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The Journal of Immunology, 2001, 166: 5087-5098.
Copyright © 2001 by The American Association of Immunologists

Relaxed DM Requirements During Class II Peptide Loading and CD4+ T Cell Maturation in BALB/c Mice1

Elizabeth K. Bikoff2, Gordana Wutz, George A. Kenty, Chad H. Koonce and Elizabeth J. Robertson

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Current ideas about DM actions have been strongly influenced by studies of mutant strains expressing the H-2b haplotype. To evaluate DM contributions to class II activities in BALB/c mice, we generated a novel mutation at the DMa locus via embryonic stem cell technology. Unlike long-lived Ab/class II-associated invariant chain-derived peptide (CLIP) complexes, mature Ad and Ed molecules are loosely occupied by class II-associated invariant chain-derived peptide and are SDS unstable. BALB/c DM mutants weakly express BP107 conformational epitopes and toxic shock syndrome toxin-1 superantigen-binding capabilities, consistent with partial occupancy by wild-type ligands. Near normal numbers of mature CD4+ T cells fail to undergo superantigen-mediated negative selection, as judged by TCR V{beta} usage. Ag presentation assays reveal 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, DM requirements differ for class II structural variants coexpressed under physiological conditions in the intact animal.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The MHC class II/peptide complexes on the surface of specialized APCs guide CD4+ T cell responses toward foreign pathogens and promote selection of a useful TCR repertoire. An increasingly clear picture of diverse MHC class II/peptide complexes has come from recent x-ray crystal studies (1, 2, 3, 4), but far less is known about structural intermediates during class II maturation and export to the cell surface. In particular, the question where and when immature class II molecules become available for peptide occupancy has been intensely investigated. Numerous studies demonstrate antigenic peptides bind to empty class II molecules during extended residence inside endocytic compartment(s). In contrast to recombinant class II readily loaded with peptide ligands in vitro, production of empty class II directly accessible to peptide ligands in normal APCs is tightly controlled due to the combined actions of specific class II chaperones, the invariant (Ii)3 chain, and DM required at distinct stages during maturation and export (5, 6).

The highly conserved Ii chain coassembles with polymorphic class II subunits to prevent irreversible misfolding or aggregation and protect the nascent empty groove from association with molecular chaperones responsible for endoplasmic reticulum quality control. Subsequently, class II occupancy by diverse peptide ligand(s) requires displacement of class II-associated Ii chain-derived peptide (CLIP) sequences. Current models suggest that the nonconventional class II product DM acts inside endocytic compartment(s) to promote CLIP release in exchange for tightly bound peptide ligand(s) and to protect empty class II from functional inactivation during extended exposure to low pH. DM associations with class II/Ii chain-processing intermediates, CLIP complexes, and empty class II have all been described (7, 8, 9), but specific interactions responsible for DM function as a peptide editor remain a mystery.

The extensive polymorphism necessary to accommodate diverse peptides also influences the intrinsic stabilities of class II heterodimers and many important aspects of Ii chain and DM chaperone functions. Allelic diversity has a significant impact on Ii chain functions during subunit assembly (10), the kinetics and specificity of CLIP associations (11, 12, 13, 14, 15), and Ii chain degradation products (16, 17). In vitro experiments demonstrate allele-dependent DM associations with empty class II (9). Allele-specific DM requirements during class II Ag presentation have been described in transfected cells, but contradictory conclusions were reached by different labs (18, 19, 20, 21). It also seems likely that DM actions demonstrated via these complementation assays could be influenced by many additional experimental parameters, such as abnormal expression levels and functional contributions by recipient cell lines, making it difficult to assess the significance of these observations.

Current thinking about the class II pathway has been strongly influenced by studies of DM mutant mice (22, 23, 24). The loss of DM function disrupts conventional Ag presentation and causes complex defects with respect to selection of mature CD4+ T cells. The striking accumulation of Ab/CLIP complexes alongside severely reduced peptide-loading capabilities strongly argues DM activity is required to mediate CLIP release and promote occupancy by diverse peptides under physiological conditions in normal APCs. As judged by the gain of reactivity toward wild-type class II molecules, mature CD4+ T cells exclusively selected on Ab/CLIP complexes exhibit somewhat incomplete tolerance toward natural self-peptides (22, 23, 24). Considerable evidence suggests these animals express a semidiverse TCR repertoire (25, 26, 27, 28).

As for Ii chain contributions (29), the extent of DM requirements with respect to CD4+ T cell maturation might be dependent on the particular epitope and/or TCR specificity. Interestingly, discrete disturbances were recently described for offspring of DM mutants crossed to class II transgenic mice (30). Thus, Ek but not Ak coexpression led to rescue of CD4+ T cell development. However, complementation using this particular allelic combination also allows formation of haplotype-mismatched dimers, and thus decreases expression of particular {alpha}{beta} pairs, adding a degree of complexity with respect to functional comparisons and self-tolerance issues. In addition, because DM mutants have not as yet been extensively backcrossed to establish congenic strains, random segregation of endogenous mouse mammary tumor virus (MMTV)-encoded superantigens potentially causes selective V{beta} deletions, and may therefore also affect the outcome of these experiments.

