Interactions of HLA-B27 with the peptide loading complex as revealed by heavy chain mutations.

MHC class I heavy chains assemble in the endoplasmic reticulum with beta(2)-microglobulin and peptide to form heterotrimers. Although full assembly is required for stable class I molecules to be expressed on the cell surface, class I alleles can differ significantly in their rates of, and dependencies on, full assembly. Furthermore, these differences can account for class I allele-specific disparities in antigen presentation to T cells. Recent studies suggest that class I assembly is assisted by an elaborate complex of proteins in the endoplasmic reticulum, collectively referred to as the peptide loading complex. In this report we take a mutagenesis approach to define how HLA-B27 molecules interact with the peptide loading complex. Our results define subtle differences between how B27 mutants interact with tapasin (TPN) and calreticulin (CRT) in comparison to similar mutations in other mouse and human class I molecules. Furthermore, these disparate interactions seen among class I molecules allow us to propose a spatial model by which all class I molecules interact with TPN and CRT, two molecular chaperones implicated in facilitating the binding of high-affinity peptide ligands.

Peptide length variants p2Ca and QL9 present distinct conformations to L(d)-specific T cells.

Recent advances have provided insights into how the TCR interacts with MHC/peptide complexes and a rationale to predict optimal epitopes for MHC binding and T cell recognition. For example, peptides of nine residues are predicted to be optimal for binding to H2-L(d), although 8 mer epitopes have also been identified. It has been predicted that 8 mer and 9 mer length variant peptides bound to L(d) present identical epitopes to T cells. However, in contrast to this prediction, we demonstrate here that the 8 mer peptide p2Ca and its 9 mer length variant QL9, extended by an N-terminal glutamine, assume distinct conformations when bound to L(d). We generated self-L(d)-restricted CTL clones specific for p2Ca that recognize L(d)/QL9 poorly if at all. This result is in sharp contrast to what has been observed with L(d)-alloreactive T cells that possess a much higher affinity for L(d)/QL9 than for L(d)/p2Ca. Alanine substitutions of the N-terminal residues of the QL9 peptide rescue detection by these self-L(d)/p2Ca-specific T cells, but decrease recognition by the L(d)-alloreactive 2C T cell clone. In addition, 2C T cell recognition of the p2Ca peptide is affected by different alanine substitutions compared with 2C T cell recognition of the QL9 peptide. These data clearly demonstrate that the p2Ca and QL9 peptides assume distinct conformations when bound to L(d) and, furthermore, demonstrate that there is flexibility in peptide binding within the MHC class I cleft.

Association of ERp57 with mouse MHC class I molecules is tapasin dependent and mimics that of calreticulin and not calnexin.

Before peptide binding in the endoplasmic reticulum, the class I heavy (H) chain-beta(2)-microglobulin complexes are detected in association with TAP and two chaperones, TPN and CRT. Recent studies have shown that the thiol-dependent reductase, ERp57, is also present in this peptide-loading complex. However, it remains controversial whether the association of ERp57 with MHC class I molecules precedes their combined association with the peptide-loading complex or whether ERp57 only associates with class I molecules in the presence of TPN. Resolution of this controversy could help determine the role of ERp57 in class I folding and/or assembly. To define the mouse class I H chain structures involved in interaction with ERp57, we tested chaperone association of L(d) mutations at residues 134 and 227/229 (previously implicated in TAP association), residues 86/88 (which ablate an N-linked glycan), and residue 101 (which disrupts a disulfide bond). The association of ERp57 with each of these mutant H chains showed a complete concordance with CRT, TAP, and TPN but not with calnexin. Furthermore, ERp57 failed to associate with H chain in TPN-deficient.220 cells. These combined data demonstrate that, during the assembly of the peptide-loading complex, the association of ERp57 with mouse class I is TPN dependent and parallels that of CRT and not calnexin.

Kb, Kd, and Ld molecules share common tapasin dependencies as determined using a novel epitope tag.

