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The Journal of Immunology, 2003, 170: 3751-3761.
Copyright © 2003 by The American Association of Immunologists

DM Loss in k Haplotype Mice Reveals Isotype-Specific Chaperone Requirements 1

Chad H. Koonce*, Gordana Wutz*, Elizabeth J. Robertson*, Anne B. Vogt{dagger}, Harald Kropshofer{dagger} and Elizabeth K. Bikoff2,*

* Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; and {dagger} Roche Center for Medical Genomics, Hoffmann-La Roche, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
DM actions as a class II chaperone promote capture of diverse peptides inside the endocytic compartment(s). DM mutant cells studied to date express class II bound by class II-associated invariant chain-derived peptide (CLIP), a short proteolytic fragment of the invariant chain, and exhibit defective peptide-loading abilities. To evaluate DM functional contributions in k haplotype mice, we engineered a novel mutation at the DMa locus via embryonic stem cell technology. The present experiments demonstrate short-lived Ak/CLIP complexes, decreased Ak surface expression, and enhanced Ak peptide binding activities. Thus, we conclude that DM loss in k haplotype mice creates a substantial pool of empty or loosely occupied Ak conformers. On the other hand, the mutation hardly affects Ek activities. The appearance of mature compact Ek dimers, near normal surface expression, and efficient Ag presentation capabilities strengthen the evidence for isotype-specific DM requirements. In contrast to DM mutants described previously, partial occupancy by wild-type ligands is sufficient to eliminate antiself reactivity. Mass spectrometry profiles reveal Ak/CLIP and a heterogeneous collection of relatively short peptides bound to Ek molecules. These experiments demonstrate that DM has distinct roles depending on its specific class II partners.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class II molecules on the surface of specialized APC guide CD4+ T cell responses and select a useful CD4+ TCR repertoire. Clonal selection is mediated by both positive and negative signals during development in the thymus, and the structural features of class II/peptide/TCR associations responsible for these different outcomes have been intensely investigated (1). Diverse ligands universally adopt a rigid extended structure and share nearly identical paths through the binding cleft due to conserved interactions between class II residues and the peptide backbone. The structural differences created by allelic substitutions govern the shapes of specific class II peptide binding pockets and are thus responsible for setting the register and selection of T cell epitopes (2). The quality of atomic contacts between allele-specific residues and peptide side chains also determines the overall stability of class II/peptide complexes. Transient intermediates formed during peptide binding have been extensively studied in vitro using purified components, but we have as yet only limited understanding of the dynamic events responsible for determinant selection under physiological conditions inside professional APC.

Peptide ligands have restricted access to empty class II in vivo due to the combined activities of specific chaperones, namely the invariant (Ii) 3 chain and DM, which are required at distinct stages during maturation and export (3, 4). The Ii chain prevents misfolding or aggregation of class II subunits during early assembly and protects the nascent empty groove from irreversible associations with chaperones such as BiP and calnexin that are responsible for endoplasmic reticulum quality control. Via targeting signals in its cytoplasmic domain, the Ii chain also directs selective class II export to an endosomal compartment(s) where exposure to acidic pH and proteolytic enzymes promotes Ii chain degradation and Ag capture. The nonclassical class II molecule designated HLA-DM in humans or H-2M in mice acts subsequently to facilitate CLIP release in exchange for tightly bound peptide ligand(s) (5, 6). DM also stabilizes empty class II and enhances the selection of best-fit peptides (7, 8). The structural basis of DM associations with these various class II intermediates remains a mystery.

Class II allelic and isotypic variants promiscuously associate with the conserved Ii chain via its class II-associated Ii chain-derived peptide (CLIP) sequence (9, 10, 11, 12, 13). Allele-specific differences govern Ii chain requirements during subunit assembly (14), interactions with Ii chain cleavage fragments (15, 16), and CLIP dissociation kinetics (17, 18, 19, 20, 21). Possibly these functional effects can be explained simply by divergent CLIP binding properties, but allele-specific contacts outside the groove may also influence Ii chain chaperone activities. Class II polymorphism also has a significant impact on DM interactions with empty class II, as judged by differential abilities to enhance peptide exchange at acidic pH (8). Allele-specific DM requirements during class II maturation and Ag presentation have been previously investigated, but inconsistencies in the literature make it difficult to evaluate these observations (22, 23, 24, 25, 26). Considerable evidence suggests that the extent of Ii chain and DM functional contributions to class II presentation depends on the particular epitope, its sensitivity to proteases, and its availability inside various peptide-loading compartment(s) (27, 28, 29, 30, 31, 32, 33, 34). Indeed, class II activities mediated via Ii chain and DM-independent nonconventional pathway(s) have been extensively documented (22, 23, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36). The intrinsic stability of class II dimers (37) and thus the relative importance of these alternative modes of peptide capture probably differ for allelic variants.

