In this report we describe a system for the generation of functional, class I MHC-restricted, T-T hybridomas. The BW5147 cell line was transfected with the CD8 gene. BW5147 transfectants were obtained that stably expressed CD8 and this expression was maintained after somatic cell hybridization with activated T lymphocytes. To determine whether the stable expression of CD8 would facilitate the generation of class I MHC-specific T-T hybridomas, the transfected cells were fused with alloreactive T cells and the resultant hybrids were screened for their ability to produce lymphokines in response to antigenic stimulation. Somatic cell hybridizations with BW5147-CD8 transfectants give rise to a much higher frequency of class I MHC-specific T-T hybridomas relative to parallel fusions with BW5147. To determine whether the BW5147-CD8 transfectants would also support the generation of Ag-specific, class I MHC-restricted T-T hybridomas, they were fused with OVA-specific CTL. Several T-T hybrid clones were identified that produced lymphokines after stimulation with a transfected APC that was synthesizing OVA, or with a tryptic digest of OVA in the presence of syngeneic APC. The stimulation by Ag was MHC-restricted and mapped to the Kb molecule. An anti-CD8 mAb inhibited the stimulation of these hybridomas by Ag plus APC, whereas their stimulation by mitogen was unaffected. Cytolytic activity was not detected when several of the OVA-specific or alloreactive hybridomas were tested for their ability to kill target cells bearing the appropriate Ag. These results demonstrate that the BW5147-CD8 transfectants allow the generation of class I MHC-restricted T-T hybridomas. The potential utility of this system is discussed.
Abstract Class I heterodimers on the surface of cells are generally unreceptive to binding peptides in the absence of exogenous beta 2-microglobulin. Paraformaldehyde covalently cross-links beta 2-microglobulin to class I heavy chains in situ and stabilizes empty class I heterodimers. Functionally, this cross-linking creates receptive class I peptide binding sites by acting on beta 2-microglobulin-associated molecules. The presentation of preexisting peptide-class I complexes is also enhanced. These findings support a model whereby a structural alteration, the dissociation of beta 2-microglobulin, limits the existence of receptive class I molecules on normal cells and may control the half-life of active class I molecules.
A synthetic peptide corresponding to residues 365-380 of the influenza nucleoprotein (NP365-380) has been previously shown to associate with class I major histocompatibility complex-encoded molecules and to stimulate cytotoxic T lymphocytes [Townsend, A. R. M., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith, D. & McMichael, A. J. (1986) Cell 44, 959-968]. We find that intact Db class I heterodimers on the cell surface are unreceptive to binding this antigen. However, NP365-380 readily associates with Db molecules on the plasma membrane in the presence of exogenous beta 2-microglobulin. In addition, there is a second pathway through which this peptide associates with class I molecules that requires energy and de novo protein synthesis. These findings have implications for maintaining the immunological identity of cells and for the use of peptides as vaccines for priming cytolytic T-cell immunity.
Cytotoxic Tlymphocytes (CTL) induce in target cells a rapid, prelytic fragmentation of target cell DNA, accompanied by apoptosis. In contrast, complement and (with a few exceptions) chemical and physical means of inducing cytolysis induce necrosis, without DNA fragmentation. The function of the unusual DNA fragmentation induced by CTL remains to be elucidated. The major recognized function of CTL is in halting virus infections. Earlier, we proposed that CTL might halt virus infections prelytically, by fragmenting viral and cellular nucleic acids, and that in this case, cytolysis per se might be a less important function of CTL. We report here experiments designed to detect prelytic halt of virus replication. We employed in vivo-like conditions: fibroblast targets (difficult to lyse) were infected with herpes simplex virus (HSV), then incubated at low E/T cell ratios overnight. At the highest E/T ratios which produced less than 10% CTL-induced lysis, plaque-forming unit yield was reduced by about 50%. At higher E/T ratios which lysed 1/6 to 1/3 of the infected target cells, 3/4 to 9/10 of the virus production was prevented. The discrepancy between the level of lysis and the reduction in virus yield is evidence for significant CTL-induced prelytic halt of HSV replication. At present, it is unclear whether the antiviral effect observed involves an activity of CTL distinct from their lytic ability, such as their DNA fragmenting ability.
