T lymphocytes respond to foreign antigens by detecting peptide fragments of those antigens bound to products of the major histocompatibility complex (MHC) ()and displayed on the surface of antigen presenting cells (reviewed in (1) ). The three-dimensional structures of both class I and II MHC molecules complexed with self- or foreign peptide antigens have recently been solved, revealing much about the molecular basis of peptide binding to MHC molecules(2, 3, 4, 5, 6, 7, 8) . Structurally, both classes of MHC molecules are similar, as predicted by sequence comparison(9) ; however, they differ in fine detail, especially in the peptide-binding site(7, 8) . These differences partly account for the distinct manner in which each class of molecule binds peptide. In class I molecules, residues forming the ``ends'' of the peptide-binding site bury the termini of bound peptides(3, 4, 5, 6) . In contrast, in class II molecules, both ends of the peptide-binding site are ``open,'' allowing the termini of bound peptides to extend out of the site(7, 8) . Consequently, peptides bound to class I molecules are predominantly 8-10 residues in length(10, 11, 12, 13, 14) , whereas peptides bound to class II molecules tend to be 13-25 residues in length(15, 16, 17) .

In both classes of MHC molecules, the presence of bound peptides influences their tertiary structure and efficiency of appearance on the cell surface. The binding of self- and foreign peptides to the class I heavy chain promotes the folding, assembly, and surface expression of class I molecules by stabilizing the class I complex(18, 19) . Similarly, peptide binding to class II molecules influences the conformation, stability, and surface appearance of class II molecules (20, 21, 22, 23, 24) . Thus, detailed functional analysis of the peptide-binding sites of both classes of molecules will be necessary to understand the role of peptide binding in determining the tertiary structure, and hence, surface expression of MHC molecules and subsequent display of foreign antigens to the immune system.

We have been interested in the biochemical basis of recognition of peptide antigens bound to MHC molecules by T cells. We have characterized the T cell receptor (TCR) expressed by the murine T cell clone D5(25) , which recognizes arsonate (Ars)-conjugated peptides presented by the class II molecule I-A(26) . We have engineered single-site substitutions in the putative MHC/peptide antigen-binding site of the D5 TCR and have identified several residues that are important in recognition of the MHCpeptide complex(27, 28) . Subsequently, we sought to compensate these single-site TCR mutations with complementary mutations in the I-A molecule. In the course of these studies, we identified an ion pair, Asp-57 of the -chain and Arg-80 of the -chain of I-A, that plays an essential role in surface expression of I-A as well as peptide presentation.

MATERIALS AND METHODS

Cell Lines

DNA Constructions

Transfection of COS Cells

Flow Cytometry

Figure 1: Expression of wild-type I-A heterodimers on the surface of transfected COS cells. COS cells transfected with WT I-A - and -chain DNA or mock-transfected (no DNA) were analyzed by flow cytometry using control or I-A-specific mAb. Shown are histograms of 5000 cells stained with control mAb 14-4-4S, which recognizes I-E, or mAb MK-D6 specific for I-A, followed by phycoerythrin-conjugated secondary antibody. Bars indicate the portions of the histograms used to calculate ``fluorescence units'' as described under ``Materials and Methods.'' Fluorescence units obtained with MK-D6 staining of COS cells transfected with WT I-A were 870,000 compared to 740 obtained with mock-transfected COS.

Peptide Presentation Assay

RESULTS

We individually substituted Lys for several negatively charged residues in the -helical regions of the 1- and 1-domains of the I-A heterodimer by site-directed mutagenesis ( Table 1and Fig. 9). These helices form the ``walls'' of the peptide-binding site of MHC molecules and, when bound with foreign peptide, are recognized by the TCR. We chose to express these variant I-A molecules in COS cells because COS cells can transiently express murine class II molecules even though they fail to express the invariant chain(37) . We analyzed expression of these variant I-A molecules by flow cytometry using a variety of mAb recognizing different epitopes on I-A (Table 2). We then tested the ability of the variant class II molecules expressed on COS cells to present foreign peptides to I-A-restricted, antigen-specific T cell hybridomas.

Figure 9: Location of tested residues on a predicted map of I-A. Shown is a ribbon diagram of the top view of the peptide-binding site of HLA-DR1 bound with an influenza virus peptide (8) onto which the residues of I-A tested in this report were modeled, based on the sequence similarity shared among class II molecules(9) . Substitution of Asp-57 with Lys or Arg impaired surface expression of I-A heterodimers, whereas substitution of 10 other negatively charged residues with Lys had little or no effect on surface expression or presentation of foreign peptide to an I-A-restricted TCR. The substitution with Glu or Asp for Arg-80, predicted to form a salt-bridge with Asp-57 in I-A, also impaired surface expression of I-A and its ability to present foreign peptides.

