The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kenty, G.
Right arrow Articles by Bikoff, E. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kenty, G.
Right arrow Articles by Bikoff, E. K.
The Journal of Immunology, 1998, 160: 606-614.
Copyright © 1998 by The American Association of Immunologists

MHC Class II Expression in Double Mutant Mice Lacking Invariant Chain and DM Functions1

George Kenty*, W. David Martin2,{dagger}, Luc Van Kaer3,{dagger} and Elizabeth K. Bikoff4,*

* Department of Molecular and Cellular Biology, The Biological Laboratories, Harvard University, Cambridge, MA 02138; and {dagger} Howard Hughes Medical Institute, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Invariant (Ii) chain and DM functions are required at distinct stages during class II maturation to promote occupancy by diverse peptide ligands. The class II molecules expressed by mutant mouse strains lacking Ii chain or DM activities display discrete structural and functional abnormalities. The present report describes the cellular and biochemical characteristics of Ii-DM- doubly deficient mice. As for Ii chain mutants, their mature A{alpha}bAßb dimers similarly exhibit reduced mobilities in SDS-PAGE, and in functional assays these molecules behave as if empty or occupied by an easily displaced peptide. Additionally, the present experiments demonstrate that the production of floppy A{alpha}bAßb dimers is TAP independent. In comparison with Ii chain mutants, Ii-DM- doubly deficient cell populations exhibit increased peptide binding activities and consistently greater presentation abilities in T cell stimulation assays. These functional differences appear to reflect higher class II surface expression associated with their increased representation of B lymphocytes. We also observe defective B cell maturation in mice lacking Ii chain or DM expression, and interestingly, B cell development appears more severely compromised in Ii-DM- double mutants. These mutant mice lacking both Ii chain and DM activities should prove useful for analyzing nonconventional class II Ag presentation under normal physiological conditions in the intact animal.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Considerable progress has been made over recent years toward understanding the complex cellular and biochemical events necessary for guiding MHC class II peptide acquisition. Important contributions are made by both the invariant (Ii)5 chain and DM to facilitate surface expression of diverse peptide ligands, but these activities are required at distinct stages of class II maturation (1). MHC class II {alpha} and ß subunits are found coassembled with the Ii chain shortly after synthesis in the ER (2). In the exceptional case of A{alpha}bAßb molecules, the Ii chain is essential for production or maintenance of {alpha}ß dimers (3). This early class II association with the Ii chain probably prevents irreversible misfolding or aggregation of the subunits and protects the empty peptide groove from association with molecular chaperones such as BiP and calnexin that are responsible for ER quality control (4, 5, 6, 7, 8). The Ii chain facilitates export of correctly folded {alpha}ß dimers past the Golgi complex (9, 10) and directs their delivery to the endocytic compartment(s) (11, 12). Selective Ii chain degradation subsequently permits occupancy of the class II peptide groove (13).

The nonconventional class II product DM, originally described as a facilitator of Ag presentation in mutant human cell lines, acts later inside the endocytic compartment(s) to cause dissociation of a relatively short proteolytic product of the Ii chain corresponding to the so-called CLIP region, in exchange for tightly bound peptide ligand(s) (14, 15). Recent studies demonstrate the DM also associates with empty class II molecules (16, 17, 18) and acting in this manner may function as a peptide editor, serving to increase the overall affinities of peptide/class II complexes (19, 20). DM contains its own endosomal targeting signal(s) located in the ß chain cytoplasmic tail (21, 22), but there is also evidence suggesting that DM transport is mediated via an association with the Ii chain (21). In contrast to {alpha}ß-Ii complexes formed early during biosynthesis, far less is known about transient associations among class II {alpha}ß dimers, Ii chain degradation products, and DM molecules during peptide acquisition in the endocytic compartment(s).

The specific defects described for Ii chain (23, 24, 25) and DM (26, 27, 28) mutant mouse strains created using embryonic stem cell technology partially overlap. Both these mutations disrupt class II maturation, Ag presentation, and CD4+ T cell development. However, DM mutant spleen cells efficiently express surface A{alpha}bAßb/CLIP complexes at levels equivalent to those in wild-type A{alpha}bAßb molecules (26, 27, 28). In contrast, the loss of Ii chain function leads to markedly reduced A{alpha}bAßb surface expression, largely due to decreased rates of export (23, 24, 25). Thus in the absence of Ii chain, the vast majority of class II {alpha}ß dimers fail to acquire endoglycosidase H-resistant glycans and are rapidly degraded. The few mature A{alpha}bAßb dimers produced by Ii chain mutants exhibit reduced mobilities in SDS gels, and in functional assays these molecules behave as if empty or occupied by an easily displaced peptide. The structural basis for the exceptional abilities of these floppy A{alpha}bAßb dimers to escape ER quality control has yet to be determined.