To further evaluate possible effects of allelic diversity on DM activities during presentation of self peptides and TCR repertoire selection, we sought to generate loss of function DM mutants expressing different MHC haplotypes. Production of such animals is impractical by conventional genetic approaches due to the tight linkage of these loci within the MHC. However, this can readily be accomplished via embryonic stem (ES) cell technology. To this end, a targeting vector comprised of homologous genomic fragments isolated by long-range PCR was introduced into germline-competent BALB/c ES cells (31), and recombinant clones gave rise to genetically pure mutant progeny. In comparison with class II functional defects previously described for DM-deficient mouse strains, BALB/c DM mutants exhibit a less severely compromised phenotype. Remarkably, the loss of DM disrupts Ad Ag presentation and enhances peptide loading by Ed molecules. These experiments demonstrate for the first time that DM requirements differ for class II structural variants coexpressed under physiological conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of the targeting vector and generation of recombinant ES cell clones

To isolate BALB/c genomic subclones, we used a long-range PCR system (Boehringer Mannheim, Indianapolis, IN; catalogue 1681 834) and appropriate primer pairs derived from conserved regions shared between 129 and BALB/c sequences (32). As shown in Fig. 1Goa, the 5' arm corresponds to a 4.5-kb BamHI-ScaI fragment located immediately 5' to exon 1. The 3' arm is comprised of a HindIII-EcoRI fragment that contains 30 nt of exon 2 and extends 4 kb downstream. The amplification products were ligated into the pCR2.1 vector using the TA cloning kit (Invitrogen, San Diego, CA; catalogue K2000-40). The XhoI-HindIII fragment from pMCIneo was subcloned into the corresponding sites of pBluescript KS, and an oligonucleotide containing a ScaI site was subsequently introduced into this intermediate via its KpnI and XhoI sites. The BamHI-ScaI-digested blunt-ended 5' arm was subsequently cloned into the SmaI site. The HindIII-EcoRI-digested blunt-ended 3' arm was introduced into the NcoI site present on a second plasmid comprised of the 1.7-kb HindIII-XhoI HSV/thymidine kinase counterselection cassette inserted into the corresponding sites of pBluescript KS. The intermediate subclones containing the 5' and 3' arms were subsequently modified to destroy KpnI and HindIII sites, respectively, and ultimately joined to create the isogenic targeting vector shown in Fig. 1Goa.



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  		FIGURE 1. Generation of DM-deficient BALB/c mice via homologous recombination in ES cells. a, Schematic representations of the wild-type locus, targeting vector, and mutant allele. The positions of SacI, ScaI, BamHI, HindIII, EcoRI, Nde, and XbaI restriction sites are shown. b, Southern blot analysis of representative intercross progeny. The positions of diagnostic fragments derived from wild-type or targeted alleles are indicated by the arrows. c, 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. The positions of mutant and wild-type products are shown. d, RNase protection analysis. The DMa probe or a control probe specific for cytoplasmic actin was hybridized with total RNA (10 µg) prepared from the indicated tissues. The positions of full-length protected fragments are indicated by the arrows.

 
BALB/c ES cells (31), maintained on mitomycin-treated STOneo feeder cells, were electroporated with NotI-linearized plasmid DNA, and after a 24- to 48-h recovery period, were grown in the presence of G418 (200 µg/ml) and FIAU (0.1 µM) for 9 days. Drug-resistant colonies were picked and expanded into 96-well tissue culture trays, and split once, and one set of duplicate trays was frozen. DNA extracted from individual clones was subjected to Southern blot analysis using restriction enzyme and probe combinations shown in Fig. 1Goa to assess the integrity of both sides of the targeted allele. Subsequent hybridization with a neo probe confirmed these recombination events represent single copy integrations.

Animals

To generate chimeras, correctly targeted ES cell clones were injected into C57BL/6 blastocysts. Proven germline chimeric males were crossed to BALB/cJ females to produce genetically pure heterozygous progeny that were subsequently intercrossed to obtain homozygous mutants. To distinguish wild-type and BALB/c DMa mutant alleles, we used a PCR screen. As shown in Fig. 1Go, the common primer (5'-TCTGGACACTGGGATTTGACCTTC-3') lying at the 3' end of exon 2, in combination with a second primer upstream (5'-CACATTCCGGCACACTCTATTCTG-3') in a portion of the gene deleted by the targeting event, yields a 246-bp wild-type band. Additionally, a third primer (5'-CTTCGCCCAATAGCAGCCAGTCC-3') specific for the neo cassette in the targeting vector gives rise to the 691-bp mutant product. Reactions were conducted for 30 s at 94°C, 30 s at 59°C, and 45 s at 72°C for 30 cycles, with a final extension for 10 min at 72°C. The amplification products were resolved on a 2% agarose gel and visualized by ethidium bromide staining.