Significant progress toward understanding competitive class II interactions with the Ii chain, DM, and peptide ligands inside professional APC has come from gene targeting experiments. Regardless of MHC haplotype, Ii chain-deficient mice display dramatically reduced surface class II and fail to produce mature compact dimers tightly occupied by peptide ligands (14, 30, 38, 39). Thus, it appears that naturally processed self peptides are predominantly loaded via the conventional Ii chain-dependent pathway(s). Similarly, the DM mutant strains studied to date express long-lived class II/CLIP complexes and exhibit severely compromised peptide-loading capabilities (40, 41, 42, 43). These findings strongly argue that DM is strictly required to mediate CLIP release and promote occupancy by diverse peptide ligands. Considerable data suggest that DM plays a less critical role during peptide acquisition by other allelic products (22, 23, 24, 25, 26, 43), but the structural framework of these selective DM requirements remains ill defined.

To evaluate possible effects of allelic and isotypic diversity on DM activities, we decided to generate mutant mice expressing different MHC haplotypes via homologous recombination in embryonic stem (ES) cells. In contrast to gain-of-function approaches used previously, this strategy has allowed us to examine functional consequences in professional APC expressing appropriate ratios of class II and Ii chain isoforms under control of endogenous regulatory elements. Recent experiments demonstrate relaxed DM requirements during class II peptide loading and CD4+ T cell maturation in BALB/c mice (43). Unlike long-lived Ab/CLIP complexes, mature Ad on the surface of mutant splenocytes expresses BP107 conformational epitopes and toxic shock syndrome toxin-1 binding capabilities, properties suggestive of partial occupancy by wild-type ligands. Interestingly, functional assays revealed consistent differences for Ad- and Ed-restricted T cells. Indeed, the mutation leads to decreased peptide capture by Ad molecules and, in striking contrast, causes enhanced peptide loading by Ed molecules. Thus, isotypic variants show differences affecting DM dependency.

To further investigate allele- and isotype-specific DM actions, here we engineered a novel mutant allele in k haplotype mice. DM loss in this context offers the opportunity for side-by-side comparisons of activities mediated by Ak and Ek molecules encoded by tightly linked loci. Previous studies have suggested that Ak maturation, surface expression, and Ag presentation are DM independent (22, 23, 24, 26), consistent with its exceptionally fast CLIP dissociation rates (17, 18). The present experiments demonstrate that a quite different situation exists in vivo. Thus, our mutant mice display substantially decreased Ak surface expression and enhanced peptide binding activities, as if DM loss creates a substantial pool of empty or loosely occupied Ak conformers. In striking contrast, the mutation causes only subtle Ek defects. Indeed, the appearance of mature compact Ek dimers, near normal surface expression, and efficient Ag presentation capabilities strengthens the evidence for distinctive isotype-specific modes of peptide capture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

The targeting strategy for disruption of the DM{alpha} locus has been described previously (43). Briefly, we used long-range PCR to isolate genomic subclones comprised of a 4.5-kb BamHI-ScaI fragment immediately 5' to exon 1 and a HindIII-EcoRI fragment containing 30 nt of exon 2 and extending 4 kb downstream. These 5' and 3' arms were placed in between positive (neo) and negative (DT-A) drug selection cassettes to create the isogenic CBA/J-based targeting vector, introduced into H-2bxk F1 TT2 ES cells (44).

From 1089 drug-resistant colonies analyzed, we recovered 10 recombinant clones carrying the desired 1.7-kb deletion spanning exon 1, intronic sequences, and most of exon 2, as judged by diagnostic Southern blots with restriction digests and probes as described (43). Because the wild-type k allele contains a unique SacI site mapped to intron 1, absent from the C57BL/6 locus, the 5' probe distinguishes SacI fragments derived from wild-type k (4.6 kb), wild-type b (8 kb), or the targeted locus (7.5 kb). All 10 recombinant clones were targeted at the k locus. Four of these were injected into C57BL/6 blastocysts to generate chimeras.

Germline males were crossed to B10.BR females to generate heterozygous progeny, and subsequent intercross matings yielded homozygous mutants and wild-type littermates. We used a PCR genotyping screen using primers specific for a common sequence near the 3' boundary of exon 2 and the neo cassette or a wild-type sequence within the deletion as previously described (43). Two independent targeted clones were used to generate the H-2k DM-deficient mice described in the present report. Due to concerns about possible genetic drift contributed by heterogeneous background influences, results were confirmed by analyzing offspring derived from both targeted clones. In functional assays testing responses of mixed lymphocyte reactions, we used backcross progeny (n = 8) derived from one of the sublines. In all experiments comparisons were made between age-matched animals, and whenever possible we analyzed homozygous mutant and wild-type littermates. DM-deficient mice derived from both targeted clones gave indistinguishable results in all assays.

