymphocytes sensitized against virus-infected cells of a given haplotype will lyse virus-infected cells of the same haplotype with much higher efficiency than virus-infected cells of a different haplotype. 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 (2-11). 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.Three models can be considered, based on alternative hypothetical structures for the T-cell receptor(s). In one model, a specific molecular complex between viral protein and transplantation antigen preexists in the target membrane before interaction with the T cell and is recognized by the T-cell receptor. In the second model, a close physical association of the two antigens is stabilized only during their interaction with the Tcell receptor. In the third model, no close physical or chemical association between viral protein and transplantation antigen exists prior to, or during, interaction with the T cell. (This is the two-receptor or "dual receptor" model.) The T-cell receptor(s) involved in the secondary elicitation of CTL is presumed to be on precytotoxic T cells or on T helper cells or on both.One biophysical approach to this problem is to use fluorescently labeled membrane components so that the distribution, motion, and interaction of these components can be studied by using optical techniques. In the work described here, purified G protein (G) of vesicular stomatitis virus (VSV) and purified H-2Kk protein were fluorescently labeled an...

Allogeneic stimulation of cytotoxic T cells by supported planar membranes.

“…When the sample is bleached without a grid in place, no significant recovery occurs, indicating that diffusion coefficients measured are not an artifact of exchange equilibrium between the surface and the solution. H-2Kk in these membranes does not show measurable diffusion, unlike its behavior in liposomes, where a diffusion coefficient of 1.13 x 10-8 cm2/sec has been measured (14). The uniform appearance of the fluorescence from fluoresceinated H-2Kk or fluoresceinated Fig.…”

“…The solution was bath sonicated to clarity and then diluted into a solution containing H-2Kk in the same buffer or into buffer solution without protein. The H-2Kk concentration was [10][11][12][13][14][15][16][17][18][19][20] ,ug/ml. Reconstitution was carried out at a lipid-to-protein ratio of 10:1 (wt/wt) unless otherwise stated.…”

Supported phospholipid bilayers

“…In recent years, a number of different approaches have been used to mimic effector-cell/ responder-cell interactions by replacing the effector or responder cell with liposomes or reconstituted membranes. Especially in the field of immunology, many typical cellular responses can be triggered by model membranes (Kinsky and Nicoletti, 1977;Littman et al, 1979;Hafeman et al, 1980;Herrmann and Mescher, 1981;Cartwright et al, 1982;Balakrishnan et al, 1982;Albert et al, 1983). To investigate the contact region of the two interacting membranes at a microscopic level, a planar membrane model system was developed in this laboratory some years ago McConnell, 1981 b: Hafeman et al, 1981).…”

Lateral diffusion of wild-type and mutant Ld antigens in L cells.

“…MHC antigens are, like other membrane integral proteins, somehow constrained in their lateral diffusion. Diffusion coefficients range from <<2 to 10 x 10 -j° cm2s -l in lymphocytes and in cultured fibroblasts, while they are 50-100 x l0 -l° cm2s -l for MHC antigens in liposomes (1).…”