The DMa-deficient mice (22), on a (129 x C57BL/6)F2 background, that express the H-2b haplotype, have been maintained by brother-sister matings. The production of Ii chain mutants (33), and sublines established by backcrossing the targeted allele onto BALB/cAn (H-2d), or B.C-9, a strain congenic with C57BL/6, but expressing the Igha allotype of BALB/c, has been described (10, 34). BALB/cJ and DBA/2J mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

RNase protection assay

For analysis of DMa transcripts, we subcloned the 335-bp EcoRI-HindIII fragment spanning exons 1 and 2 from the cDNA (35), kindly provided by John Monaco (Howard Hughes Medical Institute, University of Cincinnati, Cincinnati, OH) into corresponding sites of pBluescript KS. The resultant plasmid was linearized with BamHI and transcribed using T3 polymerase to generate a 404-nt antisense probe. A mouse {beta} actin probe was used as a control. Total RNA (10 µg) was hybridized overnight at 45°C with 32P-labeled probes. Samples were digested using RNase A (40 µg/ml) and RNase T1 (2 µg/ml) for 60 min at 37°C in the case of the DMa probe, or 10 min at 25°C following incubation with the actin probe, treated with SDS and proteinase K, extracted twice with phenol/chloroform, ethanol precipitated, redissolved in buffer containing 80% formamide, and analyzed by electrophoresis in a 6% polyacrylamide denaturing gel.

Abs and peptides

Hybridomas include MKD6 specific for a private A{beta}d epitope, 25-9-17 specific for A{beta}b/d, BP107 specific for A{beta}b/d, M5/114 specific for A{beta} + E{beta}, K24-199 specific for A{alpha}d, and 14-4-4 specific for E{alpha}d. The class II chain specificities (36, 37) and Ii chain influences affecting expression of these conformational epitopes (10, 34) have been extensively discussed. Rabbit antisera specific for determinants located in the cytoplasmic tails of the {alpha}- and {beta}-chains were generously provided by Ronald N. Germain (National Institutes of Health, Bethesda, MD). The OVA323–339 (ISQAVHAAHAEINEAAGR), hemagglutinin126–138 (HNTNGVTAACSHE), IgG2ab435–451 (YFMYSKLRVQKSTWERG), bacteriophage {lambda} repressor cI peptide P12–26 (LEDARRLKAIYEKKK), SWM 129–153 GAMNKALELFRKDIAAKYKELGYQG, the truncated variant of moth cytochrome 88–103 previously described as DASP KKANELIAYLKQATK, the Ii chain 85–101 KPVSQMRMATPLLMRPM peptides, and biotin-conjugated variants were purchased from Quality Controlled Biochemicals (Hopkinton, MA).

Immunofluorescence analysis

For single-color staining, spleen cell suspensions depleted of erythrocytes by ammonium chloride-Tris treatment were incubated on ice with saturating amounts of biotinylated Abs or toxic shock syndrome toxin-1 (TSST-1; catalogue TT606-B; Toxin Technology, Sarasota, FL), followed by FITC-labeled avidin D (catalogue A-2001; Vector Laboratories, Burlington, CA). Fluorescence was analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA), and data displayed as cell number vs log fluorescence. For T cell subset analysis, suspensions of thymocytes, lymph node, or spleen cells were incubated on ice with anti-CD8 FITC, anti-CD4 PE, and biotinylated anti-TCR (PharMingen, San Diego, CA; catalogue 01044D, 01065B, and 01302D, respectively), followed by streptavidin Red 670 (Life Technologies, Gaithersburg, MD). CD4 vs CD8 dot plots are shown. For analysis of V{beta} usage, lymph node cells were incubated with anti-CD8 FITC and anti-CD4 PE, as above, in combination with biotinylated TCR mAbs from PharMingen, as follows: anti-V{beta}3 (catalogue 01402C), V{beta}5 (catalogue 01352C), V{beta}6 (catalogue 01362C), V{beta}8 (catalogue 09952C), V{beta}11 (catalogue 01372C), V{beta}12 (catalogue 01682C).

Radiolabeling, immunoprecipitations, and Western blots

Biosynthetic labeling, immunoprecipitations, and SDS-PAGE were conducted as described (17). 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 10x excess 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) 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% Na azide, and 1 µg/ml aprotinin, and then solubilized in Laemmli buffer containing 2% SDS and 2-ME by treatment either 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.

For Western analysis, sample buffer was added to detergent extracts (2 x 107 cells/50 µl) prepared as above, and lysates were divided into equal portions, and one half boiled for 5 min before fractionation on 10% polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes (catalogue BA83; Schleicher and Schuell, Dassel, Germany) for 2 h at 500 mA. Blots were rinsed in TBST and then air dried overnight. Subsequently, blots were incubated in TBST with 10% dry milk and 3% BSA and rinsed once before the addition of primary Abs diluted in TBST containing 3% BSA and 5% calf serum. Following a 60-min incubation, blots were extensively washed with TBST containing 0.1% BSA and preadsorbed HRP secondary Abs added in TBS-T containing 3% BSA for 30 min. Blots were washed with TBST and developed by chemiluminescence using ECL (catalogue RPN2106; Amersham, Arlington Heights, IL).