BALB/c DM mutants (43), H-2b DM{alpha}-deficient mice on a mixed (129 x C57BL/6)F2 background (40), and Ii chain mutant mouse strains carrying different MHC haplotypes (14, 38, 39) have been described.

Abs and peptides

Hybridomas include 10-2-16 and OX6 reactive with A{beta}k, 11-5-2 specific for A{alpha}k, 17-3-3 and M5/114 reactive with E{beta}k, and 14-4-4 specific for E{alpha}k. Rabbit antisera specific for determinants located in the cytoplasmic tails of the {alpha}- and {beta}-chains were provided by R. N. Germain (National Institutes of Health, Bethesda, MD). The peptides OVA323–339 (ISQAVHAAHAEINEAAGR), hen egg lysozyme 46–61 (HEL46–61; NTDGSTDYGILQINSR), bacteriophage {lambda} repressor cI peptide P12–26 (LEDARRLKAIYEKKK), the truncated variant of moth cytochrome 88–103 previously described as DASP(KKANELIAYLKQATK), and biotin conjugates were purchased from Quality Controlled Biochemicals (Hopkinton, MA).

Immunofluorescence analysis

Spleen cell suspensions depleted of erythrocytes by ammonium chloride-Tris treatment were incubated on ice with saturating amounts of biotinylated Abs, followed by FITC-labeled avidin D (catalogue no. A-2001; Vector Laboratories, Burlington, CA). Fluorescence was analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA), and data are displayed as cell number vs log fluorescence. Each profile represents 20,000 events.

Radiolabeling and immunoprecipitations

Biosynthetic labeling, immunoprecipitations, and SDS-PAGE were conducted as previously described (43). Briefly, spleen cells were washed with warm HBSS containing 2% FCS and antibiotics and resuspended (2 x 107/ml) in warm methionine-free DMEM supplemented with 4 mM glutamine and 5% dialyzed FCS. After 1 h at 37°C, [35S]methionine was added (250 µCi/ml) for 40 min. The cells were subsequently resuspended in a 5-fold excess volume of warm DMEM containing 15% FCS and a 10-fold excess of cold methionine, incubated at 37°C for 4 h, harvested, and then washed twice with ice-cold PBS. The cell pellet was lysed in buffer containing 1% Nonidet P-40, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, and 10 µg/ml aprotinin. After incubation on ice for 15 min, extracts were cleared of nuclei and debris by centrifugation for 30 min at 15,000 rpm. Lysates were precleared once with rabbit anti-mouse IgG (H+L) Abs (Zymed, South San Francisco, CA), twice with rabbit anti-rat IgG (H+L) Abs (Zymed), and twice with protein A-agarose (Life Technologies, Gaithersburg, MD) before the addition of specific Abs. Immunoprecipitates were washed three times with buffer containing 0.05 M Tris-HCl (pH 8), 0.45 M NaCl, 0.5% Nonidet P-40, 0.05% sodium azide, and 1 µg/ml aprotinin, and then solubilized in Laemmli buffer containing 2% SDS and 2-ME by treatment for 60 min at room temperature or by heating at 100°C for 10 min as indicated in the figure legends. Samples were analyzed by SDS-PAGE, subsequently treated with EnHance (DuPont-NEN, Wilmington, DE), dried, and exposed to x-ray film.

Ag presentation assays

The T cell hybridomas 2B6.31, 2C8.4, 2B5.1 (27, 29), 2G7.1, and 4C1.6 (45) were donated by L. Adorini (Roche Milano Ricerche, Milan, Italy). T cell clones SKK9.11 and SKK 45.1 (46) were provided by P. Marrack (Howard Hughes Medical Institute, National Jewish Center, Denver, CO), and 3A9 (47) was contributed by P. Allen (Washington University School of Medicine, St. Louis, MO.).

IL-2 production was assessed by incubating T cells (5 x 104/well) with spleen cells (2 x 105/well) in 200 µl of complete RPMI 1640 supplemented with 15% FCS, 10% NCTC109, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 15 mM HEPES (pH 7.2), 0.1 mM nonessential amino acids, 5 x 10-5 M 2-ME, 2 mM glutamine, and increasing concentrations of Ag. Supernatants were collected after 20 h and assayed for IL-2 content in a secondary culture with CTLL indicator cells in the presence of 50% primary supernatants.

For MLR, CD4+ T cells (4 x 105/well) positively selected using magnetically labeled microbeads (catalogue no. 492-01) and MACS separation columns (Miltenyi Biotec, Auburn, CA) were cultured with increasing numbers of irradiated (3300 rad) spleen cells for 72 h. The degree of proliferation was measured by a 16- to 18-h exposure to 1 µCi of [3H]thymidine. All results are expressed as the mean counts per minute of triplicate cultures.