The endoplasmic reticulum protein tapasin is considered to be a class I-dedicated chaperone because it facilitates peptide loading by proposed mechanisms such as peptide editing, endoplasmic reticulum retention of nonpeptide-bound molecules, and/or localizing class I near the peptide source. Nonetheless, the primary functions of tapasin remain controversial as do the relative dependencies of different class I molecules on tapasin for optimal peptide loading and surface expression. Tapasin dependencies have been addressed in previous studies by transfecting different class I alleles into tapasin-deficient LCL721.220 cells and then monitoring surface expression and Ag presentation to T cells. Indeed, by these criteria, class I alleles have disparate tapasin-dependencies. In this study, we report a novel and more direct method of comparing tapasin dependency by monitoring the ratio of folded vs open forms of the different mouse class I heavy chains, L(d), K(d), and K(b). Furthermore, we determine the amount of de novo heavy chain synthesis required to attain comparable expression in the presence vs absence of tapasin. Our findings show that tapasin dramatically improves peptide loading of all three of these mouse molecules.

Definition and transfer of a serological epitope specific for peptide-empty forms of MHC class I.

Nascent class I molecules have been hypothesized to undergo a conformational change when they bind peptide based on the observation that most available antibodies only detect peptide-loaded class I. Furthermore recent evidence suggests that this peptide-facilitated conformational change induces the release of class I from association with transporter associated with antigen processing (TAP)/tapasin and other endoplasmic reticulum proteins facilitating class I assembly. To learn more about the structure of peptide-empty class I, we have studied mAb 64-3-7 that is specific for peptide-empty forms of L(d). We show here that mAb 64-3-7 detects a linear stretch of amino acids including principally residues 48Q and 50P. Furthermore, we demonstrate that the 64-3-7 epitope can be transferred to other class I molecules with limited mutagenesis. Interestingly, in the folded class I molecule residues 48 and 50 are on a loop connecting a beta strand (under the bound peptide) with the alpha(1) helix (rising above the ligand binding site). Thus it is attractive to propose that this loop is a hinge region. Importantly, the three-dimensional structure of this loop is strikingly conserved among class I molecules. Thus our findings suggest that all class I molecules undergo a similar conformational change in the loop around residues 48 and 50 when they associate with peptide.

An extensive region of an MHC class I alpha 2 domain loop influences interaction with the assembly complex.

Presentation of antigenic peptides to CTLs at the cell surface first requires assembly of MHC class I with peptide and beta 2-microglobulin in the endoplasmic reticulum. This process involves an assembly complex of several proteins, including TAP, tapasin, and calreticulin, all of which associate specifically with the beta 2-microglobulin-assembled, open form of the class I heavy chain. To better comprehend at a molecular level the regulation of class I assembly, we have assessed the influence of multiple individual amino acid substitutions in the MHC class I alpha 2 domain on interaction with TAP, tapasin, and calreticulin. In this report, we present evidence indicating that many residues surrounding position 134 in H-2Ld influence interaction with assembly complex components. Most mutations decreased association, but one (LdK131D) strongly increased it. The Ld mutants, with the exception of LdK131D, exhibited characteristics suggesting suboptimal intracellular peptide loading, similar to the phenotype of Ld expressed in a tapasin-deficient cell line. Notably, K131D was less peptide inducible than wild-type Ld, which is consistent with its unusually strong association with the endoplasmic reticulum assembly complex.

Alloreactive and syngeneic CTL are comparably dependent on interaction with MHC class I alpha-helical residues.

The molecular basis for the difference in the strength of T cell responses to self vs alloantigens is unknown, but may reflect how T cells are selected in the thymus. Because T cells with a high affinity for foreign as opposed to self MHC molecules are able to mature, it has been proposed that alloreactive T cells may be more strongly dependent upon interaction with MHC residues than are self-restricted T cells. This study was undertaken to rigorously address this hypothesis. Whereas other studies have compared self vs alloantigen recognition of different MHC alleles by a single T cell clone, we have compared self vs alloantigen recognition of a single MHC allele, H-2Ld, by a large panel of self-restricted and alloreactive T cell clones. Target cells expressing Ld molecules mutated at several different potential TCR contact residues were analyzed to determine which residues are important for recognition by self-restricted vs alloreactive T cells. We unequivocally demonstrate that self-restricted and alloreactive T cells do not differ, but rather are comparably dependent on interaction with MHC residues. Importantly, both self-restricted and alloreactive T cells are dependent upon the same MHC residues as primary contacts and, in addition, share a common recognition pattern of Ld. Furthermore, our analysis enables us to provide a model for allotype-specific T cell recognition of Ld vs Kb class I molecules.