In the present study we examine class II expression in double mutant mice lacking both Ii chain and DM functions. For the most part, their cellular and biochemical defects closely parallel those observed for Ii chain mutants. Thus we found that Ii-DM- double mutants display markedly reduced class II surface expression, peptide occupancy, and CD4+ T cell development. The loss of DM function has no noticeable effect on the production of floppy A{alpha}bAßb dimers. Additionally, our experiments demonstrate that expression of floppy A{alpha}bAßb dimers is TAP independent. Compared with Ii chain mutants, Ii-DM- double-mutant spleen cells consistently display increased peptide binding activities and enhanced presentation abilities in T cell stimulation assays. This probably reflects higher levels of A{alpha}bAßb surface molecules associated with an increased representation of B lymphocytes. B cell maturation is partially defective in the absence of Ii chain or DM expression and, interestingly, appears more severely compromised in Ii-DM- double mutants. These mutant mice lacking both Ii chain and DM functions should prove useful for studying class II peptide acquisition in the intact animal.


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

TAP-1 mutant mice (29), Ii chain mutants (23), DM{alpha}-deficient mice (26), and C57BL/6 mice carrying a targeted disruption at the A{alpha} locus (30) were previously described, and have been maintained by brother-sister matings. To generate double mutants, we set up intercross matings between heterozygous F1 progeny. Genotyping for the TAP-1 mutant allele was performed by Southern blotting of tail DNA as previously described (29). The PCR genotyping assay used to distinguish wild-type and Ii chain mutants has been described (3). Additionally, to screen for the DM{alpha} mutant allele, we used a three-primer system. The common primer (5'-TCTGGACACTGGGATTTGACCTTC-3') lying at the 3' end of exon 2 used in conjunction with a second primer upstream (5'-CACATTCCGGCACACTCTATTCTG-3') in a portion of the gene deleted by the targeting event yields a 270-bp wild-type band. Additionally, a third primer (5'-ATCGCCTCCTATCGCCTTCTTCAC-3') specific for the neo cassette in the targeting vector gives rise to the 362-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. In all experiments, comparisons were made between age- and, whenever possible, sex-matched animals.

Abs and peptides

Y3P (31) and Y-Ae (32) hybridomas were provided by Charles A. Janeway, Jr. (Yale University School of Medicine, New Haven, CT), 30-2 (33) was the gift of Sasha Rudensky (University of Washington School of Medicine, Seattle, WA), BP107 (34) and M5/114 (35) were obtained from the American Type Culture Collection (Rockville, MD). The chain specificities of class II mAbs and the formation of conformational epitopes, as mentioned in the text, have been extensively discussed (3). The E{alpha}56–73 (ASFEAQGALANIVDKA) and OVA323–339 (ISQAVHAAHAEINEAGR) peptides were purchased from Quality Controlled Biochemicals, Inc. (Hopkinton, MA).

Radiolabeling and immunoprecipitation

Biosynthetic labeling, immunoprecipitations, and SDS-PAGE were conducted as previously described (36). 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 5 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 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, 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-New England Nuclear, Wilmington, DE), dried, and exposed to x-ray film.

Immunofluorescence analysis

For class II analysis, spleen cell suspensions depleted of erythrocytes by ammonium chloride-Tris treatment were incubated on ice with saturating amounts of biotin-conjugated Abs followed by FITC-labeled avidin D. Fluorescence was analyzed using a FACScan flow cytometer (Becton Dickinson Co., Mountain View, CA), and the data are displayed as cell number vs log fluorescence. For double-staining experiments, spleen or lymph node cells were incubated with PE-conjugated goat F(ab')2 anti-mouse IgM (µ) (catalogue no. M31604, Caltag, San Francisco, CA) or PE-conjugated rat anti-mouse CD45R/B220 (catalogue no. 01125A, PharMingen, San Diego, CA) as a pan-B cell marker used in combination with FITC-labeled Abs directed against the IgE Fc receptor CD23 (PharMingen catalogue no. 01234D) or surface IgD (PharMingen catalogue no. 02214D). For T cell subset analysis, suspensions of thymocytes or spleen cells were incubated with anti-CD8-FITC, anti-CD4-PE, biotinylated anti-TCR (PharMingen catalogue no. 01044D, 01065B, and 01302D, respectively) followed by streptavidin-Red 670 (Life Technologies, Gaithersburg, MD). CD4 vs CD8 dot plots are shown.