Role of Negatively Charged Residues in Surface Expression of I-A and Presentation of Foreign Peptide

COS cells expressing WT I-A and the 11 variant Lys-substituted heterodimers listed in Table 1were analyzed by flow cytometry using several mAb reactive against either the 1- or 1-domains of I-A (Table 2). The relative efficiency of surface expression of WT and variant I-A molecules was estimated by calculating fluorescence units for each combination of - and -chains (Fig. 2). As shown in Fig. 2, of the 11 Lys substitutions, only one, D57K, prevented surface expression of I-A, since neither the -chain-reactive mAb K24-199 nor any of the -chain-reactive mAb recognized COS cells transfected with this variant. The remaining 10 Lys substitutions did not affect surface expression of I-A since they all reacted with -chain mAb K24-199 and at least one -chain mAb. Of these, seven (E51K, E59K, E70K, E74K, D76K, E84K, and E87K) had no effect on mAb reactivity: COS cells expressing these variant heterodimers stained with all mAb tested at intensities within a factor of three compared to COS cells expressing WT I-A. In contrast, the remaining three substitutions (E59K, E66K, and E69K) eliminated recognition by one or more of the -chain mAb. The E59K heterodimer failed to stain with mAb BP107 and N22, the E66K heterodimer failed to stain with mAb 34-5-3S, and the E69K heterodimer failed to stain with mAb MK-D6, 34-5-3S, BP107, 28-16-8S, and M5/114. Therefore, Glu-59, Glu-66, and Glu-69 are likely to be solvent accessible in I-A, in agreement with the orientation of the corresponding residues in the human class II molecule HLA-DR1(7, 8) . The loss of reactivity of mAb with these variant I-A molecules agrees with previous reports demonstrating that the N-terminal half of the -helix of the 1-domain of I-A molecules contributes to the epitopes of several mAb (for example, see Refs. 36, 52). In addition, our results agree with previous epitope mapping and provide new or more detailed information regarding the reactivity of several mAb (see Table 2).

Figure 2: Serological analysis of wild-type and variant I-A heterodimers expressed on the surface of transfected COS cells. COS cells transfected with plasmids encoding the indicated WT or variant - and -chains of I-A were analyzed for surface expression using control (14-4-4S) or several I-A-reactive mAb (Table 2). I-A mAb K24-199 is -chain specific, whereas the remaining I-A mAb are -chain specific. Fluorescence units were calculated for staining of the COS cells and are representative of several independent experiments.

We determined whether any of the 10 substitutions that were tolerated with respect to surface expression of I-A affected the presentation of foreign peptides to an I-A-restricted T cell hybridoma. Fig. 3A shows representative experiments indicating that COS cells expressing each of these variant heterodimers were able to present Ars-Ova(33-50) to D5h cells. Fig. 3B summarizes the results of several such experiments. When the slight differences in surface expression between the variant and WT I-A heterodimers are taken into account (see Fig. 2), each of these 10 variant heterodimers were within 10-fold as efficient at peptide presentation as WT I-A (Fig. 3B). These results demonstrate that although three of the 10 substitutions, including E59K, E66K, and E69K, completely eliminated recognition by at least one mAb, none profoundly affected peptide presentation.

Figure 3: Response of D5h T cell hybridoma to antigenic peptide presented by COS cells expressing wild-type or variant I-A heterodimers. In A, COS cells expressing WT or the indicated - (top) or -chain (bottom) variant I-A were tested for the ability to present the antigenic peptide Ars-Ova(33-50) to the I-A-restricted T cell hybridoma D5h. The difference in the potency of peptide presented by WT I-A in the two experiments is due to variation in transfection efficiency and the response of D5h and IL-2 indicator cells. In B, the relative ability of variant heterodimers to present antigenic peptide to the D5 TCR in several experiments was calculated as described under ``Materials and Methods'' and is represented by solid circles (WT = 1.0). Bars represent the geometic mean of these independent experiments.

Mutations at Asp-57 Diminish Surface Expression of I-A and Presentation of Foreign Peptides

Figure 4: Surface expression of wild-type or variant I-A containing substitutions at Asp-57 in COS cells. COS cells transfected with plasmids encoding the indicated WT -chain and WT or variant -chains were analyzed by flow cytometry using control (14-4-4S) or I-A-reactive mAb. In B and C, cells were co-transfected with control (C) 2B4- or invariant chain (Ii) DNA. In C, COS cells co-transfected with I-A and 2B4- DNA were stained with TCR -chain-specific mAb H57-597 to detect surface 2B4- molecules; in this experiment, cells co-transfected with WT -chain and the invariant chain serve as control cells for staining with mAb H57-597. These results are representative of several independent experiments.

