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The mechanism underlying the apparent differences in the capacity of murine and human class I MHC molecules to function as signal transducing structures in T cells was examined. Cross-linking murine class I MHC molecules on splenic T cells did not stimulate an increase in intracellular calcium ([Ca2+]i) and failed to induce proliferation in the presence of IL-2 or PMA. In contrast, modest proliferation was induced by cross-linking class I MHC molecules on murine peripheral blood T cells or human class I MHC molecules on murine transgenic spleen cells, but only when costimulated with PMA. Moreover, cross-linking murine class I MHC molecules or the human HLA-B27 molecule on T cell lines generated from transgenic murine splenic T cells stimulated only modest proliferation in the presence of PMA, but not IL-2. On the other hand, cross-linking murine class I MHC molecules expressed by the human T cell leukemic line, Jurkat, transfected with genes for these molecules, generated a prompt increase in [Ca2+]i, and stimulated IL-2 production in the presence of PMA. The results demonstrate that both murine and human class I MHC molecules have the capacity to function as signal transducing structures, but that murine T cells are much less responsive to this signal.

Certain class I major histocompatibility complex molecules expressed on live cells have been shown to bind exogenous peptide ligands. However, it remains controversial whether this binding occurs by peptide exchange or to empty surface class I molecules. In this report we compare the surface binding and dissociation of two virus-derived ligands of the Ld class I molecule of the mouse. The peptide ligands were previously identified in immune responses to cytomegalovirus or lymphochoriomeningitis virus as immunodominant, optimally sized, and Ld restricted. Ligand dissociation was monitored on live cells indirectly by measuring the surface turnover of Ld-peptide complexes or directly by using labeled peptides. The cytomegalovirus-derived and lymphochoriomeningitis virus-derived peptides appeared to dissociate relatively rapidly; however, the cytomegalovirus-derived peptide had a more rapid off-rate than the lymphochoriomeningitis-derived peptide. Furthermore, these rates of dissociation appear to span that seen with endogenous Ld-associated peptides expressed by cells at 37 degrees C. Exploiting the extraordinary accessibility of the surface Ld ligand binding site we developed an assay to quantitate peptide ligand exchange. Cells were precoated with saturating amounts of unlabeled peptide by overnight incubation and were then tested for secondary binding of labeled peptides in a 4-h assay. Our results unequivocally demonstrate the potential for surface class I molecules to undergo peptide exchange. Furthermore, peptide exchange was found to be largely independent of exogenous beta 2-microglobulin. This result implies that beta 2-microglobulin association and not beta 2-microglobulin exchange is the critical factor in peptide exchange by surface class I molecules. Because of the exquisite ability of T cells to discriminate different amounts of ligand bound to class I, the binding of exogenous peptides could play a critical role in normal or aberrant immune responses.

To determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular Ld molecules can be detected in an alternative conformation designated Ldalt. Ldalt molecules are non-ligand associated and show weak if any beta 2-microglobulin (beta 2m) association. We report here that Ld molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs Ld surface half-life. By contrast, Ldalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with Ld, live cells were incubated with iodinated peptides and Ld molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface Ld molecules was found not to depend upon exchange with exogenous beta 2m, but did correlate with the level of beta 2m association. To study the intracellular interaction of peptide with Ld, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded Ld. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to Ld was found to affect almost exclusively immature (Endo Hs) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Ldalt molecules are precursors awaiting constituents such as peptide and beta 2m that influence folding, whereas surface Ldalt molecules appear refractory to association with peptide, beta 2m, and consequent folding.

To better understand the biological implications of the association of ligand with major histocompatibility complex class I molecules, we have studied the Ld molecule of the mouse. The culturing of various nonselected cell lines with three different known Ld peptide ligands resulted in a two- to fourfold specific increase in surface Ld expression as detected by 10 of 11 different monoclonal antibodies (mAbs) recognizing Ld epitopes. These findings suggest that Ld molecules are not saturated with endogenous peptide ligands and thus have accessible binding sites. Exploiting this feature of Ld we demonstrate that the physical association of Ld with ligand is exquisitely specific, indicating that they function in determinant selection. In addition, a non-peptide-bound antigenic variant of Ld was specifically detected with an exceptional mAb designated 64-3-7. In comparison with other Ld molecules, 64-3-7+ Ld molecules are not peptide ligand inducible, are more susceptible to proteolysis, lack beta 2 microglobulin association, and display a slower rate of oligosaccharide maturation. In spite of their deficiencies, the non-ligand-associated 64-3-7 Ld molecules were detected on the surface of all cell types tested; however, they appear not to be recognized by alloreactive cytotoxic T lymphocytes.

Newly synthesized major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum are thought to bind peptides of foreign and endogenous antigens. Several lines of evidence indicate that beta-2 microglobulin (beta 2m) and/or peptide ligand participate in the intracellular transport and surface expression of class I molecules, but the nature of their involvement is still unclear. Here we present evidence that culturing non-mutant cells (fibroblast, thymoma or mastocytoma) with a peptide ligand specific for the Ld class I molecule of the mouse leads to a dramatic (fourfold) and specific induction of Ld surface expression. Surprisingly, this peptide ligand-induced expression of Ld does not result in an increased intracellular association of Ld with beta 2m. These findings demonstrate that the previously reported decrease in surface expression of Ld results from its failure to be saturated with endogenous self-peptide ligands. This unique feature of Ld could also contribute to the fact that several virus-specific cytotoxic T cell responses have been found to be Ld-restricted.

Data are presented that support the hypothesis that beta 2m controls the folding of the ligand binding site of newly synthesized class I molecules. This conclusion was indicated by comparisons of two antigenic forms of the Ld molecule separated by sequential immunoprecipitation. Whereas, mAb 30-5-7+ Ld molecules were found to exist either as free H chains or associated with beta 2m, 30-5-7- Ld molecules showed no beta 2m association. Chemical comparisons showed 30-5-7- Ld molecules to be highly sensitive to proteolysis relative to 30-5-7+ Ld molecules. Experiments employing a construct with the Ld gene juxtaposed to the inducible metallothionein promoter indicated that the ratio of the antigenic forms of Ld was determined by the relative synthesis of beta 2m vs class I proteins. Pulse-chase experiments demonstrated that the two antigenic forms of Ld do not share a precursor-product relationship, but do display disparate rates of intracellular transport. beta 2m dissociation or exchange at the cell surface was found not to affect the ratio of the two antigenic forms of Ld. In contrast to these findings with Ld, the Dd and Ddml molecules were not detected in alternative conformations, thus mapping this property to the N-terminus of the class I molecule. These findings support the notion that beta 2m induces conformation on the alpha 1/alpha 2 domains of Ld molecules during de novo synthesis and once beta 2m-conformed, the class I structure is fixed and irreversible.