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The Journal of Immunology, 1998, 161: 2961-2967.
Copyright © 1998 by The American Association of Immunologists

The Inability of the Nonobese Diabetic Class II Molecule to Form Stable Peptide Complexes Does Not Reflect a Failure to Interact Productively with DM1

Mary Peterson and Andrea J. Sant2

Department of Pathology, Committees on Immunology and Cancer Biology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Sequence variability in MHC class II molecules plays a major role in genetically determined susceptibility to insulin-dependent diabetes mellitus (IDDM). It is not yet clear whether MHC class II polymorphism allows selective binding of diabetogenic peptides or regulates some key intracellular events associated with class II-restricted Ag presentation. In this study, we have employed gene transfer techniques to analyze the intracellular events that control peptide acquisition by the unique class II molecule expressed by nonobese diabetic mice (I-Ag7). This structurally unique class II molecule fails to demonstrate stable binding to antigenic peptides and fails to undergo the conformational change associated with stable peptide binding to class II molecules. The experiments reported here demonstrate that I-Ag7 can productively associate with two protein cofactors important in class II-restricted Ag presentation, invariant chain (Ii) and DM. DM participates in the removal of the Ii-derived class II-associated Ii chain peptide and the p12 degradation product from the I-Ag7 molecule. In addition, I-Ag7 undergoes a conformational change when DM is expressed within the APC. Finally, DM can mediate accumulation of peptide/class II complexes on the surface of APCs. Collectively, our experiments indicate that the failure of the I-Ag7 molecule to stably bind peptide cannot be attributed to a failure to interact with the DM or Ii glycoproteins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Insulin-dependent diabetes mellitus (IDDM)3 is a complex autoimmune disease that ultimately leads to the T cell-mediated destruction of pancreatic ß cells (reviewed in Refs. 1 and 2). Susceptibility to IDDM is controlled in part by the genes within the class II region of the MHC. The important role that MHC class II molecules play in the etiology of the disease is supported by data demonstrating that CD4 T cells can transfer disease to otherwise disease-free individuals and that depletion of CD4 cells prevents disease (3, 4, 5, 6, 7). It has been speculated that a key event in the initiation of the disease is the breaking of tolerance to the islet-specific Ags that gain access to host MHC class II molecules by an unknown mechanism. MHC-restricted presentation of peptides derived from these Ags leads to the activation of autoreactive CD4 T cells.

Epidemiologic analyses of humans suffering from IDDM have indicated in most populations that the strongest association between disease susceptibility and resistance maps to allelism in HLA-DQ (1, 8, 9, 10, 11, 12). One feature that has been noted in those DQ molecules that predispose individuals to diabetes is polymorphism at position 57 of the ß-chain. This feature is shared by the class II molecule that is expressed by the nonobese diabetic (NOD) mouse (13), which is a well-defined animal model for IDDM (14, 15, 16, 17, 18). A change at position 57 (Asp to Ser or other uncharged amino acids) is thought to be of particular significance, because such a change leads to the loss of the potential for an interchain salt bridge with the class II {alpha}-chain (19, 20, 21). A common structural feature shared by many diabetogenic class II molecules suggests the possibility that there is a generalized feature in the biochemistry of those class II molecules that contributes to the autoimmune process.

There are two general types of biochemical events that polymorphism in class II might affect. First, sequence-dependent differences in the peptide-binding pocket of class II may select for diabetogenic peptides. Alternatively, these class II molecules may have atypical intracellular processing that allows them unique access to autoantigens or to defects in tolerance induction. In this study, we have evaluated the intracellular events that control peptide acquisition by class II and have focused on the ability of the NOD class II molecule to associate with DM glycoproteins. DM is thought to function primarily within the compartments of the endocytic pathway. The major known function of this protein is to release the invariant chain (Ii)-derived peptide segments that normally occupy the peptide-binding pocket of class II and to facilitate the peptide loading of class II in endosomal compartments (reviewed in Refs. 22 and 23). Recent studies indicate that DM may also facilitate the release of self or antigenic peptides from the class II molecule (24, 25, 26, 27), allowing for the selective accumulation of peptides that have a very stable interaction with the class II molecule. Because DM is a critical modulator of peptide binding to class II, it is important to determine whether the diabetes-prone class II molecules interact normally with this protein.