Since the invariant chain has been demonstrated to regulate expression of class II molecules in vivo(53) and enhance surface expression of certain class II molecules containing haplotype-mismatched - and -chains(40) , we tested whether co-expression of the invariant chain could enhance expression of WT or variant I-A molecules on the surface of transfected COS cells. In agreement with previous reports(40, 54) , co-transfection with the invariant chain had little effect on surface staining of WT I-A by mAb MK-D6 and M5/114 but reproducibly enhanced staining of WT I-A by mAb K24-199 by 2.8-fold (± 0.7-fold, n = 4) (Fig. 4B). Co-transfection of the invariant chain reproducibly enhanced surface staining of variant heterodimers D57S and D57A by mAb K24-199, MK-D6, and M5/114. Enhancement of staining by mAb K24-199 was 14-fold (± 7-fold, n = 4) for D57S and 13-fold (± 5-fold, n = 4) for D57A; by MK-D6 was 3.2-fold (± 0.7-fold, n = 4) for D57S and 2.3-fold (± 0.5-fold, n = 3) for D57A; and by M5/114 was 3.6-fold (± 1.0-fold, n = 4) for D57S and 3.8-fold (± 1.1-fold, n = 4) for D57A. In fact, when the invariant chain was co-transfected, surface staining of these variant heterodimers was nearly as intense as observed with WT I-A (Fig. 4B); for D57S and D57A staining was 89% (± 15%, n = 4) and 78% (± 26%, n = 4), respectively, of wild-type levels using mAb M5/114. Co-expression of the invariant chain reproducibly enhanced expression of variants D57K and D57R to levels slightly above background (Fig. 4B); however, this effect was not observed in experiments in which the efficiency of COS cell transfection was low. Co-transfection with plasmid DNA encoding the chimeric cell surface molecule 2B4- (38) in place of invariant chain DNA did not restore surface expression of I-A variants substituted at Asp-57 (Fig. 4B).

The relative efficiency of transfection of WT or variant DNA in these experiments was monitored by co-transfection of DNA encoding 2B4-. Surface expression of the reporter molecule 2B4- was approximately equivalent when it was co-transfected with either WT or variant I-A DNA (Fig. 4C), demonstrating that the decreased expression of these variants was not due to nonspecific effects such as toxicity or inhibition of the expression of cell surface molecules.

We tested whether the D57S or D57A substitution had any effect on the presentation of foreign peptides to I-A-restricted T hybridomas in experiments in which surface expression of these variants was made equivalent to WT I-A by co-transfecting the invariant chain. Fig. 5shows that COS cells expressing variant heterodimers D57S or D57A were at least 25-fold less efficient at presentation of Ars-Ova(33-49) peptide to the D5h T hybridoma compared to WT I-A. These substitutions also diminished presentation of Ova(323-339) peptide to T hybridoma 3DO-54.8 and cI(12-26) peptide to 9C127 (data not shown), both of which recognize peptides presented by I-A. These results demonstrate that the identity of the side chain at position 57 in the I-A -chain not only plays a role in surface expression but also influences peptide presentation.

Figure 5: Response of D5 T cell hybridoma to antigenic peptide presented by COS cells expressing wild-type or variant I-A containing substitutions at Asp-57. In A, COS cells co-transfected with WT or variant I-A D57S and D57A heterodimers and the invariant chain were tested for the ability to present Ars-Ova(33-49) to D5h cells. Relative levels of surface expression of WT and variant heterodimers determined by flow cytometry are shown in the inset. In B, the relative ability of these variant heterodimers to present antigenic peptides to D5h in independent experiments is summarized as in Fig. 3B.

Charge Reversal at Arg-80 Diminishes Surface Expression of I-A and Presentation of Foreign Peptide but Compensates Charge Reversal at Asp-57

Figure 6: Flow cytometric analysis of wild-type or variant I-A substituted at Arg-80 transfected into COS cells. COS cells transfected with WT -chain alone or WT or variant - and -chains, co-transfected with control (C) or invariant chain (Ii) DNA, were analyzed by flow cytometry using control (28-14-8S) or I-A-reactive mAb. Surface expression was calculated as fluorescence units.

We tested whether charge reversal of Arg-80 could compensate the defect in surface expression of the charge-reversed variants of Asp-57. Fig. 6shows that surface expression of the variant D57R was partly restored when it was paired with the variant R80E, even in the absence of the invariant chain. Surface expression of the variant D57R paired with R80E was further enhanced in the presence of the invariant chain. Similar results were obtained when R80E was transfected along with D57K or when R80D was transfected with D57K or D57R (data not shown). Thus, the defect in surface expression of I-A chains containing individual charge reversals at Asp-57 or Arg-80 can be partially reversed by expression of the partner charge-reversed chain, suggesting direct interaction between Arg-80 and Asp-57.