Mass spectrometry

MHC class II-associated peptides were analyzed by mass spectrometry (MALDI-MS) as previously described (48). Briefly, spleens were homogenized, and lysates were prepared in 1% Triton X-100. MHC class II molecules were bound to Sepharose beads conjugated to mAbs H116-32 specific for A{alpha}k or 14-4-4 specific for E{alpha}k, and as a control we used Tris-conjugated Sepharose beads. Peptides were eluted by incubation in 0.1% trifluoroacetic acid and analyzed on a REFLEX III mass spectrometer (Bruker, Bremen, Germany). Eluates from equal amounts of purified class II were compared.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of k haplotype DM mutant mice

To create DM mutant mice expressing the H-2k haplotype, we constructed a completely homologous CBA/J-based vector. Taking advantage of long-range PCR technology, we obtained 5' and 3' regions of genomic homology and placed these arms in between positive and negative drug selection cassettes exactly as previously described (43). The targeting construct was transfected into H-2bxk F1 TT2 ES cells (44), and two independent clones targeted at the k locus were injected into C57BL/6 blastocysts to generate chimeras. Germline males crossed to B10.BR females gave rise to heterozygous progeny, and subsequent matings yielded homozygous mutants. These animals appear healthy and are indistinguishable from wild-type littermates. As judged by RNase protection assays using probes spanning both 5' and 3' exons (43), this targeting event eliminates DM{alpha} transcripts (data not shown).

DM loss causes isotype-specific maturation defects

Ak and Ek expression was analyzed in pulse-chase experiments. As expected in unheated samples, a substantial portion of Ad, Ak, Ed, and Ek molecules produced by wild-type cells migrate as compact dimers at ~56 kDa (49). Both Ad/CLIP and Ed/CLIP strongly expressed by BALB/c DM mutants readily dissociate in SDS gels, and CLIP appears just ahead of the dye front (Fig. 1, a and b). In contrast, our k haplotype mutants express barely detectable Ak/CLIP complexes. Similar results were obtained using conformation-independent, chain-specific rabbit Abs and conformationally dependent mAbs directed against nonoverlapping epitopes contributed by both chains (Fig. 1, a and b, and data not shown). Unlike Ed-associated CLIP variants (43) or heterogeneous CLIP sequences bound to Ek/b molecules (26), we found no evidence for expression of Ek/CLIP complexes. Instead, DM mutant splenocytes expressed near normal amounts of mature compact Ek dimers. Thus, the targeted allele selectively disrupts Ak maturation and reveals isotype-specific DM contributions under physiological conditions.



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  		FIGURE 1. DM mutant mice express short-lived Ak/CLIP complexes alongside mature compact Ek dimers. Lysates were prepared from spleen cells pulsed for 40 min with [35S]methionine and chased for 4 h (a and b) or different times as indicated (c and d) and extensively precleared with rabbit anti-mouse IgG (H+L) Abs before immunoprecipitation with chain-specific rabbit Abs or conformationally dependent mAbs as indicated. Complexes were boiled (B; a and b; all samples, c and d) or solubilized at room temperature (not boiled (NB); a and b) and analyzed on 10% (a–c) or 15% (d) polyacrylamide gels under reducing conditions. Recovery of electrophoretically distinct Ed-associated CLIP variant(s) with 14-4-4 mAb has been reported previously (43 ). Representative data from one of three similar experiments are shown.

 
The DM mutant strains studied to date strongly express class II/CLIP complexes at steady state, as proved to be the case for Ab, Ad, Ed, and Ek/b molecules (26, 40, 41, 42, 43). On the other hand, the results reported above suggest that, similar to recombinant A(g7) peptide binding properties (21), Ak/CLIP may rapidly dissociate at endosomal pH. To investigate allelic differences affecting CLIP stabilities in vivo, DM mutant splenocytes were pulse-labeled and subsequently chased for increasing periods of time (Fig. 1, c and d). Regardless of MHC haplotype, class II subunits and Ii chain display similar rates of transport past the Golgi, as judged by characteristic increases in chain mobilities and acquisition of endoglycosidase H-resistant glycans. The sequential appearance of Ii chain degradation intermediates followed well-described endosomal proteolysis pathways (50, 51). Initially we observed p23–25 Iif (LIP), then p12Iif (SLIP), and subsequently CLIP, suggestive of a precursor product relationship. Ii chain fragments produced by different strains display minimal size differences, indicative of limited structural heterogeneity. These results demonstrate that oligomeric complexes with intact Ii chain are probably processed via a universal pathway(s). CLIP recoveries initially showed roughly similar kinetics in all three strains. However, in contrast to long-lived Ab/CLIP readily detected after 8 h of chase, we found that Ak-associated CLIP has an exceptionally short half-life. Despite extensive analysis, we failed to observe Ek/CLIP complexes at any time point.