Ag presentation assays

T hybridomas used in this study include BO97.1 specific for I-Ab/OVA (37), provided by Philippa Marrack (Howard Hughes Medical Institute, National Jewish Center, Denver, CO), and 1H3.1 specific for I-Ab/E{alpha}52–68 (38), given to us by Sasha Rudensky (University of Washington, Seattle, WA). IL-2 production was assessed by incubating T cells (5 x 104/well) with irradiated (3300 rad) 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 as indicated in Figure 4Go. Supernatants were collected after 20 h and assayed for IL-2 content in a secondary culture using CTLL indicator cells. [3H]thymidine incorporation was measured in the presence of 50% primary supernatant. Responses were measured after a 48-h culture by a 16- to 18-h exposure to 1 µCi of [3H]thymidine. All results are expressed as mean counts per minute of triplicate cultures.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 4. Ag presentation activities. Cultures contained spleen cell populations (2 x 105), I-Ab-restricted T hybridomas (5 x 104), and increasing concentrations of peptides or protein Ag, as indicated. Representative data from one of four identical experiments with similar results are shown.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DM- and TAP-independent expression of floppy A{alpha}bAßb dimers

Recent experiments suggest that DM associates with empty class II molecules to prevent their irreversible misfolding or aggregation after CLIP release (16, 17, 18). A strong argument can be made that floppy A{alpha}bAßb conformers produced by Ii chain mutants lack peptide ligand(s) (3, 23). It was therefore of interest to test the possibility that DM activity may be necessary for expression of floppy A{alpha}bAßb dimers. To this end, we set up matings between Ii chain and DM mutant strains. Their doubly heterozygous offspring were subsequently intercrossed to produce homozygous Ii-DM- mutants. Additionally, to examine possible contributions made by the class I peptide loading pathway, we generated TAP-Ii- doubly deficient animals. We then compared these double-mutant spleen cells for their expression of floppy A{alpha}bAßb dimers in immunoprecipitation experiments.

As shown in Figure 1Go after 5 h of chase, virtually all mature A{alpha}bAßb molecules produced by wild-type splenocytes migrated as compact dimers at approximately 56 kDa, whereas, in contrast, floppy A{alpha}bAßb dimers produced by Ii chain mutants displayed reduced mobilities on SDS-PAGE. Consistent with previous results, in the nonboiled samples we found abundant A{alpha}bAßb/CLIP complexes migrating at an intermediate position expressed by DM mutant spleen cells. In comparison with Ii chain mutants, roughly equal amounts of floppy A{alpha}bAßb dimers were produced by both Ii-DM- and Ii-TAP- double-mutant spleen cells. Moreover, these floppy A{alpha}bAßb conformers displayed equivalent reactivities with both Y3P ({alpha}+ß) and M5/114 (ß-specific) mAbs directed against conformational determinants. Thus, expression of floppy A{alpha}bAßb dimers is independent of both DM and TAP functions.



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 1. DM- and TAP-independent production of floppy A{alpha}bAßb dimers. Cytoplasmic extracts prepared from spleen cells labeled with [35S]methionine for 40 min and chased for 5 h were immunoprecipitated with the indicated mAbs. Complexes solubilized at 100°C (B) or at room temperature (NB) were analyzed on 10% polyacrylamide gels under reducing conditions. C and F indicate the positions of compact and floppy dimers, respectively. Under these conditions, the CLIP peptide migrates just ahead of the dye front.