The findings that the I-Ag7 molecule does not form SDS-stable dimers, binds antigenic peptide in an unstable way, and rapidly turns over within cells (28) all suggest that this molecule may fail to interact productively with DM and consequently may not accumulate stably bound peptides. In the experiments presented in this paper, we have used gene transfer techniques to construct cells that express the NOD (I-Ag7) class II molecule and differ only in the expression of genetically defined Ii and DM proteins. This experimental design has allowed us to evaluate the contribution of MHC class II allelic polymorphism to the biochemical events that are associated with peptide acquisition by the class II molecule.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Reagents and cell lines

Ltk- cells were transfected as we have described previously (29) by CaPO4 precipitation with genes encoding the I-Ad {alpha} and I-Ag7 ß (NOD), I-Ad, or I-Ak. Cloned cells from these transfections were screened for class II surface expression with mAb and FACS, and positive clones were subsequently transfected with a murine Ii (gIi) construct and/or the cDNAs for the Ma and Mb-1 chains of H2-DM as we have described previously (24, 30). Clones from these transfections were screened for the presence of Ii by Western blotting and for DM mRNA by RT-PCR. All cells were maintained at 37°C and 10% CO2 in DMEM containing 10% bovine calf serum plus 10 mM HEPES, 4 mM glutamine, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate (Life Technologies, Gaithersburg, MD) as well as selective drug to maintain the expression of the transfected gene(s).

mAbs and flow cytometry

mAbs reacting with I-Ag7 and I-Ak (10.2.16) and with I-Ad (MKD6 and M5114.15.2) were obtained from the American Type Culture Collection (Manassas, VA). Cells secreting 40M, 39E, 40F, 40L, and 40B (31) as well as K24-199 (32) and the anti-Ii Ab In-1 (33) were provided by Jim Miller (University of Chicago). The cell surface expression of the MHC class II molecules was evaluated by flow cytometry as described previously (24, 30). Briefly, cells were incubated successively with mAbs and subsequently with secondary-step FITC-conjugated goat anti-mouse Igs (IgA, IgG, and IgM) (FITC-GAM) (Sigma, St. Louis, MO); cells were then analyzed on a FACScan cytofluorometer (Becton Dickinson, Sunnyvale, CA). Background fluorescence was determined by incubating cells with media alone or with an irrelevant mAb and then with FITC-conjugated goat anti-mouse Ig.

RT-PCR

Measurements of DM-specific mRNA were accomplished by RT-PCR as we have described previously (30). Briefly, cells were solubilized in YRLB buffer (0.5 M NaCl, 0.2 M Tris (pH 7.5), 10 mM EDTA, and 1% SDS) and then extracted twice with phenol/chloroform/isoamyl alcohol. RNA was precipitated with a 2.5-fold excess of 100% ethanol at -20°C overnight, pelleted, and dissolved in distilled water. A total of 2 µg of RNA was used in the reverse transcription reaction to obtain the cDNA. One-twentieth of the cDNA was used in the PCR; this amount is a substrate concentration that is within the linear range of amplification for both actin and H2-DM.