Peptide ligand structure influences the exchange of beta2-microglobulin by cell surface Kb.

We have studied the interactions which occur between the peptide ligand and beta2-microglobulin (beta2m) components of the class I MHC complex by analysing the process of beta2m exchange. We have previously shown that the rate of beta2m exchange on a cell-surface class I MHC complex varies with the peptide ligand to which it is bound. It remains unclear, however, whether the ability of peptide ligand to alter beta2m/heavy-chain association is related to peptide affinity, peptide structure, or both. In this article, we examine the effects of variations in peptide ligand structure on the rate of beta2m exchange by cell surface Kb complexes. Using a panel of alanine substituted variants of the MCMV peptide (YPHFMPTNL), we show that single amino acid changes in peptide sequence can have dramatic effects on the rates of beta2m exchange. The observed changes in beta2m exchange rates are directly due to modification of the peptide ligand structure as they do not reflect changes in peptide affinity. These findings suggest that peptide ligand structure can induce conformational changes in the Kb heavy chain which alter the rates of cell surface beta2m exchange, and provide further evidence for peptide-dependent fluidity of the class I heavy chain.

Biochemical and functional characterization of soluble multivalent MHC L(d)/Fc gamma 1 and L(d)/Fc mu chimeric proteins loaded with specific peptides.

Central to the specificity of the immune system is the interaction between the T cell receptor and the major histocompatibility complex (MHC)-peptide ligand complex. To better understand the nature of this interaction, and to investigate possible avenues for specific therapeutic intervention, we have produced soluble recombinant molecules that can modulate antigen-specific T cells. Our approach involved the construction of recombinant murine genes composed of the MHC class I gene H-2L(d) and the Fc portion of immunoglobulin (Ig) heavy chain genes mu or gamma1. Stable transfectants of these L(d)/Fc gamma1 and L(d)/Fc mu genes generated correctly spliced transcripts and were capable of secreting chimeric protein. Immunoprecipitation analyses demonstrated the presence of chimeric L(d)/ Fc gamma1 and L(d)/Fc mu monomers of approximately 69 kDa and 90 kDa, respectively, as well as chimeric dimers under nonreducing conditions. The capacity of L(d)/Ig molecules to bind specific peptide ligands was demonstrated using radiolabeled peptides or with monoclonal reagents that specifically identify peptide-induced conformational changes in the L(d) ligand binding site. Soluble divalent L(d)/Fc gamma1 molecules were loaded with the murine cytomegalovirus-derived peptide and other L(d)-specific peptide ligands and subsequently isolated and purified. Peptide-loaded L(d)/Fc gamma1 molecules were capable of inhibiting the response of class I-restricted T cells in vitro in a peptide-specific fashion. The development of soluble multivalent chimeric proteins that possess unique properties of both the MHC class I and Ig molecules provides a valuable reagent for the study of potential mechanisms of in vitro and in vivo immune modulation.

The mechanisms of peptide exchange and beta 2-microglobulin exchange on cell surface Ld and Kb molecules are noncooperative.

It has previously been shown that the presence of exogenous beta 2-microglobulin (beta 2m) can dramatically enhance the binding of exogenous peptide to cell surface class I MHC. However, the mechanism by which this enhancement takes place is unknown. Two models have been proposed to explain this phenomenon. In the first model, the exchange of peptide and the exchange of beta 2m are cooperative processes. In the alternative model, beta 2m stabilizes free class I heavy chains to increase the total number of peptide binding sites available. In this report, we have examined the relationship between peptide exchange and beta 2m exchange. Comparisons of Ld and Kb complexes formed with peptides possessing widely disparate affinities revealed a reciprocal correlation between the peptide off-rate and the rate of beta 2m exchange. This result indicates that peptide exchange and beta 2m exchange are noncooperative processes that may, in fact, antagonize one another. These findings provide the first demonstration of peptide-specific influences on the rate of beta 2m exchange and suggest that exogenous beta 2m promotes peptide binding by maintaining class I heavy chains in a peptide-receptive state.