In vitro exposure to low pH in the absence of peptide causes irreversible Ak aggregation, probably due to denaturation of the binding cleft (52). If spontaneous CLIP release in the absence of DM creates empty Ak conformers inside the endocytic compartment(s), are these converted to inactive isomers or maintained in a peptide receptive state (53, 54, 55)? To test this, mutant spleen cells were pulse labeled and subsequently chased in the presence of HEL46–62 peptide. As shown in Fig. 2, a and b, exogenously added peptide ligand rescued the production of mature compact Ak dimers, detectable using conformation-dependent mAbs directed against distinct epitopes contributed by both chains. These results demonstrate that not only Ii chain, but also DM mutant splenocytes, produce empty Ak molecules readily available for peptide occupancy.



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  		FIGURE 2. Exogenously added HEL46–61 peptide ligand rescues expression of mature compact Ak dimers. Lysates prepared from spleen cells pulsed for 40 min with [35S]methionine and chased for 4 h in the presence of HEL46–61 peptide (100 µM) or, as a control, medium alone, were extensively precleared with rabbit anti-mouse IgG (H+L) and immunoprecipitated with the indicated mAbs. Complexes were solubilized at 100°C for 10 min (B) or at room temperature for 60 min (NB) and analyzed on 10% polyacrylamide gels under reducing conditions. C, Position of compact dimers.

 
To assess surface expression by k haplotype DM mutants, spleen cells were stained with mAbs and analyzed by FACS. To control for conformational effects, we used a panel of mAbs specific for distinct epitopes contributed by both chains. Unlike mutant strains described previously (40, 41, 42, 43), DM loss caused markedly reduced levels of surface Ak (Fig. 3). Because similar results were obtained with all these Abs, a strong argument can be made that this reflects decreased amounts of total surface Ak rather than a serological change. These findings suggest that empty or loosely occupied Ak molecules are inefficiently exported to the cell surface or possibly have a reduced life span. Because cytoplasmic staining experiments demonstrate roughly 2-fold less total Ak, and B cell representation is unaffected (data not shown), we conclude that DM loss selectively compromises Ak stability. In striking contrast the mutation has hardly any impact on Ek surface expression.



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  		FIGURE 3. Reduced levels of Ak and near normal Ek surface expression. Splenocytes from 1) wild-type, 2) DM-deficient, or 3) Ii chain mutants were stained with biotin-conjugated mAbs, as indicated, followed by FITC-conjugated avidin. Representative data from one of six identical experiments with similar outcomes are shown.

 
Isotype-specific DM contributions during Ag presentation and peptide capture

DM function in BALB/c mice is essential for Ad-restricted Ag presentation, but in contrast, Ag capture by Ed molecules appears to be DM independent (43). To evaluate functional defects in k haplotype mutants, we tested Ak- and Ek-restricted T cell responses. As shown in Fig. 4, DM markedly enhances Ak-restricted presentation, especially at suboptimal Ag concentrations. In contrast, DM mutant splenocytes efficiently stimulate Ek-restricted T cells, strengthening evidence for isotype-specific DM requirements. As for Ii chain mutants, we also observed here enhanced responses toward HEL46–61 peptide, suggesting that Ak conformers are empty or occupied by easily displaced ligand(s). As for BALB/c mutants, these functional experiments also demonstrate isotype-specific Ii chain requirements. Thus, responses of Ek-restricted, but not Ak-restricted, T cells strictly depend on Ii chain coexpression.



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  		FIGURE 4. DM mutants display isotype-specific Ag presentation defects. Wild-type, DM-deficient, or Ii chain mutant splenocytes were incubated with T cell hybridomas and increasing concentrations of intact protein Ags or HEL46–61 peptide, as indicated. Supernatants were collected after 20 h, and IL-2 content was measured in secondary cultures with CTLL indicator cells by addition of 1 µCi of [3H]thymidine. All results are expressed as the mean counts per minute of triplicate cultures. Data are representative of three independent experiments.

 
To further evaluate isotype-specific DM actions during peptide capture, splenocytes were incubated with biotinylated peptides, stained with FITC-avidin, and analyzed by FACS (56). We used a panel of well-defined peptides with previously assigned class II specificities (57). Thus OVA323–339 selectively binds to Ad, and HEL46–61 exclusively binds to Ak molecules, whereas {lambda} repressor cI peptide P12–26 and the truncated variant of moth cytochrome 88–103 (DASP) bind to Ek molecules. Consistent with previous results, H-2b DM mutants exhibit severely compromised peptide loading capabilities (40, 41, 42). As shown in Fig. 5, wild-type and Ii chain-deficient splenocytes expressing three different MHC haplotypes display predicted allele-specific binding activities. Thus, OVA323–339 binds preferentially to Ad and weakly to Ab and fails to give a detectable signal with H-2k splenocytes. On the other hand, HEL46–61 exclusively binds H-2k wild-type and Ii chain mutant splenocytes. Surprisingly we observed that H-2k DM mutants display markedly enhanced peptide binding capacities, surpassing not only wild-type but also those observed for Ii chain mutant splenocytes, and even bind OVA323–339. Similarly, BALB/c DM mutants (H-2d) gain reactivity toward HEL46–61. As for Ed peptide capture in BALB/c mutants, we also found here that the mutation leads to markedly enhanced Ek peptide binding activities (Fig. 5). Thus both Ak and Ek surface molecules display increased reactivity toward exogenously added peptides, probably due to the absence of a tightly associated ligand(s).