 
Next, we examined Ii-DM- double mutants for their class II surface expression. As judged by staining with Y3P ({alpha}+ß) mAb, Ii-DM- splenocytes expressed reduced amounts of total surface A{alpha}bAßb roughly equal to Ii chain mutants (Fig. 2Go). As expected, we found that DM mutant spleen cells express 30-2 epitopes, whereas Ii-DM- double-mutant spleen cells fail to react with 30-2 mAb specific for surface A{alpha}bAßb/CLIP complexes (33). In contrast with diverse I-Ab/peptide complexes expressed on the surface of wild-type spleen cells, the I-Ab/CLIP complexes produced by DM mutants showed no reactivity toward BP107 (ß-specific) mAb (27, 28). Consistent with earlier results, we observed that A{alpha}bAßb surface molecules expressed by DM mutant spleen cells selectively lacked BP107 epitopes. Surprisingly, in contrast, we found that Ii-DM- double-mutant splenocytes gained reactivity with BP107 mAb. Thus, loss of DM function appears to promote surface display of selected Aßb conformational determinants, possibly due to binding site occupancy by a specific peptide ligand(s).



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 2. A{alpha}bAßb surface expression. Splenocytes from +/+ +/+ (1), DM- +/+ (2), +/+ Ii- (3), or DM-Ii- (4) mice were stained with biotin-conjugated mAbs followed by FITC-conjugated avidin.

 
Ii-DM- spleen cells exhibit increased functional activities

To test Ii-DM- double mutants for their peptide binding capabilities, we used Y-Ae mAb reactive with A{alpha}bAßb/E{alpha}56–73 complexes (32). Splenocytes were cultured with E{alpha}56–73 peptide and then analyzed in surface staining experiments. As expected, DM mutant spleen cells displayed severely compromised peptide loading abilities, reflecting their expression of stable A{alpha}bAßb/CLIP complexes (Fig. 3Go). We also observed markedly increased amounts of E{alpha}56–73 peptide bound by Ii chain mutant spleen cells. The Ii-DM- double mutants consistently displayed increased percentages of Y-Ae-positive cells, unaccompanied by an upward shift in fluorescence intensity. Surface staining with Y3P mAb was increased to the same degree, suggestive of changes affecting representation of B lymphocytes. As shown in Figure 3Go (Expt. 1), peptide acquisition by TAP-Ii- double mutants closely paralleled that observed for Ii-deficient mice.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 3. Peptide binding capabilities. Splenocytes from +/+ +/+ (1), DM- +/+ (2), +/+ Ii- (3), DM-Ii- (4), or TAP-Ii- (5) mice were cultured for 5 h at 37°C in the presence of E{alpha}56–68 and then stained with biotin-labeled mAbs followed by FITC-avidin. Control spleen cells cultured in medium alone gave no detectable Y-Ae staining above background.

 
Next, we analyzed Ag presentation abilities in T cell stimulation assays. As expected, DM mutant spleen cells appeared relatively ineffective for presentation of already processed peptides or intact protein Ags (Fig. 4Go). Ii chain mutant spleen cells were defective for presentation of native protein Ags and displayed markedly enhanced abilities to present already processed peptides. We found that Ii-DM- spleen cells similarly lacked the ability to present intact protein Ags, and they consistently had marginally higher responses in the presence of already processed peptides. These enhanced functional activities potentially reflect increased percentages of B lymphocytes expressing class II surface Ags, as discussed below.

B cell development is severely compromised in Ii-DM- double mutants

A recent study describes defective B cell maturation caused by the loss of Ii chain expression (39). The results of functional assays presented above suggest that Ii-DM- double mutants contain increased percentages of B lymphocytes. For these reasons, we decided to analyze B cell subpopulations present in our mutant strains. As a control, we also tested class II- mutant mice for their representation of B cell subsets. As a marker for mature B cells, we examined the expression of CD23, the low affinity IgE Fc receptor (40, 41). Spleen and lymph node IgM+ B cells were also analyzed for their coexpression of surface IgD. As expected in wild-type cells, the predominantly mature IgM+ B cells coexpressed both IgD and CD23 surface markers (Fig. 5Go). Consistent with previous results, we found here increased percentages of immature B cells lacking surface IgD and CD23 expression present in Ii chain mutants. As shown in Figure 5Go, DM mutants similarly displayed defective B cell maturation. The representation of immature B cells was markedly increased in Ii-DM- double mutants. Interestingly, we observed a striking reduction in the total number of IgM+ B cells in lymph node populations from mutants lacking either Ii chain or class II expression. Similar conclusions were reached in double-staining experiments using CD45R/B220 as the pan-B cell marker (data not shown). These findings support the idea that Ii chain expression is necessary to promote B cell maturation. Moreover, our results strongly suggest that class II expression also contributes to B cell development.