Chromosomal translocation in a child with SLI and apraxia.

A case study is presented of a 5-year-old boy who was classified as preschool handicapped and was assessed as having a specific expressive language impairment with verbal apraxia. Chromosomal studies revealed a de novo (new) balanced translocation between first and second chromosomes. Results of the neurological, speech/language, cognitive, and play evaluations revealed a child with a severe expressive speech-language deficit but good nonverbal cognitive and communicative skills. The hypothesis of a relationship between a chromosomal translocation and speech/language disorders is explored.

Peptide-induced rescue of serologic epitopes on class I MHC molecules.

To monitor conformational changes in MHC class I structure induced by interaction with peptide or beta 2-microglobulin (beta 2-m), we have taken a serologic approach. Previous studies by us and others have defined circumstances wherein specific peptides can decrease serologic recognition of class I molecules. However, such blocking of serologic epitopes has often been interpreted as steric hindrance by peptide side chains. In this paper, we describe peptide-induced gains in recognition by mAbs 30-5-7, 34-1-2, and B22/249. In experiments with mAb 30-5-7, impaired reactivity, which resulted from an Ld loop mutation, was specifically rescued by the binding of a beta-galactosidase-derived peptide to the Ld mutant. In studies with mAb 34-1-2, poor Ld detection was enhanced by mutations in Ld at beta 2-m interaction sites or by changes within the peptide-binding groove. To evaluate whether known peptides in the Ld groove could influence 34-1-2 recognition, we tested six peptide ligands, four of which increased the reactivity of 34-1-2 with the Ld-expressing cell to various degrees (up to 14-fold). It is of interest that Ld mutations at position 9 and 95/97 made significant differences in the ranking of the peptides in regard to their ability to increase recognition by 34-1-2 and B22/249. This finding suggests that mutations in the binding groove can alter peptide conformation and result in secondary changes in class I structure. On the basis of the cumulative serologic data, we propose that the class I molecule displays considerable fluidity, and is structurally influenced by both beta 2-m and peptide.

Model for the in vivo assembly of nascent Ld class I molecules and for the expression of unfolded Ld molecules at the cell surface.

To characterize the process of class I assembly and maturation, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of intracellular and surface Ld molecules can be detected in an alternative conformation designated Ldalt1. Nascent Ldalt molecules are non-peptide ligand associated and are weakly associated with beta 2-microglobulin (beta 2m). Unexpectedly, when monoclonal antibodies were added directly to the lysis buffer, significant amounts of Ldalt/beta 2m heterodimer were detected, suggesting that beta 2m association is not necessarily sufficient to induce Ld conformation. By contrast, addition of peptide to cell lysates rapidly induced the folding of beta 2m-associated Ldalt to conformed Ld. Furthermore, the time course and dynamics of this conversion correlated precisely with peptide binding to Ld. The precursor-product relationship of Ldalt and conformed Ld was also visualized in vivo by pulse-chase analysis of BALB/c splenocytes. To investigate the factors that regulate intracellular transport of class I molecules, expression of Ld was studied in the peptide transport-deficient cell line, RMA.S-Ld, and in beta 2m-/- splenocytes. In contrast to wild-type cell lines, both Ldalt and conformed Ld are poorly expressed at the cell surface of RMA.S-Ld and beta 2m-/- splenocytes. Therefore, surface expression of Ldalt is dependent upon the concomitant expression of conformed Ld molecules. To determine whether surface Ldalt molecules can result from melting of conformed Ld molecules, surface Ld molecules were loaded with several different known Ld peptide ligands. Complexes of Ld with different ligands were found to have dramatically disparate surface half-lives. Importantly, the Ld peptide complexes that turned over the most rapidly resulted in the most gain in surface Ldalt, implying that peptide dissociation can induce the accumulation of nonconformed Ld heavy chains at the cell surface.

Binding of peptides lacking consensus anchor residue alters H-2Ld serologic recognition.