Ag presentation assays

The T cell hybridomas F1.2, 1H11.31, and 1E5.11 2G12.1, 2D7.12, 1F5.2, and 1F10.2 were generously given to us by Luciano Adorini (Roche Milano Ricerche, Milan, Italy). T cell clones AODH7.1, 3DT52.5.8, 3D054.8, DO11.10, 3D018.3, and 3D026.1 were kindly provided by Philippa Marrack (Howard Hughes Medical Institute, National Jewish Center, Denver, CO). 7B7.3 was from Malcolm Gefter (Massachusetts Institute of Technology, Cambridge, MA). The IgG2ab-specific hybrids C5-10 and C5-45 were produced in this lab.

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 mixed lymphocyte reactions, CD4+ T cells (4 x 105/well) positively selected using magnetically labeled microbeads (catalogue 492-01) and MACS separation columns (Miltenyi Biotec, Auburn, CA.) were cultured with increasing numbers of irradiated (3300 R) 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 mean cpm of triplicate cultures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of BALB/c DM mutant mice

Distinct structural properties of MHC class II allelic variants are known to influence disease susceptibility and immune protection, but information about DM actions in vivo as yet comes from studies of mutant mice expressing the H-2b haplotype. To analyze DM contributions to class II activities in the context of the H-2d haplotype, we decided to generate a novel mutation at the DMa locus in germline-competent BALB/c ES cells (31). Initial attempts to target the locus using a nonisogenic 129-based vector yielded no recombinant clones, making it necessary to design a completely homologous construct. Taking advantage of available BALB/c genomic sequence information (32), and long-range PCR technology, we obtained 5' and 3' regions of genomic homology and placed these arms in between positive (neo) and negative (HSV/thymidine kinase) drug selection cassettes. As shown in Fig. 1Goa, the desired recombination event creates a 1.7-kb deletion spanning exon 1, intronic sequences, and most of exon 2. From 1076 drug-resistant colonies, we recovered four correctly targeted clones giving the expected patterns on Southerns. Proven germline males derived from two independent clones were crossed to BALB/cJ females to generate genetically pure heterozygotes, and subsequent intercross matings yielded homozygous mutants and wild-type littermates. Next we analyzed steady state levels of mRNA using an RNase protection assay. As shown in Fig. 1God, wild-type lymphoid tissues efficiently express DMa transcripts, whereas in contrast RNA from homozygous mutants gave no detectable signal. Results obtained with this 5' probe spanning the deletion were confirmed using a 3' probe specific for exon 5 (data not shown). Targeted disruption of the BALB/c locus thus results in the absence of DMa gene expression.

Partially disrupted class II conformational maturation

Selective loss of class II conformational epitopes in human somatic variants (38) and mice lacking DM function (23, 24, 39) closely correlates with CLIP occupancy. In contrast, considerable evidence suggests DM activities are nonessential for conformational maturation of other class II allelic products, but there are also contradictory results in the literature (18, 19, 20, 21, 30). To further evaluate allele-specific DM requirements under physiological conditions, class II surface expression by BALB/c mutants and control wild-type spleen cells was tested using a panel of mAbs. As shown in Fig. 2Go, MKD6 (A{beta}d-specific), 25-9-17 (A{beta}b/d-specific), K24-199 (A{alpha}d-specific), and 14-4-4 (E{alpha}d-specific) mAbs demonstrate mature properly folded class II is efficiently exported to the cell surface. Surprisingly, surface expression of BP107 (A{beta}b/d-specific) epitopes was substantially above background levels, in contrast to the complete loss of BP107 epitopes observed for H-2b DM mutants (23, 24, 39). Similarly, mature Ad produced by DM mutants exhibits detectable TSST-1-binding capabilities (Fig. 2Gof), whereas in contrast Ab/CLIP complexes produced by H-2b mutants entirely lack TSST-1 reactivity (data not shown). This diagnostic serological profile was consistently observed for homozygous mutants independently derived from two targeted BALB/c ES cell clones. Interestingly, DM- Ii- double-mutant splenocytes gain expression of BP107 epitope(s), coincident with the loss of CLIP occupancy. It is possible that these structural changes may in part reflect a poor fit of Met98 in the P9 pocket (15), but to our knowledge the precise residues responsible for BP107 reactivity have not been mapped. The TSST-1 contact site(s) partially overlaps with the class II peptide groove (40). These findings strongly suggest that the BALB/c mutant allele permits limited occupancy by diverse self peptides structurally similar to wild-type ligands.



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  		FIGURE 2. BALB/c DM mutants weakly express BP107 epitopes and TSST-1-binding capabilities. Splenocytes from 1) wild-type, 2) DM-deficient, 3) Ii chain mutants, or 4) double mutants lacking both DM and Ii chain functions were stained with biotin-conjugated mAbs or TSST-1, as indicated, followed by FITC-conjugated avidin. Representative data from one of three identical experiments with similar outcomes are shown.