Western blot analysis

Cells were lysed in 6 mM CHAPS (3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate), 150 mM NaCl, and 50 mM Tris (pH 7.8) plus 50 mM iodoacetamide and protease inhibitors (34). Postnuclear supernatants were incubated with class II mAbs that were bound to protein A-Sepharose (Pharmacia, Piscataway, NJ). Immunoprecipitated proteins were eluted from the protein A-Sepharose with 2% SDS, 10% glycerol, and 0.0625 M Tris (pH 6.8) at room temperature and were divided into two aliquots that were either maintained at room temperature or boiled for 3 min. Samples were electrophoresed on SDS-10% PAGE and transferred to nitrocellulose as described previously (34). Nitrocellulose membranes were blocked with a solution containing 5% dry milk in water and then probed with the mouse anti-I-Ak and I-Ag7 mAb (10.2.16) and/or the rat anti-I-Ad M5/114 mAb in 5% milk overnight. Bound Abs were detected by incubating the membranes with a mixture of anti-mouse- and/or anti-rat-conjugated horseradish peroxidase-linked secondary Abs. The blots were developed by chemiluminescence using LumiGLO (Kirkegaard & Perry Laboratories, Gaithersburg, MD). For Tris-Tricine immunoblots, the samples were fractionated on a 16.5% acrylamide gel for 20 h at 90 V as described previously (35) and then treated as described above, except that In-1 was used to detect Ii.

Metabolic labeling

For analysis of class II-associated Ii chain peptide (CLIP) binding to class II, cells were labeled overnight with 250 µCi/ml [35S]methionine (Amersham, Arlington Heights, IL), and class II molecules were isolated from detergent lysates by immunoprecipitation. Immunoprecipitated proteins were fractionated by SDS-12.5% PAGE; gels were treated successively for 20 min each with 30% methanol/10% acetic acid, H2O, and Flouro-Hance (Research Products, Mt. Prospect, IL), dried, and exposed to film at -70°C.

Ag-presentation assays

The OVA-specific, I-Ag7-restricted T cell hybridomas were derived by the fusion of OVA-specific T cells to the TCR-negative variant of the T cell lymphoma BW5147. The T cells that were used for fusion were derived by in vivo priming with 50 µl of 1 mg/ml OVA in CFA (Sigma) and maintained by subsequent restimulations in vitro with Ag and syngeneic splenocytes for 10 days. Fusion was performed at 3 days after antigenic stimulation. After cloning, Ag-specific cells were identified by coculture with OVA and NOD-derived spleen cell APCs. T cell Ag-presentation assays using I-Ag7-expressing transfectants were performed as described previously (29, 30). Briefly, 25,000 L cell transfectants were incubated with graded doses of OVA. A total of 50,000 T cell hybridomas were added to a final volume of 200 µl in flat-bottom, 96-well plates. After overnight coculture, supernatants were harvested, frozen, and tested for IL-2 content using the CTLL indicator and an MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to detect viable cells after 16 h of culture of CTLL with the test supernatants.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The I-Ag7 class II molecule is unable to form SDS-stable dimers

The NOD mouse expresses a unique MHC class II I-A molecule (I-Ag7) that is composed of a unique ß-chain (17) that has some structural similarity to I-Aßk and I-Aßd (with the IDDM-associated change at Asp 57) and an {alpha}-chain identical with I-A{alpha}d. Previous experiments have indicated that the I-Ag7 class II molecule binds peptides in a relatively unstable manner (28). One measure of stable peptide binding to the class II molecule is a resistance to SDS-induced chain dissociation (36, 37). "SDS-stable" dimers can be detected in unboiled samples as a 58- to 60-kDa protein form. Upon boiling, the noncovalently linked {alpha}- and ß-chains dissociate and migrate as free 33-kDa and 28-kDa forms, respectively.

We analyzed I-Ag7 class II molecules isolated from NOD spleen cells for their ability to form SDS-stable dimers. The structurally related I-Ak molecule, which shares some mAb epitopes with I-Ag7, was isolated from C3H spleen cells and examined in parallel. Figure 1Go shows the results of this analysis. While I-Ak molecules form readily detectable levels of SDS-stable dimers, the I-Ag7 molecules isolated from NOD spleen cells were detected only as free chains in both the boiled and unboiled samples and are thus primarily in an SDS-unstable form. This finding is in agreement with previous results indicating that the I-Ag7 class II molecules have low (38) or undetectable (28) levels of SDS-stable dimers when expressed in NOD (28, 38) or F1 animals (28) that express other class II allelic products that display readily detectable SDS-stable dimers.