We tested the consequences of charge reversal at Arg-80 or Asp-57 on the presentation of foreign peptide. Fig. 7shows that the singly substituted variants R80E or R80D paired with the WT -chain were 50-100-fold less efficient at presentation of Ars-Ova(33-49) peptide to D5h cells than WT I-A when expressed on the cell surface in COS cells co-transfected with the invariant chain. In addition, when matched for surface expression with WT I-A, the doubly substituted variant R80E/D57R was also 50-100-fold less efficient at peptide presentation than WT I-A (Fig. 7). These single- and double-site substitutions also decreased presentation of Ova(323-339) peptide to 3DO-54.8 cells (data not shown). Therefore, as do substitutions at Asp-57, substitutions at Arg-80 affect surface expression of I-A and its ability to present foreign peptides.

Figure 7: Response of D5h T cell hybridoma to antigenic peptide presented by COS cells expressing variant I-A heterodimers substituted at Arg-80 and Asp-57. In A, COS cells co-transfected with the invariant chain and WT or variant I-A as indicated were tested for the ability to present Ars-Ova(33-49) peptide to T cell hybridoma D5h. In the insets, relative levels of surface expression of I-A heterodimers were determined by flow cytometry and calculated as fluorescence units. In B, the relative ability of these variant heterodimers to present antigenic peptide to D5h cells in independent experiments is summarized as in Fig. 3B.

Surface Expression of Mutant I-A Heterodimers Is Restored at Reduced Temperature

Fig. 8shows that singly substituted variant class II heterodimers containing charge reversal at Asp-57 or Arg-80, which were expressed poorly at 37 °C in the absence of the invariant chain, appeared on the surface of COS cells cultured at room temperature. After room temperature incubation, the level of surface expression of singly substituted charge-reversed variants R80E or D57R was identical to expression of the doubly substituted charge-swapped variant R80E/D57R and WT I-A heterodimers, as determined by staining with mAb MK-D6 (Fig. 8A) and - and -chain reactive mAb (Fig. 8B). The increase in fluorescence of singly substituted charge-reversed variants at room temperature was due to an increase in both the number of cells expressing I-A as well as the intensity of those cells (Fig. 8A). On the other hand, the decrease in fluorescence of R80E/D57R and WT heterodimers (Fig. 8B) was primarily due to the reduction of cells expressing I-A at room temperature, rather than a decrease in the intensity of staining (Fig. 8A). It is important to note that the pattern of staining of heterodimers containing WT or variant - and -chains expressed in COS cells at room temperature and at 37 °C was similar when tested with the - and -chain-specific mAb (Fig. 8B), demonstrating that the differences in temperature did not cause selective changes in the epitopes or conformations of I-A molecules detectable by these mAb. These results demonstrate that surface expression of mutant class II molecules expressed poorly at 37 °C in the absence of the invariant chain can be enhanced at reduced temperature.

Figure 8: Surface expression of mutant I-A heterodimers in COS cells cultured at reduced temperature. COS cells transfected with WT or variant - and -chain DNA of I-A were cultured for 2 days at 37 °C (top row) or 1 day at 37 °C followed by 1 day at room temperature (RT, bottom row), and analyzed by flow cytometry using I-A-reactive mAb. In A, results are expressed as histograms of 5000 living cells analyzed by staining with mAb MK-D6. Bars indicate regions used to calculate fluorescence units. In B, fluorescence units were calculated for staining with control (28-14-8S) or I-A-reactive mAb. These results are representative of two separate experiments.

DISCUSSION

We have conducted extensive mutational analysis of negatively charged residues in the peptide/TCR-binding domain of the class II molecule I-A, which has been modeled onto the structure of HLA-DR1 (Fig. 9). We have demonstrated the importance of the ion pair Arg-80 and Asp-57 in both surface expression of and peptide presentation by class II heterodimers. Our results suggest that interactions in the peptide-binding site, including the formation of the salt-bridge between Arg-80 and Asp-57 and possibly peptide binding, are important for folding and surface expression of class II molecules at 37 °C. These results agree with previous studies demonstrating that residues pointing into the peptide-binding site within the 1- and 1-domains influence surface expression of class II molecules(32, 36, 57, 58) . The mutant class II molecules described here resemble haplotype-mismatched class II - and -chains, which are partially blocked in egress to the medial Golgi compartment(40) , and mutant heterodimers that are blocked in a relatively late stage of transport to the cell surface(57) . We hypothesize that an important step in the transport of class II molecules to the cell surface can be blocked by disrupting the salt-bridge between Arg-80 and Asp-57 or the hydrogen bonds between these residues and bound peptides.