t *Division of Lymphocyte Biology Dana-Farber Cancer Institute Boston, Massachusetts 02115 tDepartment of Pathology Harvard Medical School Boston, Massachusetts 02115 Summary A large pool of free class I heavy chains is detected in situ on the plasma membrane of living cells. These chains are present on cells of different MHC genotypes and appear to exist under physiological conditions in vivo. These molecules arise from the dissociation of previously assembled class I heterodimers at the cell surface. The ratio of intact to dissociated heterodimers is strongly affected by the occupancy of the peptide- binding site of the class I molecule. Upon dissociation of the heterodimer, the class I molecule is functionally inactive. These findings may help to explain why class I molecules on the cell surface are unreceptive to bind- ing peptides yet readily associate with peptides in the presence of exogenous P2-microglobulin. These re- sults have implications for understanding the distinct functions of class I versus class II molecules and how the immunological identity of cells is preserved. Introduction Class I major histocompatibility complex (MHC) hetero- dimers are composed of a MHC-encoded, transmembrane heavy chain that is associated noncovalently with a non- transmembrane protein, fin-microglobulin. In contrast, class II MHC heterodimers are composed of two trans- membrane chains, both of which are encoded in the MHC. Despite these differences in primary structure, both class I and class II MHC molecules bind and present peptides to T lymphocytes (Townsend and Bodmer, 1989; Allen et al., 1987). Furthermore, these two classes of molecules bind similar and, in some cases, identical peptides (Cor- nette et al., 1989; Sweetser et al., 1989; Perkins et al., 1989; Hickling et al., 1990). It has been suggested that the tertiary structure of the peptide-binding site of class I and class II MHC molecules is similar (Brown et al., 1988). Although the configuration of the peptide-binding site may be similar for both classes of MHC molecules, exoge- nously added peptides readily associate with class II but not class I MHC molecules on the surface of cells (Rock et al., 1990b, 1991; Vitiello et al., 1990; Kozlowski et al., 1991). The molecular basisforthisdifference is not known. However, exogenous peptides in the presence of added m Vitiello et al., 1990; Kozlowski 1991). These results imply that the peptide-binding sites of class I and class II MHC molecules have different functional proper- ties and that the former is influenced by the dissociation and/or reassociation of Pn-microglobulin. Here we show that a substantial fraction of free murine class I heavy chains is present on the surface of cells and arises from the dissociation of be-microglobulin from previously assembled class I heterodimers. These free heavy chains appear to be unable stably associate with and/or present immunogenic peptides to T cells. Dissocia- tion of the class I MHC heterodimer will thereby limit the activity of this molecule and may account for the paucity of free class I peptide-binding sites on the cell surface. Results The Association of Exogenous Pz-Microglobulin with the Cell Surface Is Not Dependent on Added Peptides Immunogenic peptides associate with class I MHC mole- cules on antigen-presenting cells (APCs) in the presence of free human firmicroglobulin (Rock et al., 1990b, 1991; Vitiello et al., 1990; Kozlowski 1991). The present studies were initiated to directly measure the binding of human I%-microglobulin to class I molecules in situ. For this purpose we incubated murine EL4 cells with purified Pn-microglobulin of human origin in the presence or ab- sence of peptides. The presence of human m these peptides associate with Kb and Db, respectively in the presence of human P2-microglobulin (Moore et al., 1988; Townsend and Bodmer, 1989; Rocket al., 1990b, 1991). Theassocia- tion of exogenous P2-microglobulin with the cell surface is also not dependent on cellular metabolism as it inhibited by azide (30 mM) (data not shown). The binding of human BP-microglobulin is not affected by prior washing of cells (data not shown). We next examined the kinetics of binding BP-mi- croglobulin to cells. The association of &-microglobulin with EL4 cells is rapid. All cells have bound the human light chain after only 5 min of incubation (Figure 2A). After 15 min of incubation amount Prmicroglobulin that is bound continues to increase but at a slower rate.