 
T cell repertoire selection

Ii chain mutant mice expressing the H-2b haplotype contain 10–20% of the normal numbers of mature CD4+ T cells in the thymus and periphery (33, 41). In contrast, relatively efficient CD4+ maturation has been described in BALB/c Ii chain mutant mice (34). To evaluate CD4+ T cell selection in BALB/c DM-deficient animals, we analyzed T cell subpopulations using three-color flow cytometry. As shown in Fig. 3Goa, mutant thymi contain roughly one half the number of mature CD4+ T cells. As for Ii chain mutants, BALB/c DM-deficient mice consistently display near normal numbers of mature CD4+ T cells in the periphery. The less severe CD4 maturation defect observed in this study for BALB/c mutants does not simply reflect changes in animal health status over time, because age-matched H-2b DM mutants analyzed in the same experiments consistently display a more striking phenotype. Thus, in the context of the BALB/c background, peripheral expansion of CD4+ T cells is efficiently mediated via DM-independent pathways. In all likelihood, distinct self peptide ligands are responsible for selection of CD4+ T cells in Ii chain and DM mutants. Consistent with this possibility, CD4+ T cell development is more severely compromised in DM- Ii- double mutants (Fig. 3Goa).



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  		FIGURE 3. CD4+ T cell repertoire selection. a, Thymus, spleen, and lymph node cell suspensions were stained for CD4 and CD8 expression and analyzed by flow cytometry. The numbers refer to the percentages of total cells within the indicated gates. Representative data from one of eight independent experiments are shown. b, Lymph node cells were stained for CD4, CD8, and TCR V{beta} expression. The numbers refer to the percentages of CD4 or CD8 single-positive T cells costained with the indicated V{beta} mAbs in individual animals. As a control, we used B.C-9 (H-2b), a strain congenic with C57BL/6 but expressing the Igha allotype. c, Purified CD4+ T cells (4 x 105/well) were cultured in the presence of increasing numbers of irradiated (3300 R) spleen cells, as indicated. Proliferative responses were measured by a 16-h exposure to 1 µCi of [3H]thymidine. All results are expressed as mean cpm of triplicate cultures.

 
Superantigen-mediated clonal deletion has an important impact on selection of the CD4+ T cell repertoire, but possible role(s) for DM during this process has yet to be elucidated. The BALB/c mouse strain carries well-defined MMTV-encoded superantigens (42). Three loci corresponding to endogenous MMTV proviruses are associated with clonal deletion of T cells with V{beta}3, 5, 11, and 12. To evaluate DM-dependent reactivity toward endogenous superantigens, we examined the percentages of T cells carrying these V{beta} segments, and as a control, V{beta}6 and 8 representation was analyzed in parallel. As shown in Fig. 3Gob, T cell clones expressing TCR V{beta}3, 5, 11, and 12 were efficiently eliminated in wild-type BALB/c mice. In contrast, these TCR V{beta} segments are well represented in Ii chain and DM mutants. Thus, we conclude DM plays an essential role during presentation of endogenous superantigens.

The CD4+ T cells selected in DM mutants display tolerance toward autologous stimulators, but parental wild-type cells elicit vigorous proliferative responses (22, 23, 24). Anti-self reactivity appears to be specific for diverse low abundance self peptides encoded by unknown loci (25, 28). To further evaluate DM contributions shaping the TCR repertoire, we similarly examined the autoimmune status of residual CD4+ T cells in genetically pure BALB/c mutants. As shown in Fig. 3Goc, CD4+ T cells from BALB/c DM-deficient mice consistently gave relatively weak proliferative responses directed toward wild-type stimulators. As a third-party control, we used B.C-9 (H-2b) spleen cells, a congenic strain identical with C57BL/6 but expressing the Igha allotype. Interestingly, the degree of stimulation was markedly enhanced in the presence of H-2d-compatible DBA/2J stimulators possibly due to increased reactivity toward divergent self-peptides. Thus, residual CD4+ T cells produced by BALB/c DM mutants seem to display partial tolerance toward self-peptides naturally presented via DM-independent pathways.

Absence of mature compact dimers coincident with appearance of SDS-unstable Ad/CLIP and Ed/CLIP complexes

To further examine class II structure, we performed immunoprecipitation experiments. As expected, we found Ab/CLIP migrates just slightly behind wild-type compact dimers, and in heated samples, CLIP appears ahead of the dye front. In contrast, Ad/CLIP and Ed/CLIP complexes are unstable in SDS gels (Fig. 4Go, a and b). Surprisingly, in the case of Ed-associated CLIP, we found evidence for structural heterogeneity because different Abs yielded electrophoretically distinct CLIP species (Fig. 4Gob). Are unstable dimers detectable after a 4-h chase, loosely occupied by CLIP and/or other mediocre peptides, displaced over time by mature class II bound to tightly associated ligand(s)? To evaluate possibly improved quality of class II peptides at steady state, we also examined constitutive expression of compact dimers via Western blot analysis. Under conditions appropriate for detection of Ab/CLIP complexes, mature Ad expressed by BALB/c DM mutants readily dissociates in SDS-PAGE, consistent with findings reported for Ad transformants (19, 20, 21). Results obtained with M5/114 (A{beta} + E{beta}-specific) mAb similarly demonstrate SDS-unstable Ed molecules (Fig. 4God). Thus, the mutation disrupts selection of tightly bound self-peptides.