The ion pair Asp-57 and Arg-76, which corresponds to Arg-80 in murine class II -chains, is present in nearly all class II molecules (9) and is likely to represent an important universal structural feature of class II molecules(8) . Based on the structure of HLA-DR1 (8) , the formation of this salt-bridge replaces the hydrogen-bonding network found at the end of the peptide-binding site of class I molecules in which the C termini of bound peptides are buried. Asp-57, present in the -chain of most class II molecules, occupies a position analogous to Thr-143 conserved in all class I 2-domains; similarly, Arg-80, conserved in all -chains of class II molecules, occupies a position analogous to Tyr-84, conserved throughout all class I 1-domains(9) . Together Tyr-84 and Thr-143 in class I heavy chains form hydrogen bonds with C termini of foreign and self-peptides(3, 4, 5, 6) . The side chains of Asp-57 and Arg-80 in HLA-DR1 occupy roughly the same position as the side chains of Tyr-84 and Thr-143 in class I molecules, yet are located about 2 Å lower in the class II molecule due to slight changes in the -helices(7) . This lowering ``opens'' this end of the peptide-binding site of class II molecules, allowing longer peptides to bind and extend out of the site(7, 8) . In addition, Asp-57 and Arg-80 form hydrogen bonds with amide hydrogen and carbonyl oxygen atoms of bound peptides.

The interchain salt-bridge formed between Asp-57 and Arg-80 and the hydrogen bonds they form with the backbone of peptides bound to class II heterodimers might contribute a great deal to the stability of the class II heterodimer and its ability to bind peptides. The reversal of charge in singly substituted mutants D57R, D57K, R80E, and R80D would bring together in close proximity similarly charged residues in the peptide-binding domains of assembling class II heterodimers, potentially destabilizing a portion of the interface between the - and -chains and preventing these mutant class II heterodimers from adopting a conformation such that they can bind peptides efficiently. Similarly, the substitution of Ser or Ala for Asp-57 disrupts the salt-bridge, but unlike charge reversal at Asp-57, this substitution might be less disruptive because only one charged residue, Arg-80, remains unpaired. However, since the remaining unmutated charged residue is still available to hydrogen bond to the main chain of a bound peptide, the singly substituted mutant class II molecules might be stabilized to some degree when provided with peptide. The pairing of charge-swapped -chains R80E or R80D with -chains D57R or D57K restores a salt-bridge between - and -chains; however, these variants might not bind peptides efficiently because hydrogen bonding between residues at these positions and carbonyl oxygen and amide hydrogen atoms of bound peptides is disrupted. Alternatively, the new charge-swapped salt-bridge might not be as strong as the original WT salt-bridge because the I-A microenvironment that stabilizes the WT salt-bridge might destabilize the charge-swapped salt-bridge, as predicted on theoretical grounds(59) .

Because the substitutions for Arg-80 and Asp-57 affect both suface expression of I-A and presentation of peptides to the TCR, we propose that the defect in efficient surface expression of the variant heterodimers results from intracellular editing of the heterodimers as incorrectly folded proteins. Class II - and -chains are synthesized initially in the endoplasmic reticulum as heterodimers complexed with the invariant chain and devoid of tightly bound peptides(21) . The invariant chain forms multimers with class II heterodimers(60) , precludes the binding of endogenous peptides (61, 62) and may, like low affinity peptide binding, prevent the aggregation and denaturation of peptide-free class II heterodimers at physiological temperature(24) . The invariant chain directs the intracellular transport of class II heterodimers(63) , which includes passage through the Golgi complex and subsequent entry into the endocytic pathway. In specialized endocytic compartments(64, 65, 66) , the invariant chain is proteolytically processed and dissociates from class II heterodimers, resulting in the stable binding of peptides to class II molecules and their transport to the cell surface. Class II heterodimers that fail to acquire peptide undergo aggregation and denaturation, are presumably recognized as mis-folded proteins, and are blocked from transport to the cell surface(23) . The class II mutants described in this report that are defective in surface expression might be inactivated by aggregation, denaturation, or degradation more rapidly than WT I-A heterodimers. In the presence of the invariant chain, surface expression of these variants is partly restored, perhaps because the invariant chain stabilizes the association of the nascent polypeptides, provides a peptide to the peptide-binding site, or enhances the transport of the variant heterodimers to a subcellular compartment where peptides can be supplied.

In addition, the mutant class II molecules described here resemble class I molecules that fail to appear on the surface of RMA-S cells at physiological temperature but reappear at the cell surface at reduced temperature(56) . It is likely that at reduced temperature, both class I and II MHC molecules do not require bound peptide for surface expression because, when expressed in insect cells cultured at reduced temperature, they appear on the cell surface apparently devoid of bound peptide(22, 55) . Perhaps at reduced temperature, rates of inactivation of peptide-deficient MHC molecules and the mutant class II molecules described here are decreased. Our results support the hypothesis that both class I and II molecules might be best suited for surface expression at physiological temperature only when bound with peptide.