CTL recognize oligopeptides bound to MHC class I molecules. Immunization of animals with antigenic peptides has often failed to stimulate CTL responses. We confirm that immunizations with several peptides, including natural and optimally active antigenic sequences, do not prime cytotoxic immunity in mice. However, immunization with peptides together with human beta 2-microglobulin primes Ag-specific CTL. Priming is observed when animals receive injections either i.v. with ex vivo peptide/beta 2-microglobulin-pulsed cells or s.c. with an admixture of peptide and beta 2-microglobulin. beta 2-Microglobulin promotes the priming of CTL immunity if it is added with peptide, but not if it is added after cells are exposed to peptide. Synthetic peptides and mixtures of peptides from enzymatically cleaved Ag are immunogenic. When a tryptic digest of OVA or the synthetic peptide (OVA258-276) are used as immunogens, the CTL that respond recognize the endogenously processed epitope presented by an OVA-transfected target cell. The peptide + beta 2-microglobulin-primed CTL are CD4-CD8+ and are class I MHC restricted. Using the immunization protocol with beta 2-microglobulin, we have primed CTL responses with peptides from OVA, Sendai virus, and vesicular stomatitis virus. These results may explain previous failures to prime CTL with peptides in vivo and provide a novel approach for developing peptide-based vaccines for viral diseases.
Soluble antigens (Ags) in the extracellular fluids are excluded from the class I major histocompatibility complex (MHC)-restricted pathway of Ag presentation in most cells. However, an exogenous Ag can be internalized, processed, and presented in association with class I MHC molecules on specialized Ag-presenting cells (APCs). These APCs express class II molecules and can simultaneously present exogenous Ags to both class I and class II MHC-restricted T cells. These APCs may be important participants in the regulation of host immune responses. This APC activity may explain several phenomena of cytotoxic T lymphocyte (CTL) priming in vivo and might be exploited for eliciting CTL responses to protein vaccines.
Abstract Ag in the extracellular fluids can be internalized, processed, and presented in association with class I MHC molecules on specialized APC in normal spleen. We examine the fate of these APC after they present Ag to a CTL. When splenocytes present exogenous OVA to CTL, their ability to subsequently present native Ag in association with both class I and class II molecules is inhibited. CTL do not inhibit the ability of splenocytes to present processing independent peptides with class I or class II molecules. Inhibition of Ag presentation is only observed in the presence of the specific Ag recognized by the CTL. This inhibition is MHC-restricted. In the presence of specific Ag, CTL inhibit the ability of APC to present unrelated Ag. However, bystander APC are not affected by activated CTL. Taken together these results indicate that when APC present exogenous Ag to CTL, they are inhibited or killed. The CTL that mediates this activity has a conventional CD4-CD8+ phenotype and utilizes a TCR-alpha beta. The potential significance of these findings and their possible relationship to phenomena associated with Ts cells are discussed.
Abstract Exogenous Ag in the extracellular fluids do not gain access to the class I Ag-presenting pathway in most cells. However, there is an APC resident in spleen that can process and present exogenous Ag in association with class I molecules. We characterize the phenotype of this cell. This APC is of low buoyant density, is adherent to Sepharose and glass, and expresses both class II molecules and FcR. This phenotype identifies this APC as a macrophage. Resident, peptone- and thioglycolate-induced peritoneal macrophages also display this Ag-presenting activity. Analysis with CTL clones suggest that this Ag-presenting pathway may be active in only a subset of macrophages. A similar Ag-presenting activity is also present in dendritic cell-enriched populations from spleen although we cannot rule out the possible involvement of contaminating macrophages. In contrast, B and T cells that are resident in spleen and LPS blasts are unable to present exogenous Ag in association with class I molecules. The presentation of exogenous OVA with class I molecules is not inhibited by the inhibitors of thiol proteases, leupeptin, and antipain. The presence of gelonin, a ribosomal inactivating protein, in the extracellular fluids inhibits the ability of these APC to present exogenous OVA. Under identical conditions, gelonin does not inhibit Con A-stimulated T cell proliferation, or LPS-stimulated B cell proliferation and Ag presentation. These results are discussed in relation to the potential pathways through which an Ag in the extracellular fluids is presented with MHC class I molecules.