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  		FIGURE 4. Loss of mature compact dimers alongside the appearance of Ad/CLIP and Ed/CLIP complexes. a and b, Lysates from spleen cells labeled with [35S]methionine for 40 min and chased for 4 h were immunoprecipitated with mAbs or chain-specific rabbit Abs, as indicated. Complexes solubilized at 100°C (B) or at room temperature (NB) were analyzed by SDS-PAGE under reducing conditions. c and d, Western blot analysis fails to demonstrate conformationally distinct Ad or Ed compact dimers expressed at steady state. Lysates were divided in half, boiled for 5 min (B), or kept on ice (NB), and samples subsequently resolved by SDS-PAGE were transferred onto nitrocellulose membranes, and blots probed with class II-specific Abs, as indicated.

 
Isotype-specific DM activities during Ag presentation and peptide capture

DM mutants carrying the H-2b haplotype display severely compromised class II activities (22, 23, 24). However, a complex picture has emerged with respect to DM and Ii chain contributions during functional maturation of other class II allelic variants. To further explore allele-specific DM and Ii chain requirements under physiological conditions, mutant spleen cells were tested for their abilities to stimulate a diverse panel of Ad- and Ed-restricted T cell hybridomas (Table IGo). Unlike DM-independent activity observed for transfected cell lines (19, 20), in this study we found presentation of intact protein Ags to Ad-restricted T cell clones strictly requires DM coexpression (Fig. 5Goa). In addition, DM leads to enhanced responses toward already processed peptides (Fig. 5Gob), whereas Ii chain contributions depend on the particular epitope and/or TCR affinity. In striking contrast, Ed-restricted T cell clones strictly require Ii chain coexpression, and are partially independent of DM functional activities (Fig. 5Goc), consistent with recent evidence suggesting that DM enhances presentation of the immunodominant Ed-associated hen egg lysozyme 107–116 epitope (43). However, unlike fibroblasts transfected with class II, Ii chain, and DM expression constructs, in this study we found Ad- and Ed-restricted T cells consistently exhibit distinct Ii chain and DM requirements, suggestive of isotype-specific maturation pathway(s).


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  		Table I. Ag-specific T cell hybridomas used in the present study1

 


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  		FIGURE 5. Partially disrupted Ag presentation activities. Wild-type, DM, or Ii chain mutant splenocytes were cultured with T cell hybridomas, as indicated, in the presence of intact protein Ags (a and c), synthetic peptides (b), or medium alone (d). IL-2 production was measured by a 16-h exposure to 1 µCi of [3H]thymidine. All results are expressed as mean cpm of triplicate cultures. Data are representative of three independent experiments.

 
To extend these observations, we also compared the abilities of mutant spleen cells to stimulate T cells directed toward closely overlapping Ad- and Ed-associated epitopes derived from endogenous naturally processed self {beta}2-microglobulin ({beta}2m) (44). Consistent with results above, Ad-restricted T cells display enhanced responses in the absence of Ii chain and a complete loss of functional activity in the presence of DM-deficient splenocytes, and in contrast Ed-restricted clones require Ii chain, but not DM functions (Fig. 5God). As a control, these spleen cell populations stimulate roughly equivalent responses in the presence of H-2Dd-specific T cells. Thus, class II associations with self {beta}2m similarly display isotype-specific Ii chain and DM requirements.

Experiments above demonstrate a subpopulation of mature Ad expresses BP107 epitopes and TSST-1-binding capabilities, consistent with detectable levels of CLIP release and leaky peptide loading. To directly evaluate peptide-binding capabilities, mutant and control wild-type splenocytes were incubated with biotinylated peptides, stained with FITC avidin, and analyzed by FACS, as described (45). As shown in Fig. 6Goa, DM-deficient spleen cells exhibit markedly reduced activities in the presence of both hemagglutinin126–138 and OVA323–339, known to be good Ad binders, whereas Ii chain mutant splenocytes display enhanced binding capabilities, as expected for empty class II and/or molecules occupied by easily displaced ligand(s). Interestingly, Ii 85–101 gave similar results with H-2b haplotype mice, but contrary to expectations, we found in the absence of DM, BALB/c spleen cells bound exogenously added CLIP peptide with increased efficiency (Fig. 6Goa). DM mutant splenocytes similarly display enhanced binding capabilities in the presence of several Ed-specific peptides surpassing not only wild-type, but also remarkably those observed for Ii chain mutant splenocytes (Fig. 6Gob). These experiments demonstrate isotype-specific DM actions during peptide capture.