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  		FIGURE 1. I-Ag7 molecules isolated from the spleen fail to form SDS-stable dimers. Spleen cells from NOD or C3H mice were solubilized in CHAPS and postnuclear supernatants were incubated with the mAb 10.2.16 to isolate the I-A molecules. Immunoprecipitated proteins (corresponding to 2 x 107 cell equivalents/lane) were fractionated by SDS-10% PAGE and transferred to nitrocellulose for Western blot analysis using 10.2.16 to detect the class II molecule. The positions of the SDS-stable dimers and the free ß-chain are shown.

 
The failure of the NOD class II molecule to form SDS-stable dimers could be due to several factors, including 1) a structural feature inherent to the I-Ag7 molecule that allows SDS to access the contact site(s) between the {alpha}- and ß-chains, 2) an inability of the I-Ag7 peptide-binding pocket to stably bind peptide, or 3) a defect in intracellular events involved in stable peptide binding to class II. To address the possibility that the intracellular environment of NOD spleen cells is not conducive to SDS-stable dimer formation, we assessed the biochemical properties of I-Ag7 in isolation from the other NOD-derived cellular processes. To accomplish this, we introduced the genes encoding I-Ag7 into a homogeneous cell line expressing the components that are known to allow Ag presentation and SDS-stable dimer formation for other allelic forms of class II. Previously, we have described a transfection model in which class II-negative L cell fibroblasts are reconstituted successively with genes encoding the class II molecule and subsequently with the known protein cofactors that participate in class II-restricted Ag presentation (24, 30). For most allelic forms of class II, SDS-stable dimer formation requires the participation of Ii and DM glycoproteins (34, 39, 40, 41, 42). The genes encoding A{alpha}d and Aßg7 were introduced into L cells and then supertransfected with genes encoding the murine Ii and/or murine DM molecules. This experimental design allowed us to independently assess the function of Ii and DM proteins on the I-Ag7 molecule.

Figure 2Go shows the characterization of the cells used in this study. Figure 2GoA represents surface staining of the derived cells, in which the mAb 10.2.16 was used to assess the plasma membrane expression of the I-Ag7 class II molecule. This analysis indicated that the cells used in our studies varied by less than twofold in the density of the class II molecules expressed at the cell surface. The results of Western blotting with an Ii-specific Ab are shown in Figure 2GoB. The Ii glycoprotein is not detectably expressed in the original cells used for transfection but is expressed in the cells that were transfected with the gene encoding this protein. Similarly, the RT-PCR analysis shown in Figure 2GoC indicates that the mRNA expression of DM could be detected in cells that had been transfected with cDNA constructs encoding the {alpha}- and ß-chain genes.



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  		FIGURE 2. Characterization of the cells used in this study. Cells transfected with genes encoding I-Ag7 alone (NOD) or that additionally express Ii (NODgIi) or Ii and DM (NODgIDM) were analyzed for their surface expression of class II molecules using the mAb 10.2.16 (A), for the Ii glycoprotein by Western blotting using the In-1 mAb (B), or for the mRNA levels of Mb-1 or Ma (encoding DM) by RT-PCR (C).

 
Cells expressing I-Ag7 with Ii and DM were then examined for their expression of SDS-stable class II dimers. Cells similarly derived that expressed two alternate alleles of class II were examined in parallel. The I-Ak and I-Ad allelic forms of class II were chosen for comparison due to structural similarity or identity with the NOD ß and {alpha}-chains, respectively. The MHC class II molecules expressed by the transfectants were isolated from detergent lysates using mAb that was specific for the class II dimer and were analyzed by Western blotting for the presence of SDS-stable dimer in the unboiled sample (Fig. 3Go). High levels of SDS-stable dimers were recovered in L cells that expressed I-Ak. Similarly, I-Ad expressed readily detectable quantities of SDS-stable dimers. In contrast to the results obtained with these nondiabetogenic class II molecules, our experiments indicated that the I-Ag7 molecules recovered from the transfected L cells were exclusively in the SDS-unstable form. Even after long exposures of gels to film, SDS-stable dimers were not detected in these cells. The results of this experiment indicate that the failure to form an SDS-stable {alpha}ß dimer reflects a structural element controlled by polymorphic sequences within the I-Ag7 molecule itself rather than altered cellular Ag processing machinery expressed within NOD spleen cells.