View larger version (92K):
[in this window]
[in a new window]
  		FIGURE 5. B cell maturation. Spleen and lymph node cell suspensions from 12- to 15-wk-old animals were stained using PE-conjugated anti-IgM in combination with FITC-conjugated anti-IgD or anti-CD23 and analyzed by flow cytometry. The numbers refer to the percentages of total cells within the indicated gates. Representative data from one of four identical experiments with similar results are shown.

 
Decreased CD4+ and CD8+ T cell maturation in the absence of DM expression

Mutants lacking either Ii chain (23, 24, 25) or DM (26, 27, 28) expression display partially defective CD4+ T cell maturation. It was therefore of interest to examine the extent of CD4+ T cell development in Ii-DM- double mutants. Consistent with previous results we also observed that Ii chain and DM mutants contained reduced numbers of mature CD4+ T cells in the thymus and periphery (Fig. 6Go). Doubly deficient Ii-DM- mutants similarly lacked mature CD4+ T cells, and their CD4+ percentages often appeared slightly reduced compared with those observed for Ii chain or DM mutants. Interestingly, Ii-DM- double mutants did not contain increased percentages of peripheral CD8+ T cells as observed for Ii chain mutants. Thus, we conclude that Ii-DM- double mutants exhibit decreased T cell maturation potentially due to their lack of surface A{alpha}bAßb/CLIP complexes.



View larger version (80K):
[in this window]
[in a new window]
  		FIGURE 6. T cell subsets. 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 six identical experiments with similar results are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Ii chain is an essential class II chaperone, guiding selective peptide capture in the endocytic pathway (1, 2). In its absence, the vast majority of class II molecules fail to acquire endoglycosidase H-resistant glycans and are rapidly degraded; class II surface expression is markedly reduced, but is not completely eliminated (23, 24, 25). As for floppy conformers generated upon exposure of compact dimers to low pH in the absence of peptide (42), the few mature A{alpha}bAßb molecules expressed by Ii chain mutant mice display reduced mobilities in SDS gels and by functional criteria appear to lack self peptide ligand(s) (3, 23, 25). To test whether production of floppy A{alpha}bAßb dimers depends on DM or TAP functions, we examined class II structure in Ii-DM- and TAP-Ii- double mutants.

It is possible that truly empty A{alpha}bAßb molecules escape ER quality control. On the other hand, considerable data suggest that binding site occupancy is essential for class II export through the secretory pathway (43, 44, 45). In the absence of an Ii chain, the empty groove of A{alpha}bAßb dimers potentially associates with peptides available inside the ER lumen. According to this way of thinking, the production of floppy A{alpha}bAßb conformers may depend on activities contributed by the class I peptide transporter (TAP). Recent studies demonstrate that TAP preferentially interacts with short peptides of approximately 8 to 10 residues, an appropriate length for class I binding (46). However, longer peptides up to 40 residues in length also function as TAP substrates and are thus available for class II loading (47). To determine whether the expression of floppy A{alpha}bAßb dimers depends on TAP activity, we generated TAP-Ii- double-mutant mice. Similar TAP-Ii- double mutants were recently described by Tourne et al. (48). They also found roughly equal amounts of mature A{alpha}bAßb dimers produced by Ii chain mutants and double mutants lacking TAP function. Additionally, the present experiments demonstrate that these floppy A{alpha}bAßb dimers are equally reactive with mAbs directed against distinct conformational epitopes contributed by both {alpha}- and ß-chains. Perhaps floppy A{alpha}bAßb conformers devoid of peptide exit the ER and are transported to the cell surface. On the other hand, previous studies also describe class I loading of signal sequence-derived peptides (49, 50). Thus, TAP-independent expression of floppy A{alpha}bAßb dimers potentially reflects transient association with signal peptides or perhaps intact ER polypeptides (51, 52).

Recent studies suggest that DM associates with empty class II molecules to prevent their irreversible misfolding or aggregation in endocytic compartments (16, 17, 18). The cytoplasmic tails of class II ß-chains also contain an endosomal targeting signals(s) (53). Thus, generation of floppy A{alpha}bAßb dimers could also be caused by peptide loss upon exposure to acidic pH inside the endocytic compartment(s) and thus be dependent on DM chaperone functions. The present experiments demonstrate that this is not the case. Roughly equal amounts of floppy A{alpha}bAßb dimers were produced by Ii chain and Ii-DM- double mutants. Thus, structurally distinct A{alpha}bAßb dimers lacking tightly bound self peptide ligand are probably exported via the constitutive secretory route.