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  		FIGURE 6. DM mutants exhibit decreased Ad and enhanced Ed peptide-loading 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 medium alone, were stained with FITC-labeled avidin and analyzed by FACS. Representative data from one of three identical experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The extensive polymorphism necessary to accommodate diverse peptides also restricts partner choices during {alpha}- and {beta}-chain pairing and imposes allele-specific influences with respect to Ii chain and DM associations. The Ab product displays several exceptional properties. Other class II molecules readily dissociate in SDS gels, whereas greater than 90% of mature Ab migrates as compact dimers (46). Similarly as a general rule, class II dimers efficiently assemble in the absence of Ii chain, but Ab subunit association strongly depends on Ii chain function (10). Recent experiments demonstrate DM actions required for CLIP release and Ab functional activities (22, 23, 24). However, DM-independent class II Ag presentation has also been extensively documented (18, 19, 20, 30). We wondered to what extent these discordant observations truly reflect allelic differences, and whether strict DM requirements might prove to be Ab specific.

To further investigate allele-specific DM actions during class II maturation, we created mutant mice carrying the H-2d haplotype via homologous recombination in BALB/c ES cells. Unlike gain of function experiments, this targeted mutagenesis approach has allowed us to examine functional consequences in a completely homologous system in professional APCs with appropriate ratios of class II, Ii chain, and the putative DM chaperone DO, all coexpressed under control of endogenous regulatory elements. This genetic background also offered the opportunity for side by side comparisons of Ad and Ed functional activities. Coexpression of structurally divergent class II surface Ags more closely approximates the situation in humans in which several homologous gene products are encoded by tightly linked loci. Several independent spontaneous mutations in mice selectively disrupt expression of the E subregion product, but interestingly strains with a reciprocal expression pattern (E subregion only) do not appear to exist. The null allele we engineered results in decreased peptide capture by Ad molecules, and in striking contrast causes enhanced peptide loading by Ed molecules. Thus, the functional role of DM differs for class II structural variants coexpressed in normal APCs.

Structurally diverse class II molecules display distinctive CLIP-binding properties in biochemical assays (11, 12, 13, 14, 15, 47, 48). In the case of CLIP bound to DR3, the highly conserved methionine side chains tightly fit inside the P1 and P9 pockets (1). A quite different situation exists with respect to the overall shape of the Ad peptide-binding groove (4). Although its P1 pocket appears large enough to accommodate a methionine, the P9 pocket is smaller, so Met98 is predicted to sit suboptimally. This relatively poor fit could potentially lead to enhanced spontaneous CLIP release and subsequent displacement by better binders. Possibly empty Ad survives well on its own in the absence of DM influences. In vitro studies have described empty class II conformers with distinct peptide-binding properties (49, 50), but we have as yet only vague ideas about the structure and fate of empty class II molecules inside normal APCs. As previously shown (33) and confirmed in this study, class II constitutively expressed by Ii chain mutants has superior peptide-loading capabilities, but it remains unclear whether these molecules are truly empty or occupied by easily displaced ligand(s) that permits properly folded class II to escape endoplasmic reticulum quality control. Recent evidence suggests empty class II molecules are selectively produced by immature dendritic cells (51), possibly due to down-regulated Ii chain expression (52, 53). Interestingly, allele-specific Ii chain requirements also appear to regulate dendritic cell functional activities (54). The extent to which allelic and isotypic differences influence intrinsic class II stabilities under physiological conditions has yet to be elucidated.

Another possible scenario is that allele- and isotype-specific contacts give rise to structural variants of CLIP with different dissociation kinetics. Sequence data reported for Ab- and Ad-associated peptides suggest this is not the case (47, 55). However, natural DM substrate(s) may well include additional portions of Ii chain extending outside the groove. Consistent with this way of thinking, it appears that the amino-terminal segment of CLIP destabilizes class II complexes (56, 57, 58), whereas C-terminal portions of Ii chain enhance class II associations (58, 59, 60). The relative importance of these additional Ii chain binding sites could perhaps differ for class II structural variants. Allele- and isotype-specific motifs also potentially influence the alignment of CLIP residues within the groove (14). Interestingly, Ek-associated CLIP produced by DM mutant spleen cells displays greater sequence diversity in comparison with Ab/CLIP (30). Similarly in this study, conformationally distinct CLIP products were recovered using different Ed-specific Abs. Sequence comparisons of these various class II-associated Ii chain proteolytic fragment(s) should help to provide a clearer understanding of Ii chain cleavage pathways.

Discrete Ad as opposed to Ed peptide-binding preferences can be explained in the simplest terms as being entirely due to selective binding motifs inside the groove (2, 4). However, the present data suggest additional complexities. Side by side comparisons analyzing presentation of Ad- and Ed-associated epitopes to a wide panel of T cell clones demonstrate striking isotype differences with respect to Ii chain and DM requirements. Thus, we found Ad-restricted T cell clones strictly require DM coexpression and only partially depend on Ii chain, whereas, in contrast, Ed-restricted T cell clones require Ii chain activities, but show relatively relaxed DM requirements. Presentation of {beta}2m epitopes with Ed requires acidic pH and increased concentrations, in comparison with binding properties of closely overlapping Ad peptides (44). Similarly at neutral pH, CLIP efficiently associates with Ad molecules, and interactions with Ed are greatly enhanced at mildly acidic pH (47, 48). Consistent with these observations, the structure of Ek reveals a network of hydrogen bonds with a conserved water molecule and a buried cluster of acidic residues in the binding groove, in all likelihood available only under low pH conditions such as those inside endocytic vesicles (2). Obviously, intact Ii chain efficiently forms oligomeric complexes with Ed at neutral pH and guides its post-endoplasmic reticulum export, possibly, as suggested above, via isotype-specific contact site(s) outside the peptide-binding cleft.