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  		FIGURE 3. The I-Ag7 allele of I-A fails to form an SDS-stable dimer. Transfected L cells expressing Ii and DM with either I-Ad (LADgIiDM), I-Ag7 (NODgIiDM), or I-Ak (LAKgIDM) were lysed and immunoprecipitated with anti-class II mAb (10.2.16 for I-Ak and I-Ag7 or MKD6 for I-Ad). Immunoprecipitated samples were eluted in SDS-sample buffer, divided in half, and either left at room temperature (unboiled (UB)) or boiled for 3 min (B) before their application to SDS-PAGE gels. Different input cell numbers were used to allow for similar exposure times for the different class II allelic products. We used 2 x 106, 1 x 106, and 3.5 x 106 cell equivalents per lane from LADgIiDM, LAKgIiDM, and NODgIiDM, respectively. Class II molecules were identified by Western blotting using a mixture of anti-Aßd and anti-Aßk/g7 mAbs (M5114 and 10.2.16, respectively). The positions of the SDS-stable {alpha}ß dimer and the free ß-chain are indicated by the top and bottom arrows, respectively.

 
Biochemical and serologic evidence for DM/I-Ag7 interactions

The preceding results indicate that the I-Ag7 molecule fails to undergo the conformational changes upon peptide binding that lead to resistance to SDS-induced denaturation. This phenotype is reminiscent of the phenotype displayed by the class II molecules expressed in the absence of the DM or Ii glycoproteins. Previous experiments (28, 38) have indicated that the I-Ag7 molecule does assemble with Ii. Our experiments (Fig. 4GoB and data not shown) confirm this observation and show that both p41 and p31 forms of Ii assemble with the I-Ag7 molecule. Therefore, we considered the possibility that the NOD class II molecule is unable to form SDS-stable dimers because it fails to interact productively with DM. It now appears evident that DM functions by direct interaction with the class II molecule (43) at a site in close proximity to the peptide-binding pocket (44). Thus, the failure to form SDS-stable dimers might reflect a structural feature in the I-Ag7 molecule that precludes an efficient interaction with DM. This was a particularly important question based on the finding that the peptides bound to the NOD class II molecule displayed unusually rapid off-rates (28). A failure to accumulate high-affinity peptides could be accounted for by a failure to interact productively with DM.



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  		FIGURE 4. DM facilitates removal of Ii-derived CLIP and p12 from I-Ag7. A, LADgI, LADgIiDM, NODgIi, or NODgIDM (see the legend to Fig. 2Go) were radiolabeled, and lysates were immunoprecipitated with either MKD6 (I-Ad cells) or 10.2.16 (I-Ag7 cells). Autoradiographs of SDS-12% PAGE gels in which the position of CLIP is indicated are shown. B, 10.2.16 immunoprecipitates were made from NODgIi or NODgIDM and probed in Western blots for Ii using In-1. The positions of Ii p31 and Ii p12 are shown. C, Radiolabeled cells expressing the indicated genes were lysed and immunoprecipitated with either 40M or 10.2.16. Autoradiographs of SDS-12% PAGE gels in which the positions of class II/Ii or CLIP are indicated are shown.