In functional experiments, Ii-DM- double mutants consistently had slightly higher responses compared with mice lacking Ii chain alone. Similar results were obtained in both peptide binding and T cell stimulation assays. Thus, we observed higher percentages of Y-Ae-positive cells following incubation with E{alpha}56–73 peptide, for the most part unaccompanied by an upward shift in peak fluorescence intensity. Because surface staining with Y3P mAb was increased to the same degree, it seemed likely that Ii-DM- double mutants might simply contain increased percentages of B lymphocytes. To examine this possibility, we compared B cell representation in Ii chain, DM, and double mutant strains. As described in original reports (23, 24, 25), we also observed here for Ii chain-deficient splenocytes, only slightly reduced B cell percentages in surface staining experiments using IgM or CD45R/B220 as a pan-B cell marker. Recent experiments analyzing spleen, bone marrow, and Ag-primed lymph node cell populations suggest that Ii chain function is required for B cell maturation (39). It was therefore of interest to evaluate the extent of B cell development in our mutant mouse strains. Consistent with recent data, we also found here that Ii chain-deficient mice contain higher percentages of immature B cells. Interestingly, mutant mice lacking DM function also display defective B cell maturation, and Ii-DM- double mutants exhibit a more severe phenotype. In contrast to results obtained by analyzing splenocytes, we found that the total representation of IgM+ B cells was markedly reduced in lymph nodes from mutants lacking Ii chain or class II expression.

The possible relationship(s) between B cell development and class II surface expression has been intensely investigated. Surface class II appears late during B cell development coincident with the onset of surface IgD expression (54) and is up-regulated as a consequence of cross-linking Ig surface receptors (55). Ag uptake via surface Ig facilitates class II Ag presentation (56, 57). Interestingly, class II expression distinguishes B cell developmental pathways during ontogeny (58, 59). However, experiments to date collectively argue that B cell development is independent of class II surface expression. For example, there is general agreement that class II mutant mice exhibit a normal Ab response to T-independent Ags (30, 60, 61, 62). The lack of responses directed toward T-dependent Ags and the absence of germinal centers in class II-deficient mouse strains have been attributed to the near-complete elimination of mature CD4+ T cells (30, 60, 61, 62). Initial observations suggested that these animals lack mature IgM+IgD+ B cells (60), but subsequent reports demonstrate normal B cell development in class II mutant strains (39, 62). In contrast, recent experiments suggest that Ii chain function is necessary for B cell maturation and production of T-independent Abs (39).

The present experiments demonstrate that Ii chain, DM, and class II mutant mice all exhibit B cell defects. It is possible that such discrepancies reflect strain differences, because these targeted mutations were independently established on a mixed (129 x C57BL/6)F2 genetic background and have been separately maintained in different laboratories. Moreover, the present study describes defective B cell maturation caused by targeted disruption of the A{alpha}b gene (30); in contrast, other investigators analyzed class II-deficient mice created by targeting the Aßb locus (39, 60, 61). Perhaps normal B cell development observed in these mice reflects their expression of mixed A{alpha}Eß heterodimers. These contradictory results may also reflect variable animal health status in different labs. The underlying mechanism for defective B cell maturation remains unclear. Adoptive transfer experiments reported by Shachar and Flavell (39) are consistent with an intrinsic block to B cell development. Thus, B cell survival in the competitive follicular environment may be compromised due to the loss of intracellular signaling via surface class II. Alternatively, class II surface display of diverse peptides may be necessary for thymic development and activation of mature CD4+ helper T lymphocytes producing inductive cytokines. Additional experiments are needed to distinguish between these possibilities.