Finally, the importance of Arg-80 and Asp-57 in surface expression and the function of I-A might explain why these residues are conserved throughout nearly all class II molecules. Several naturally occurring class II molecules have been identified that contain substitutions at position -57, which are analogous to the variants D57S and D57A described in this report. It is noteworthy that these alleles have been implicated in autoimmune disease. Deviations from Asp-57 in human class II molecule -chains have been observed in alleles associated with insulin-dependent diabetes mellitus, including DQw3.2, DQw2, DQw1.1, and DQw1.AZH, in which Ser, Val, or Ala has been substituted for Asp-57(67) . In the I-A -chain of non-obese diabetic mice, Ser is substituted for Asp-57(68) . Although such alleles have other substitutions in addition to the change for Asp-57 that might account for the association of these alleles with diabetes, the identity of the side chain at -chain position 57 appears to determine susceptibility and resistance to diabetes: the presence of Asp appears to be protective against diabetes(67) . Expression of these variant forms of class II molecules might result in the presentation of an abnormal array of self- or foreign peptides or an altered conformation of class II molecule on the cell surface. Indeed, self-peptides eluted from I-A molecules of non-obese diabetic mice are reportedly distinct from other peptides that bind class II molecules in that they possess acidic C-terminal residues(69) . Thus, antagonists directed at the peptide-binding site of the variant class II molecule might be designed that selectively inhibit presentation of these abnormal polypeptides.

FOOTNOTES

*

This investigation was supported by National Institutes of Health Grant AI22900 (to A. R.) and a Post-doctoral Fellowship from the Medical Research Council of Canada (to K. T. Y. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Small Molecule Drug Discovery Group, Genetics Institute, Cambridge, MA 02140.

¶

To whom correspondence should be addressed: B465, Dana-Farber Cancer Institute, Boston, MA 02115. Tel.: 617-375-8265. Fax: 617-375-8308.

()

The abbreviations used are: MHC, major histocompatibility complex; TCR, T cell antigen receptor; Ars-, arsonate; mAb, monoclonal antibody; HPLC, high performance liquid chromatography; IL, interleukin; WT, wild-type.