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  		FIGURE 5. DM loss reveals enhanced peptide binding capabilities. 1) Wild-type, 2) DM-deficient, or 3) Ii chain mutant splenocytes cultured for 5 h at 37°C with biotin-conjugated peptides as indicated or with medium alone were stained with FITC-labeled avidin and analyzed by FACS. The capabilities of these well-defined peptides to interact with class II allelic and isotypic variants have been previously studied (57 ). Thus, OVA323–339 selectively binds Ad, HEL46–61 exclusively binds Ak molecules, whereas {lambda} repressor cI peptide P12–26 and the truncated variant of moth cytochrome 88–103 (DASP) bind Ek molecules. Representative data from three identical experiments are shown.

 
To directly assess the complexity of endogenous class II-associated peptides, acid eluates from affinity-purified Ak and Ek molecules were analyzed by MALDI-MS. As shown in Fig. 6, four CLIP truncation variants were found associated with mature Ak produced by mutant, but not wild-type, splenocytes, and these closely resemble the Ab- and Ad-associated Ii chain-derived sequences characterized in previous experiments (41, 58, 59). Apart from CLIP, hardly any other Ak-associated peptides were recovered. In striking contrast, Ek profiles from DM mutants reveal a broad distribution of heterogeneous peptides, on the average slightly shorter and comprised of 14–17 mers, compared with those found with wild-type Ek molecules, ranging from 15–20 residues in length. This shift is reminiscent of findings reported for transfected human cell lines showing that DM promotes acquisition of longer variants (6). Ek/CLIP was present at barely detectable levels.



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  		FIGURE 6. Mass spectrometry profiles of class II/self peptide complexes expressed by DM mutant splenocytes. The predominant CLIP variants found associated with purified Ak molecules in the absence of DM are indicated by the arrows. In contrast, acid eluates of purified Ek molecules from mutant splenocytes are comprised of a heterogeneous collection of peptides including only two minor CLIP species. In this case, DM loss influences peptide length selection, since the average size (14–17 mers) is markedly reduced compared with self peptides bound to wild-type Ek molecules ranging from 15–20 residues in length.

 
DM peptide editing is nonessential for self tolerance

Relatively efficient CD4+ maturation has been described in both BALB/c Ii chain and DM-deficient strains, consistent with leaky peptide loading via an alternative pathway(s) (39, 43). Similarly, here we observe in k haplotype DM mutants, near normal numbers of mature CD4+ T cells (data not shown). To evaluate DM functional contributions shaping the repertoire in k haplotype mice, we tested mature CD4+ T cells for antiself reactivity. Due to concerns about possible contributions from stimulatory Mls loci encoded by CBA/J background genes, these experiments awaited the generation of backcross (n = 8) progeny, and subsequent intercross matings yielded our B10.BR nearly congenic subline. As shown in Fig. 7, in striking contrast to mutant phenotypes described previously (40, 41, 42, 43), CD4+ T cells from k haplotype DM-deficient mice displayed no detectable proliferative response directed against wild-type stimulators. Thus, we conclude that peptide ligands selected in the absence of DM adequately represent the wild-type repertoire and effectively eliminate self-reactive clones. Ek maturation and peptide loading may efficiently promote the development of mature CD4+ T cells. As a positive control, we assessed BALB/c (H-2d) CD4+ T cell responses. Interestingly as for d (43), but in contrast to b, haplotype mice (40, 41, 42), DM mutant splenocytes proved to be effective stimulators due to partial occupancy by wild-type ligands.



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  		FIGURE 7. DM peptide editing is nonessential for self tolerance. Purified CD4+ T cells (4 x 105/well) were cultured in the presence of increasing numbers of irradiated (2500 rad) spleen cells as indicated. Proliferative responses were measured by a 16-h exposure to 1 µCi of [3H]thymidine. All results are expressed as the mean counts per minute of triplicate cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Current thinking about DM as an essential class II chaperone necessary to promote occupancy by diverse peptide ligands has been strongly influenced by studies of mutant mouse strains created using ES cell technology. DM mutants described to date strongly express long-lived class II/CLIP complexes and exhibit defective peptide-loading activities; under these circumstances, negative selection is at best incomplete. Consequently, mature CD4+ T cells display vigorous antiself reactivity directed against wild-type ligands (40, 41, 42, 43). The present analysis of k haplotype mutants reveals a remarkably different phenotype. Here we observe spontaneous CLIP release and enhanced peptide binding capabilities. Parental wild-type stimulators fail to elicit CD4+ T cell proliferative responses. Thus, it appears in k haplotype mice that DM selection of best-fit peptides is not essential for the development of self tolerance.