Numerous reports describe Ii chain-independent presentation of selected T cell epitopes (24, 53, 63, 64, 65, 66, 67, 68, 69). Similarly, previous studies document class II peptide loading via an alternative DM-independent pathway (67, 68, 69, 70, 71). Self peptides available within the constitutive secretory pathway may associate with empty class II molecules inside the ER (45, 72, 73), during transport through the Golgi, or at the cell surface. Additionally, mature recycling class II molecules can present selected epitopes in an Ii chain- and DM-independent manner (53, 67, 68). On the other hand, it is also known that association with the Ii chain prevents class II peptide occupancy (13, 74, 75, 76) and blocks presentation of selected epitopes (66). Similarly, DM facilitates Ag presentation via the conventional pathway and inhibits presentation of a significant fraction of endogenous self peptides (77, 78). Consistent with these findings, we observed that DM mutants fail to express BP107 epitopes, but Ii-DM- double mutants display BP107 reactivity. These results strengthen the idea that Ii chain and DM both positively and negatively influence class II surface display of self peptide ligands by an as yet poorly understood mechanism(s).

Previous studies characterizing Ii chain and DM functional activities have extensively used established cell lines. The relative expression levels of class II, Ii chain, and DM are clearly an important factor determining the outcome of these experiments. Moreover, transfection recipients used in functional experiments may differ in their content of organelles, proteases, and molecular chaperones, and recent experiments demonstrate that these cell type-specific differences have a significant impact on the intracellular distribution of class II molecules (79). Particularly in the case of heterologous expression systems comprised of introducing murine constructs into human mutant cell lines and vice versa, subtle structural differences affecting transient associations among class II {alpha}ß dimers, Ii chain, and DM are likely to have a significant impact on the diverse array of self peptides presented at the cell surface. Recent experiments also demonstrate the important influence contributed by endogenous peptides (77, 78). Mutant spleen cell populations described in this report are identical in every respect, except for their DM and Ii chain expression. Nonetheless, their surface display of BP107 epitopes exhibits a complex pattern of regulation.

MHC class II, Ii chain, and DM genes show similar tissue-specific patterns of expression and are coordinately up-regulated in response to cytokines. However, there is also evidence for non-co-ordinate expression of class II and Ii chain in selected cell types (80, 81, 82, 83). It seems likely that subtle imbalances affecting the relative levels of class II, Ii chain, and DM expression may influence the presentation of self peptides by nonprofessional accessory cells, such as thyroid follicular cells, astrocytes, and pancreatic ß cells under pathologic conditions, and potentially results in exposure of neopeptides that initiate autoimmune responses. Mutant mice selectively lacking Ii chain and DM activities should prove useful for analyzing nonconventional class II Ag presentation by diverse types of accessory cells in the intact animal.

Note added in proof. While this manuscript was under review, a similar study analyzing mice doubly deficient for Ii chain and H-zM complexes was published by Tourne et al. (84).


   Acknowledgments
 
We thank Liz Robertson, Ross Waldrip, and members of the lab for helpful discussions; Cyprian Gardine III for preliminary analysis of lymph node subpopulations in Ii chain-deficient mice; Horst Bluethmann for generously providing C57BL/6 mice carrying the A{alpha} null allele; Sasha Rudensky for the 30-2 mAb; Jennifer Lower for the DM{alpha} genotyping protocol; Jennifer Lower and Debbie Pelusi for valuable assistance screening mutant progeny; Patti Lewko and Mark O’Donnell for careful maintenance of the mouse colony; Pippa Marrack and Sasha Rudensky for T cell hybridomas; Carol Plunkett for secretarial assistance; 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.). Back

2 Present address: Purdue University, West Lafayette, IN 47907 Back

3 Assistant Investigator with the Howard Hughes Medical Institute. Back

4 Address correspondence and reprint requests to Dr. Elizabeth K. Bikoff, Department of Molecular and Cellular Biology, The Biological Laboratories, Harvard University, 16 Divinity Ave., Cambridge, MA 02138. Back

5 Abbreviations used in this paper: Ii, invariant; ER, endoplasmic reticulum; CLIP, class II-associated Ii chain peptide; PE, phycoerythrin. Back