ACKNOWLEDGEMENTS

REFERENCES

1. Germain, R. N. (1994) Cell 76,287-299 [CrossRef][Medline] [Order article via Infotrieve]
2. Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L., and Wiley, D. C. (1987) Nature 329,506-512 [CrossRef][Medline] [Order article via Infotrieve]
3. Madden, D. R., Gorga, J. C., Strominger, J. L., and Wiley, D. C. (1991) Nature 353,321-325 [CrossRef][Medline] [Order article via Infotrieve]
4. Fremont, D. H., Matsumura, M., Stura, E. A., Peterson, P. A., and Wilson, I. A. (1992) Science 257,919-927 [Abstract/Free Full Text]
5. Madden, D. R., Gorga, J. C., Strominger, J. L., and Wiley, D. C. (1992) Cell 70,1035-1048 [CrossRef][Medline] [Order article via Infotrieve]
6. Silver, M. L., Guo, H.-C., Strominger, J. L., and Wiley, D. C. (1992) Nature 360,367-369 [CrossRef][Medline] [Order article via Infotrieve]
7. Brown, J. H., Jardetzky, T. S., Gorga, J. C., Stern, L. J., Urban, R. G., Strominger, J. L., and Wiley, D. C. (1993) Nature 364,33-39 [CrossRef][Medline] [Order article via Infotrieve]
8. Stern, L. J., Brown, J. H., Jardetzky, T. S., Gorga, J. C., Urban, R. G., Strominger, J. L., and Wiley, D. C. (1994) Nature 368,215-221 [CrossRef][Medline] [Order article via Infotrieve]
9. Brown, J. H., Jardetzky, T., Saper, M. A., Samraoui, B., Bjorkman, P. J., and Wiley, D. C. (1988) Nature 332,845-850 [CrossRef][Medline] [Order article via Infotrieve]
10. Van Bleek, G. M., and Nathenson, S. G. (1990) Nature 348,213-216 [CrossRef][Medline] [Order article via Infotrieve]
11. Rötzschke, O., Falk, K., Deres, K., Schild, H., Norda, M., Metzger, J., Jung, G., and Rammensee, H.-G. (1990) Nature 348,252-254 [CrossRef][Medline] [Order article via Infotrieve]
12. Falk, K., Rötzschke, Stevanoviç, S., Jung, G., and Rammensee, H.-G. (1991) Nature 351,290-296 [CrossRef][Medline] [Order article via Infotrieve]
13. Jardetzky, T. S., Lane, W. S., Robinson, R. A., Madden, D. R., and Wiley, D. C. (1991) Nature 353,326-329 [CrossRef][Medline] [Order article via Infotrieve]
14. Hunt, D. F., Henderson, R. A., Shabanowitz, J., Sakaguchi, K., Michel, H., Sevilir, N., Cox, A. L., Appella, E., and Engelhard, V. H. (1992) Science 255,1261-1263 [Abstract/Free Full Text]
15. Rudensky, A. Y., Preston-Hurlburt, P., Hong, S.-C., Barlow, A., and Janeway, C. A. (1991) Nature 353,622-627 [CrossRef][Medline] [Order article via Infotrieve]
16. Chicz, R. M., Urban, R. G., Lane, W. S., Gorga, J. C., Stern, L. J., Vignali, D. A. A., and Strominger, J. L. (1992) Nature 358,764-768 [CrossRef][Medline] [Order article via Infotrieve]
17. Hunt, D. F., Michel, H., Dickinson, T. A., Shabanowitz, J., Cox, A. L., Sakaguchi, K., Appella, E., Grey, H. M., and Sette, A. (1992) Science 256,1817-1820 [Abstract/Free Full Text]
18. Townsend, A., Öhlén, C., Bastin, J., Ljunggren, H.-G., Foster, L., and Kärre, K. (1989) Nature 340,443-448 [CrossRef][Medline] [Order article via Infotrieve]
19. Townsend, A., Elliott, T., Cerundolo, V., Foster, L., Barber, B., and Tse, A. (1990) Cell 62,285-295 [CrossRef][Medline] [Order article via Infotrieve]
20. Sadegh-Nasseri, S., and Germain, R. N. (1991) Nature 353,167-170 [CrossRef][Medline] [Order article via Infotrieve]
21. Germain, R. N., and Hendrix, L. R. (1991) Nature 353,134-139 [CrossRef][Medline] [Order article via Infotrieve]
22. Stern, L. J., and Wiley, D. C. (1992) Cell 68,465-477 [CrossRef][Medline] [Order article via Infotrieve]
23. Germain, R. N., and Rinker, A. G. (1993) Nature 363,725-728 [CrossRef][Medline] [Order article via Infotrieve]
24. Sadegh-Nasseri, S., Stern, L. J., Wiley, D. C., and Germain, R. N. (1994) Nature 370,647-650 [CrossRef][Medline] [Order article via Infotrieve]
25. Rao, A., Faas, S. J., and Cantor, H. (1984) J. Exp. Med. 159,479-494 [Abstract/Free Full Text]
26. Nalefski, E. A., and Rao, A. (1993) J. Immunol. 150,3806-3816 [Abstract]
27. Nalefski, E. A., Kasibhatla, S., and Rao, A. (1992) J. Exp. Med. 175,1553-1563 [Abstract/Free Full Text]
28. Kasibhatla, S., Nalefski, E. A., and Rao, A. (1993) J. Immunol. 151,3140-3151 [Abstract]
29. Nalefski, E. A., Wong, J. G. P., and Rao, A. (1990) J. Biol. Chem. 265,8842-8846 [Abstract/Free Full Text]
30. Shimonkevitz, R., Kappler, J., Marrack, P., and Grey, H. (1983) J. Exp. Med. 158,303-316 [Abstract/Free Full Text]
31. Guillet, J.-G., Lai, M.-Z., Briner, T. J., Smith, J. A., and Gefter, M. L. (1986) Nature 324,260-262 [CrossRef][Medline] [Order article via Infotrieve]
32. Braunstein, N. S., and Germain, R. N. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,2921-2925 [Abstract/Free Full Text]
33. Glutzman, Y. (1981) Cell 23,175-182 [CrossRef][Medline] [Order article via Infotrieve]
34. Gillis, S., and Smith, K. A. (1977) Nature 268,154-156 [CrossRef][Medline] [Order article via Infotrieve]
35. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
36. Braunstein, N. S., Germain, R. N., Loney, K., and Berkowitz, N. (1990) J. Immunol. 145,1635-1645 [Abstract]
37. Miller, J., and Germain, R. N. (1986) J. Exp. Med. 164,1478-1489 [Abstract/Free Full Text]
38. Engel, I., Ottenhoff, T. H. M., and Klausner, R. D. (1992) Science 256,1318-1321 [Abstract/Free Full Text]
39. Nardone, J., Gerald, C., Rimawi, L., Song, L., and Hogan, P. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,4412-4416 [Abstract/Free Full Text]
40. Layet, C., and Germain, R. N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,2346-2350 [Abstract/Free Full Text]
41. Koch, N., Hämmerling, G. J., Tada, N., Kimura, S., and Hämmerling, U. (1982) Eur. J. Immunol. 12,909-914 [Medline] [Order article via Infotrieve]
42. Kappler, J. W., Skidmore, B., White, J., and Marrack, P. (1981) J. Exp. Med. 153,1198-1214 [Abstract/Free Full Text]
43. Ozato, K., Mayer, N., and Sachs, D. H. (1982) Transplantation 34,113-118 [Medline] [Order article via Infotrieve]
44. Symington, F., and Sprent, J. (1981) Immunogenetics 14,53-61 [CrossRef][Medline] [Order article via Infotrieve]
45. Ozato, K., and Sachs, D. H. (1981) J. Immunol. 126,317-321 [Abstract]
46. Bhattachary, A., Dorf, M. E., and Springer, T. A. (1981) J. Immunol. 127,2488-2495 [Abstract]
47. Metlay, J. P., Witmer-Pack, M. D., Agger, R., Crowley, M. T., Lawless, D., and Steinman, R. M. (1990) J. Exp. Med. 171,1753-1771 [Abstract/Free Full Text]
48. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E., and Bluestone, J. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,1374-1378 [Abstract/Free Full Text]
49. Ozato, K., Mayer, N., and Sachs, D. H. (1980) J. Immunol. 124,533-540 [Abstract]
50. Ozato, K., Hansen, T. H., and Sachs, D. H. (1980) J. Immunol. 125,2473-2477 [Abstract]
51. Kubo, R. T., Born, W., Kappler, J. W., Marrack, P., and Pigeon, M. (1989) J. Immunol. 142,2736-2742 [Abstract]
52. Buerstedde, J.-M., Pease, L. R., Bell, M. P., Nilson, A. E., Buerstedde, G., Murphy, D., and McKean, D. J. (1988) J. Exp. Med. 167,473-487 [Abstract/Free Full Text]
53. Viville, S., Neefjies, J., Lotteau, V., Dierich, A., Lemeur, M., Ploegh, H., Benoist, C., and Mathis, D. (1993) Cell 72,635-648 [CrossRef][Medline] [Order article via Infotrieve]
54. Peterson, M., and Miller, J. (1990) Nature 345,172-174 [CrossRef][Medline] [Order article via Infotrieve]
55. Jackson, M. R., Song, E. S., Yang, Y., and Peterson, P. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,12117-12121 [Abstract/Free Full Text]
56. Ljunggren, H.-G., Stam, N. J., Öhlen, C., Neefjes, J. J., Höoglund, P., Heemels, M.-T., Bastin, J., Schumacher, T. N. M., Townsend, A., Kärre, K., and Ploegh, H. L. (1990) Nature 346,476-480 [CrossRef][Medline] [Order article via Infotrieve]
57. Griffith, I. J., Nabavi, N., Ghogawala, Z., Chase, C. G., Rodriguez, M., McKean, D. J., and Glimcher, L. H. (1988) J. Exp. Med. 167,541-555 [Abstract/Free Full Text]
58. Lechler, R. I., Sant, A. J., Braunstein, N. S., Sekaly, R., Long, E., and Germain, R. N. (1990) J. Immunol. 144,329-333 [Abstract]
59. Hwang, J.-K., and Warshel, A. (1988) Nature 334,270-272 [CrossRef][Medline] [Order article via Infotrieve]
60. Roche, P. A., Marks, M. S., and Cresswell, P. (1991) Nature 354,392-394 [CrossRef][Medline] [Order article via Infotrieve]
61. Roche, P. A., and Cresswell, P. (1990) Nature 345,615-618 [CrossRef][Medline] [Order article via Infotrieve]
62. Teyton, L., O'Sullivan, D., Dickson, P. W., Lotteau, V., Sette, A., Fink, P., and Peterson, P. A. (1990) Nature 348,39-44 [CrossRef][Medline] [Order article via Infotrieve]
63. Lotteau, V., Teyton, L., Peleraux, A., Nilsson, T., Karlsson, L., Schmid, S., Quaranta, V., and Peterson, P. A. (1990) Nature 348,600-605 [CrossRef][Medline] [Order article via Infotrieve]
64. Amigorena, S., Drake, J. R., Webster, P., and Mellman, I. (1994) Nature 369,113-120 [CrossRef][Medline] [Order article via Infotrieve]
65. Tulp, A., Verwoerd, D., Dobberstein, B., Ploegh, H. L., and Peters, J. (1994) Nature 369,120-126 [CrossRef][Medline] [Order article via Infotrieve]
66. West, M. A., Lucocq, J. M., and Watts, C. (1994) Nature 369,147-151 [CrossRef][Medline] [Order article via Infotrieve]
67. Todd, J. A., Bell, J. I., and McDevitt, H. O. (1987) Nature 329,599-604 [CrossRef][Medline] [Order article via Infotrieve]
68. Acha-Orbea, H., and McDevitt, H. O. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,2435-2439 [Abstract/Free Full Text]
69. Reich, E.-P., von Grafenstein, H., Barlow, A., Swenson, K. E., Williams, K., and Janeway, C. A. (1994) J. Immunol. 152,2279-2288 [Abstract]

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