CTL recognize class I MHC/peptide complexes on the surface of target cells. Crystallographic and serologic data have indicated that peptide ligands can influence the conformation of class I molecules and hence T cell recognition. How the binding of peptides with disparate sequence motifs affects the conformation of distinct regions within a class I molecule remains unknown. A series of site-directed mutants of the murine class I molecule H-2Ld was studied to address this question. These mutants were generated by in vitro mutagenesis and used to map the serologic epitopes recognized by a panel of Ld-reactive mAb. The influence of six different ligands on serologic recognition by these mAb was then examined. Of 12 mAb tested, only one, B22/249, was found to be significantly influenced by the bound peptide. Peptide discrimination by B22/249 was observed at the cell surface and in immunoprecipitates of Ld after incubation with two of the six ligands. The two peptides that caused suboptimal B22/249 recognition of Ld/peptide lack a proline at position 2, which is present in the other four peptides and has previously been defined as an anchor residue for Ld ligands. The epitope on Ld detected by mAb B22/249 includes residues 63 to 70 on the alpha 1 domain helix. Two of these residues are in pocket B, which computer modeling predicts to be in contact with the second residue of Ld-binding peptides. Therefore, these data imply that a mAb to a class I molecule can distinguish peptides with different motifs, possibly reflecting peptide-dependent conformational changes in the class I molecule.

Extensive peptide ligand exchange by surface class I major histocompatibility complex molecules independent of exogenous beta 2-microglobulin.

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.

Disparate interaction of peptide ligand with nascent versus mature class I major histocompatibility complex molecules: comparisons of peptide binding to alternative forms of Ld in cell lysates and the cell surface.

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.

The specific binding of peptide ligand to Ld class I major histocompatibility complex molecules determines their antigenic structure.

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.

Molecular evidence that the H-2D and H-2L genes arose by duplication. Differences between the evolution of the class I genes in mice and humans.

To resolve issues regarding the evolution of D region class I MHC genes and their relationship to other class I-encoding regions of the mouse, as well as man, we characterized the class I genes from the Dq region of the B10.AKM mouse strain. The Dq region was selected because it was known to express multiple gene products, yet two of the products previously characterized have structural features in common with the Ld molecule. Since DNA hybridization data defined similarities between the Dd and Dq regions, we used low-copy genomic or oligonucleotide probes derived from the Dd region of BALB/c (H-2d) to screen a B10.AKM cosmid library. Cosmid clones containing Dq, D2q, D3q, D4q, Lq, and Q1q genes have been isolated and aligned with the corresponding genes of the BALB/c MHC, thus demonstrating a similar gene organization. The two classical transplantation genes, Dq and Lq were found to be strikingly similar to each other such that exons 1-3 of Dq and Lq, are approximately 97% homologous, and exons 4-8 are identical. Furthermore, the implied amino acid sequences of both Lq and Dq molecules show considerable homology to Ld, particularly in regions presumed to be involved in ligand binding. These comparisons suggest not only that the Dq and Lq genes arose from the duplication of an Ld-like progenitor, but also that there is a selective advantage for the maintenance of an Ld-like structure. In addition, the 5' portion of the D4q gene was sequenced and found to have a 13-bp deletion and a 4-bp insertion within the alpha 2 exon. These result in a frame shift that creates a premature termination codon and potential polyadenylation site, respectively. Thus, D4q does not encode a typical class I molecule. Sequence comparisons suggest that the D4q gene did not arise from a duplication event involving an Ld-like gene such as Dq and Lq. Interestingly, the D4q molecule, if produced, would have amino acid residues in common with K and/or Q molecules that differ from those observed in D/L molecules. These findings, in conjunction with hybridization data, provide evidence that the D2, D3, and D4 genes were derived from Q genes by an unequal crossover event. Additional hybridization data using low-copy D region probes suggest that several different D region gene organizations exist among mice of different haplotypes. These and other recent molecular studies provide multiple examples of expansion and contraction of the class I genes in the D region.(ABSTRACT TRUNCATED AT 400 WORDS)

Peptide ligand-induced conformation and surface expression of the Ld class I MHC molecule.

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.