I-E molecules are known to associate with virally encoded superantigens in a manner distinct from conventional peptide ligands (34). Proteolytic processing of these type II transmembrane glycoproteins is required for class II binding, and thus engagement of specific TCR V{beta} segments (61, 62). Selective presentation with E but not A subregion products cannot simply be explained due to preferential association per se because the orf product binds equally well to A molecules (61). The present experiments demonstrate for the first time that DM influences presentation of MMTV-encoded superantigens. Additionally, we found that Ed molecules expressed by DM mutants exhibit markedly increased peptide-binding capabilities, even in comparison with those produced by Ii chain-deficient spleen cells. These findings suggest in the absence of DM-editing functions that Ed molecules become stably occupied by mediocre binders. According to this way of thinking, the self peptide repertoire displayed by BALB/c DM mutants potentially includes novel low affinity ligands such as those thought to provoke autoimmune disease.

Targeted mutagenesis aimed to cause loss of gene function may in some cases also result in the accumulation of intermediates with distinctive effects. A striking example comes from studies of class II mutant strains in which null alleles introduced at either the {alpha}- or {beta}-chain locus effectively disrupt class II surface expression, Ag presentation abilities, and CD4+ T cell maturation, but these strains display marked differences with respect to B cell characteristics. Defective B cell development observed for {alpha}- but not {beta}-chain mutants can best be explained due to selective overproduction of {beta}-chain aggregates (34, 39, 63). According to current models, DM associates with various substrates, including CLIP-occupied and empty class II molecules. Additionally, DM activities may be down-regulated in selected cell types, due to the formation of DM/DO complexes (64, 65, 66, 67, 68). Ii chain associations also influence DM stability (69). The recently solved structure of DM failed to reveal an open binding cleft able to accommodate peptide ligand (70, 71). A mutagenesis screen recently mapped DM interactions broadly to the lateral surface of DR nearest P1 as opposed to the P9 pocket (72), but we have as yet only rudimentary insights into specific contact site(s) responsible for CLIP release, peptide editing, and DO associations. The BALB/c DM mutant mice described in the present work may prove useful for analysis of allele- and isotype-specific structural features guiding class II peptide loading via these complex oligomeric assemblies in specialized APCs.


   Acknowledgments
 
We thank Nancy Noben-Trauth for BALB/c ES cells; Luciano Adorini and Pippa Marrack for T cell hybridomas; Luc Van Kaer and Ross Waldrip for 129 genomic subclones and restriction maps; Jen Lower for RNase protection probes; Ron Germain for chain-specific rabbit Abs; Debbie Pelusi for valuable assistance in screening mutant progeny; Patti Lewko, Joe Rocca, and Shanda Porter for careful maintenance of the mouse colony; Kara McClellan for secretarial assistance; and Renate Hellmiss for preparing the figures.


   Footnotes
 
1 This work was supported by Grant AI-19047 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; MMTV, mouse mammary tumor virus; TSST-1, toxic shock syndrome toxin-1; {beta}2m, {beta}2-microglobulin. Back

Received for publication December 4, 2000. Accepted for publication February 12, 2001.


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N. K. Nanda and E. K. Bikoff
DM Peptide-Editing Function Leads to Immunodominance in CD4 T Cell Responses In Vivo
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C. H. Koonce and E. K. Bikoff
Dissecting MHC Class II Export, B Cell Maturation, and DM Stability Defects in Invariant Chain Mutant Mice
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K. Honey, K. Forbush, P. E. Jensen, and A. Y. Rudensky
Effect of Decreasing the Affinity of the Class II-Associated Invariant Chain Peptide on the MHC Class II Peptide Repertoire in the Presence or Absence of H-2M1
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C. Alfonso, G. S. Williams, J.-O. Han, J. A. Westberg, O. Winqvist, and L. Karlsson
Analysis of H2-O Influence on Antigen Presentation by B Cells
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R. C. Doebele, A. Pashine, W. Liu, D. M. Zaller, M. Belmares, R. Busch, and E. D. Mellins
Point Mutations in or Near the Antigen-Binding Groove of HLA-DR3 Implicate Class II-Associated Invariant Chain Peptide Affinity as a Constraint on MHC Class II Polymorphism
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C. H. Koonce, G. Wutz, E. J. Robertson, A. B. Vogt, H. Kropshofer, and E. K. Bikoff
DM Loss in k Haplotype Mice Reveals Isotype-Specific Chaperone Requirements
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C. Alfonso, J.-O. Han, G. S. Williams, and L. Karlsson
The Impact of H2-DM on Humoral Immune Responses
J. Immunol., December 1, 2001; 167(11): 6348 - 6355.
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