Evidence that class II universally binds the Ii chain via its CLIP sequence came from peptide elution experiments, since the same Ii chain-derived residues 81–104 in humans and 85–101 in mice were consistently represented among naturally processed ligands regardless of haplotype (9, 10, 58, 59). Studies to date have failed to demonstrate similar CLIP truncation variants associated with mature Ak or Ek expressed by wild-type or DM-deficient cells (26, 60, 61). Here we characterized short-lived Ak/CLIP complexes in pulse-chase experiments and via MALDI analysis. However, transiently expressed Ek/CLIP produced by mutant splenocytes was hardly detectable. These differences could simply reflect CLIP binding affinities, but Ak/CLIP has exceptionally fast dissociation kinetics (17) and is therefore predicted to display enhanced CLIP release relative to Ek, unlike the situation here. Other investigators reported that Ek/CLIP has the faster off rate (18), suggesting that technical aspects, such as affinity purification, assay conditions, and discrete CLIP sequence differences, affected the outcome of previous in vitro experiments measuring kinetic stabilities.

The question of how the class II cleft becomes CLIP-associated during early coassembly and subsequently undergoes subtle structural transitions to accommodate better binders has been the focus of intense investigation. Peptide mapping studies and nuclear magnetic resonance methods demonstrate that the class II binding segment of Ii chain encompasses residues 72–110 (62, 63), but the peptide groove accommodates only the core CLIP sequence comprised of residues 91–99 (64). Considerable data suggest that adjacent regions of the Ii chain modulate class II associations (65, 66). The segment N-terminal to Met91 appears to bind an effector site outside the groove and may act allosterically to enhance peptide exchange and CLIP release (48). Interestingly, Ii chain residues 63–78 were recently described as Ak-associated (67). In contrast, MALDI profiles demonstrate that CLIP variants recovered with Ak closely resemble Ab- and Ad-associated sequences spanning residues 83–104 (58, 59). It is of course possible that additional loosely bound fragments dissociated during isolation procedures, but taken at face value our results strongly argue that exceptional Ak/CLIP instability reflects intrinsic features of the complex.

In the case of CLIP bound to DR3, the highly conserved methionine side chains tightly fit inside the P1 and P9 pockets (64). A quite different situation exists with respect to the Ad binding cleft (68). Although its P1 pocket appears large enough to accommodate a methionine, the P9 pocket is smaller, so Met98 is thought to sit suboptimally. This situation leads to enhanced CLIP release in vitro (19) and probably accounts for partial displacement by wild-type ligands observed in BALB/c DM mutants (43). In comparison, the recently solved Ab structure reveals relatively empty P1, P4, P6, and P9 pockets because its specific ligand has only small Ala side chains at these positions (69). Multiple additional hydrogen bond contacts appear to compensate for the loss of stabilizing anchors. Long-lived Ab/CLIP complexes have instead bulky Met side chains inside both P1 and P9 pockets, so it is hardly surprising that competitive binding by self peptide ligands is undetectable in b haplotype mutants.

DM loss of function mutants in the context of b and d haplotypes express surface class II at wild-type levels (40, 41, 42, 43). In contrast, here we observed markedly decreased Ak surface expression. These results demonstrate for the first time that DM selection of best-fit peptides enhances surface expression under physiological conditions in professional APC. How is quality control mediated? It has long been known that peptide ligands extend intracellular class II half-lives (70). Recent evidence suggests that empty or loosely occupied class II is susceptible to proteolytic degradation inside endocytic compartments and at the cell surface (71, 72). Allele- and isotype-specific sensitivities to acid-induced denaturation of empty dimers may also selectively influence the elimination of unstable complexes (8, 52).

BALB/c DM mutants express Ed/CLIP complexes sensitive to SDS-induced denaturation, but, in contrast, here DM loss fails to disrupt the formation of mature compact Ek dimers. These findings are especially surprising, since both molecules share extensive sequence homology (73, 74) and peptide binding motifs (57), and CLIP affinities also appear quite similar (17). Perhaps the intrinsic stability of these empty dimers differs, and Ek displays enhanced peptide-loading capabilities due to its extended half-life inside endocytic compartments. Peptide binding by empty Ek has been extensively assessed in vitro (54, 75, 76, 77, 78, 79), but less is known about Ed conformational changes during peptide capture. A buried cluster of acidic residues surrounding the P6 pocket is conserved in all mouse I-E and human DR molecules, and it seems likely that these contacts are available only inside endocytic compartments (80). On the other hand, numerous experiments demonstrate that the Ii chain efficiently oligomerizes with both Ed and Ek at neutral pH shortly after synthesis in the ER (14, 49). Wiley and co-workers (64) previously noted that the conserved CLIP Ala94 and Pro96 side chains hardly fit inside the DR3 P4 and P6 acceptor pockets, but these small residues avoid destabilizing contacts in the central portion of the groove. Interactions with adjacent residues outside the groove, possibly class II contacts mapped to transmembrane domains (81, 82), potentially strengthen associations necessary for coassembly of oligomeric complexes.