 
Several biochemical assays were performed to probe the potential for and consequences of an interaction of DM with the I-Ag7 molecule. The most extensively described activity of DM is the removal of the terminal degradation product of Ii (CLIP) (reviewed in Refs. 22 and 23). Therefore, we tested for the presence of CLIP in class II immunoprecipitates that had been prepared from radiolabeled I-Ag7 cells expressing Ii alone (NODIi) or Ii and DM (NODgIiDM). Cells expressing I-Ad were examined in parallel. Radiolabeled CLIP can be detected at or just ahead of the dye front on SDS-PAGE gels (44). Figure 4GoA shows that, similar to I-Ad, I-Ag7 molecules in cells expressing Ii alone have readily detectable CLIP in association with the class II molecule. In the presence of DM, CLIP can no longer be recovered in the class II immunoprecipitate that was prepared from the I-Ag7 cell lysate. This finding indicates that DM can interact with the I-Ag7 molecule and catalyze the release of CLIP from the class II-binding pocket. We subsequently tested whether DM could remove a larger proteolytic fragment of Ii from the I-Ag7 molecule. We have shown that DM can facilitate the removal of a naturally occurring processing intermediate of Ii (p12) from the class II molecule (30). Class II immunoprecipitates prepared from lysates of I-Ag7 cells expressing Ii alone or Ii and DM were tested for the presence of Ii-p12 by immunoblotting. As can be seen in Figure 4GoB, conventional Ii and p12 can be detected in the class II immunoprecipitate that was prepared from the I-Ag7 cell expressing Ii alone, but the p12 fragment is absent when the cells additionally express DM. The results of these biochemical studies suggest that both I-Ag7/CLIP and I-Ag7/p12 are substrates for DM.

DM expression in APCs has been shown to affect class II structure, which can be detected by changes in mAb epitopes (45, 46, 47). To evaluate whether the structure of the I-Ag7 molecule is influenced by DM, we tested the I-Ag7 transfectants for their reactivity with a panel of anti-class II mAbs. Table IGo shows a summary of the flow cytometry results; the reactivity of the mAb with I-Ak is shown for comparison. Three cross-reactive mAbs, 10.2.16, 40M, and 39E, reacted strongly with the I-Ag7 molecule, as did an anti-A{alpha}d mAb (K24–199). Other Abs that react with Aßk (40F and 40L) or cross-react with many allelic and isotypic forms of class II (40B) did not react with the I-Ag7 molecule (data not shown). This serologic analysis was consistent with protein sequence data (17) indicating that the NOD class II molecule is related to and yet distinct from other class II molecules. When the flow cytometry data was examined for relative reactivity of the mAb for the I-Ag7 molecule, we found that 40M (and 39E) displayed higher staining relative to 10.2.16 (which recognizes a linear sequence in many class II ß-chains) in cells that express Ii and DM. In the experiment shown, calculating the ratio of the mean fluorescence intensity (MFI) obtained with 40M to that obtained with 10.2.16 results in a value of 0.9 for the cells expressing Ii alone, whereas the ratio for the cells expressing DM with Ii is 1.20. In seven experiments where these cells were examined in parallel by flow cytometry, the relative expression of these epitopes on the DM/Ii-positive I-Ag7 cells was 1.3-fold higher (± 0.076) than that obtained with the cells that expressed Ii but lacked DM. This analysis suggested that a conformational change was induced in I-Ag7 when the Ii-positive cells expressed the DM protein.


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  		Table I. Effect of Ii and DM on mAb epitopes expressed by I-Ag71

 
To understand the relationship between CLIP removal from I-Ag7 and the change in the mAb epitopes detected in the presence of DM, we asked whether 40M would recognize the CLIP/class II complex. Cells expressing I-Ag7 with Ii (NODgIi) or I-Ag7 with Ii and DM (NODgIDM) were radiolabeled and lysates were immunoprecipitated with either 10.2.16 or 40M. Figure 4GoC shows that CLIP can only be detected in the 10.2.16-immunoprecipitated material from NODgI, and not in the 40M immunoprecipitate. This result suggests that CLIP binding to I-Ag7 disrupts the epitope that is recognized by 40M, and that the DM-mediated removal of CLIP accounts for enhanced 40M staining.

Role of DM in accumulation of peptide/class II complexes

Despite the productive interactions with Ii and DM that are evident in the preceding assays, SDS-stable I-Ag7 dimers were not detected in cells expressing these class II protein cofactors. There are several possibilities that could explain this result. First, the interactions between the I-Ag7 ß-chain and the I-Ad {alpha}-chain may be inherently less stable than other class II molecules and may allow SDS-induced dissociation irrespective of peptide occupancy. Alternatively, this class II molecule may indeed have a generalized deficiency in stable peptide binding. With regard to this last possibility, it is important to note that the biochemical and serologic data provided thus far provide insight into only one of two functions that are displayed by DM in vitro, which is removal of Ii fragments. Although this is the most well-documented function of DM, there is now increasing evidence that DM functions to enhance peptide loading onto class II, which allows for the accumulation of stably bound peptides (25, 26, 27, 48). DM is often likened to an enzyme (25), and as such it is unusual in that it readily facilitates both the forward (removal of CLIP) and the "backward" reaction (reloading of peptide) with most class II substrates.

To explain the failure of I-Ag7 to acquire SDS-stability despite its apparent ability to interact with DM, we considered the possibility that the interaction of DM with the I-Ag7 class II allows the release of unstable peptides such as CLIP while not concomitantly allowing accumulation of stably bound peptides. Such an interaction would account for the low levels of the SDS-stable dimers detectable for this class II allele. To examine this possibility, we sought to test the role of DM in class II-restricted Ag presentation by the I-Ag7 class II molecule. To accomplish this, Ag-specific T cells were derived that were restricted to the I-Ag7 molecule. NOD mice were immunized with chicken OVA (cOVA) in CFA. After priming, lymph node T cells were reactivated in vitro with Ag and then fused to the BW5147 thymoma to allow for the immortalization of the Ag-specific T cells. OVA-specific, I-Ag7-specific T cell hybridomas were identified after subcloning and were tested for reactivity on the panel of APCs that varied only in Ii and DM expression. Typically, at least two APC clones of a given genotype were analyzed in any given assay. Figure 5Go shows a representative result from these analyses. Two types of reactivity were identified in these experiments. One set of T cells, exemplified by 83.9 and 35.3 (Fig. 5Go, A and B), recognized peptides whose expression with class II was not detectably affected by Ii and DM expression within the APC. A separate set of T cells, represented by 97.4 and 96.4 (Fig. 5Go, C and D), recognized epitopes whose expression with the NOD class II was enhanced when the APC expressed Ii and were further enhanced by additional expression with DM. Compared with the cell lacking the expression of Ii and DM, the DM/Ii-positive cells demonstrated a 500-fold shift in their Ag dose-response curve. Also worthy of note is the ability of DM expression alone to facilitate Ag presentation for these clones. These results indicate that DM can facilitate the accumulation of at least some peptides on the NOD class II molecule.



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  		FIGURE 5. DM and Ii can facilitate the accumulation of peptide/class II complexes on the surface of APCs. T cell hybridomas that are reactive with cOVA were derived after in vivo priming and in vitro restimulation. Cloned, I-Ag7-restricted, cOVA-specific T cell hybridomas were tested for their ability to recognize APCs expressing I-Ag7 alone (cross), I-Ag7 plus DM (•), I-Ag7 plus Ii (squares), or I-Ag7 plus Ii and DM (triangles) upon the addition of graded doses of OVA in culture. After the overnight culture of T cells with APCs and graded doses of cOVA, supernatants were collected and tested for IL-2 content by coculture with CTLL. The results of a MTT assay that reflects the viability of CTLL after 24 h of culture are shown. Two independent APC clones were tested in each assay and are represented by the same symbol.