DM has been viewed as an enzyme that catalyzes peptide exchange, but the structural basis of its activities remains ill defined. Our experiments strongly suggest that, similar to the Ii chain, DM has both allele- and isotype-specific contacts. Recent studies demonstrate that DM binds empty or loosely occupied class II at an exposed surface of DR {alpha}1 near the P1 pocket (83). These residues probably lie buried underneath intact Ii chain, but become accessible upon cleavage N-terminal to CLIP. Allele- and isotype-specific substitutions mapped to this site potentially influence DM associations. DM editing functions seem to cooperatively strengthen multiple contacts along the length of the groove. DM was previously shown to influence peptide size selection and promote the acquisition of longer ligands (6). The present results extend these observations and provide conclusive evidence that this mode of DM action affects peptide capture under physiological conditions in normal APC. Thus, the average size of Ek-associated peptide ligands was significantly reduced in DM mutants compared with those bound to wild-type Ek molecules. Our findings argue that these short Ek peptides fit suboptimally in the cleft, since they are readily displaced by better binders. Partial occupancy by short variants avoids destabilizing side chain interactions, but the longer peptides selected by DM potentially have additional hydrogen bond contacts at both ends as well as better anchors buried inside the central portion of the groove. Increased peptide length thus offers more extensive atomic contacts over a greater surface area.

The strength of TCR signaling is influenced by the stability of class II/peptide complexes on the surface of APC (1). Structural studies demonstrate that CD4+ T cells bind diagonally across the central portion of the class II/peptide surface (84, 85). Interestingly peptides loosely bound at one end of the cleft select a distinct subset of CD4+ T cells directed toward DM-independent epitopes (86). Loosely bound Ak ligands and relatively short Ek peptides in our DM mutants may similarly bind toward one end of the groove, as described for short-lived complexes triggering autoimmune disease (87, 88). Previous evidence suggests that low affinity T cells may escape negative selection (89, 90). In contrast, here mature CD4+ T cells selected by empty or loosely occupied class II lack anti-self reactivity. These findings strongly suggest that weakly bound self peptide ligands expressed by k haplotype DM mutants broadly represent the diverse wild-type repertoire. It will be important to further assess the ability of these low affinity peptides to promote CD4+ T cell survival and homeostasis.

Here we describe striking isotype-specific DM actions in professional APC with appropriate ratios of class II subunits, Ii chain isoforms, and the DM chaperone, DO, all coexpressed under control of endogenous regulatory elements. Transfection recipients may differ in their content of organelles, proteases, and molecular chaperones responsible for quality control and endocytic targeting. In contrast, here distinctive DM contributions to Ak and Ek activities were revealed in identical mutant spleen cell populations. Our mutant mice should therefore prove useful for dissecting isotype- and allele-specific peptide-loading pathways in various physiological settings in the intact animal. Humans express three classical class II molecules, namely HLA-DP, -DQ, and -DR (91). DM peptide editing has been extensively studied in the context of various HLA-DR alleles, but isotypic differences affecting DM dependency have not been systematically examined. It will be interesting to learn whether distinctive isotype-specific Ii chain and DM chaperone activities such as those described in the present report are conserved across species boundaries and how these various modes of peptide capture influence host protection and disease susceptibility.


   Acknowledgments
 
This paper is dedicated to the memory of Don Wiley, whose seminal contributions helped us understand how MHC molecules use classic features of protein structure to sample the universe of Ags. We thank Shin Aizawa for TT2 ES cells; Luciano Adorini, Pippa Marrack, and Paul Allen for T cell hybridomas; Ron Germain for chain-specific rabbit Abs; Luc van Kaer for H-2b DM mutant mice; George Kenty, Shanda Porter, and Caroline Lallouet for valuable technical assistance; Patti Lewko for careful maintenance of the mouse colony; and Renate Hellmiss for preparing the figures.


   Footnotes
 
1 This work was supported by Grant AI19047 from the National Institutes of Health (to E.K.B.) and by an Erwin-Schroedinger Fellowship from the Austrian Science Foundation (to G.W.). Back

2 Address correspondence and reprint requests to Dr. Elizabeth K. Bikoff, Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138. Back

3 Abbreviations used in this paper: Ii, invariant; CLIP, class II-associated Ii chain-derived peptide; ES, embryonic stem; HEL, hen egg lysozyme; MALDI-MS, mass spectrometry. Back

Received for publication October 23, 2002. Accepted for publication January 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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