 
Collectively, our experiments demonstrate that the unique class II molecule that is expressed by the NOD mouse can productively associate with the two known protein cofactors that are associated with the class II presentation pathway, Ii and DM. DM participates in the removal of Ii proteolytic fragments from the I-Ag7 class II molecule and facilitates a conformational change in the I-Ag7 molecule. In addition to showing that DM can remove Ii-derived peptides, we have also evaluated whether DM can act positively with regard to peptide presentation by the I-Ag7 molecule. The derivation and use of Ag-specific, NOD-restricted T cells allowed us to evaluate the contribution of Ii and DM to class II-restricted Ag presentation. These studies have revealed that DM and Ii each can promote the accumulation of peptide class II complexes on the surface of APCs. As we (30, 34) and others (22, 40, 49, 50, 51, 52, 53) have noted in an analysis of the role of protein cofactors in class II-restricted presentation, the requirement for Ii and DM is peptide-specific. Although we have not yet defined the peptide specificity of the OVA T cells used in this study, we know that there are at least two peptides that are recognized. We have tested the ability of the cOVA-specific T cells to recognize the closely related turkey OVA protein (data not shown). The two T cells whose recognition is unaffected by Ii and DM (35.2 and 83.9) are able to be activated by turkey OVA, while those cells whose reactivity is enhanced by Ii and DM expression within the APCs do not recognize turkey OVA. Thus, DM-independent T cells most likely recognize a peptide that is conserved among these species, while the DM- and Ii-enhanced T cells probably recognize a species-specific peptide.

Of particular interest to us is the conformation change detected on the I-Ag7 molecule when the APCs express DM. This conformation change is most likely related directly to CLIP release and may require a substitution with structurally distinct peptides, an event that is likely to be facilitated by DM. An analysis of the crystal structure of the CLIP/class II complexes indicates that one region of DR3 is altered when it is bound by CLIP as opposed to when it is bound by antigenic peptide (54). Our serologic and biochemical studies raise the possibility that such conformational alternatives may exist for the I-Ag7 molecule depending upon the sequence of peptides occupying the binding groove of the class II molecule. We hypothesize that a structural feature of the CLIP peptide precludes the conformation that is preferred by the mAb 40M. DM-mediated loading may allow the binding of peptides offering motifs that drive a more stable interaction with the class II molecule or a conformation of the I-Ag7 molecule that is better recognized by the 40M and 39E mAbs. Peptide-sequencing (55) and peptide-binding (38, 56) studies have shown that some antigenic peptides and self peptides can accumulate on the I-Ag7 class II molecule despite the relatively unstable binding of certain antigenic peptides. In fact, peptide-sequencing studies and a sequence comparison of the peptides that are presented by the I-Ag7 molecule indicate that this class II molecule may selectively bind peptides that possess a negatively charged residue at their carboxyl terminus and consequently have the potential to neutralize the unpaired, positively charged residues that are unique to this class II molecule (38, 55). However, although such peptides may have a relatively stable interaction with I-Ag7, they apparently do not protect the I-Ag7 molecule from SDS-induced denaturation. Collectively, our results suggest that although the I-Ag7 molecule undergoes conformational changes that are induced by the exchange of CLIP for self or antigenic peptides in the presence of DM, this class II molecule may possess unique conformational features that render it particularly susceptible to SDS-induced denaturation. Our biochemical, functional, and serologic data suggest that the conformational features of I-Ag7 that are associated with weak peptide binding and with a sensitivity to SDS-induced chain dissociation do not reflect a nonproductive interaction with the DM protein.


   Acknowledgments
 
We thank Hugh McDevitt for providing us with the gene encoding the I-Ag7 ß-chain and John Monaco for the gift of the cDNAs encoding the murine DM molecule. We also thank Jim Miller, Chris Stebbins, and Patrick Noud for their critical comments regarding this manuscript. Flow cytometry was performed in the University of Chicago Cancer Center Core Facility.


   Footnotes
 
1 This work was supported by Grants PO1DK 49799 and 2R01AI34359 from the National Institutes of Health and the American Cancer Society, respectively. Back

2 Address correspondence and reprint requests to Dr. Andrea J. Sant, Department of Pathology, Committees on Immunology and Cancer Biology, University of Chicago, 5841 S. Maryland Avenue, MC1089, Chicago, IL 60637. E-mail address: Back

3 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; Ii, invariant chain; CLIP, class II-associated invariant chain peptide; cOVA, chicken OVA; MFI, mean fluorescence intensity. Back

Received for publication February 27, 1998. Accepted for publication May 14, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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