Received for publication July 16, 1997. Accepted for publication September 25, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wolf, P. R., H. L. Ploegh. 1995. How MHC class II molecules acquire peptide cargo: biosynthesis and trafficking through the endocytic pathway. Ann. Rev. Cell Dev. Biol. 11:267.[Medline]
  2. Cresswell, P.. 1996. Invariant chain structure and MHC class II function. Cell 84:505.[Medline]
  3. Bikoff, E. K., R. N. Germain, E. J. Robertson. 1995. Allelic differences affecting invariant chain dependency of MHC class II subunit assembly. Immunity 2:301.[Medline]
  4. Schaiff, W. T., Jr K. A. Hruska, D. W. McCourt, M. Green, B. D. Schwartz. 1992. HLA-DR associates with specific stress proteins and is retained in the endoplasmic reticulum in invariant chain negative cells. J. Exp. Med. 176:657.[Abstract/Free Full Text]
  5. Anderson, K. S., P. Cresswell. 1994. A role for calnexin IP90 in the assembly of class II MHC molecules. EMBO J. 13:675.[Medline]
  6. Bonnerot, C., M. S. Marks, P. Cosson, E. J. Robertson, E. K. Bikoff, R. N. Germain, J. S. Bonifacino. 1994. Association with BiP and aggregation of class II MHC molecules synthesized in the absence of invariant chain. EMBO J. 13:934.[Medline]
  7. Nijenhuis, M., J. Neefjes. 1994. Early events in the assembly of major histocompatibility complex class II heterotrimers from their free subunits. Eur. J. Immunol. 24:247.[Medline]
  8. Schreiber, K. L., M. P. Bell, C. J. Huntoon, S. Rajagopaian, M. B. Brenner, D. J. McKean. 1994. Class II histocompatibility molecules associate with calnexin during assembly in the endoplasmic reticulum. Int. Immunol. 6:101.[Abstract/Free Full Text]
  9. Layet, C., R. N. Germain. 1991. Invariant chain promotes egress of poorly expressed, haplotype-mismatched class II major histocompatibility complex A{alpha}Aß dimers from the endoplasmic reticulum/cis-Golgi compartment. Proc. Natl. Acad. Sci. USA 88:2346.[Abstract/Free Full Text]
  10. Anderson, M. S., J. Miller. 1992. Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules. Proc. Natl. Acad. Sci. USA 89:2282.[Abstract/Free Full Text]
  11. Baake, O., B. Dobberstein. 1990. MHC class II-associated invariant chain contains a sorting signal for endosomal compartments. Cell 63:707.[Medline]
  12. Lotteau, V., L. Teyton, T. Peleraux, L. Nilsson, L. Karlsson, S. L. Schmid, V. Quaranta, P. A. Peterson. 1990. Intracellular transport of class II MHC molecules directed by invariant chain. Nature 348:600.[Medline]
  13. Roche, P. A., P. Cresswell. 1991. Proteolysis of the class II-associated invariant chain generates a peptide binding site in intracellular HLA-DR molecules. Proc. Natl. Acad. Sci. USA 88:3150.[Abstract/Free Full Text]
  14. Roche, P. A.. 1995. HLA-DM: an in vivo facilitator of MHC class II peptide loading. Immunity 3:259.[Medline]
  15. Busch, R., E. D. Mellins. 1996. Developing and shedding inhibitions: how MHC class II molecules reach maturity. Curr. Opin. Immunol. 8:51.[Medline]
  16. Denzin, L. K., C. Hammond, P. Cresswell. 1996. HLA-DM interactions with intermediates in HLA-DR maturation and a role for HLA-DM in stabilizing empty HLA-DR molecules. J. Exp. Med. 184:2153.[Abstract/Free Full Text]
  17. Sanderson, F., C. Thomas, J. Neefjes, J. Trowsdale. 1996. Association between HLA-DM and HLA-DR in vivo. Immunity 4:87.[Medline]
  18. Kropshofer, H., S. O. Arndt, G. Moldenhauer, G. J. Hammerling, A. B. Vogt. 1997. HLA-DM acts as a molecular chaperone and rescues empty HLA-DR molecules at lysosomal pH. Immunity 6:293.[Medline]
  19. Kropshofer, H., A. B. Vogt, G. Moldenhauer, J. Hammer, J. S. Blum, G. J. Hammerling. 1996. Editing of the HLA-DR-peptide repertoire by HLA-DM. EMBO J. 15:6144.[Medline]
  20. van Ham, S. M., U. Gruneberg, G. Malcherek, I. Broker, A. Melms, J. Trowsdale. 1996. Human histocompatibility leukocyte antigen HLA-DM edits peptides presented by HLA-DR according to their ligand binding motifs. J. Exp. Med. 184:2019.[Abstract/Free Full Text]
  21. Lindstedt, R., M. Liljedahl, A. Peleraux, P. A. Peterson, L. Karlsson. 1995. The MHC class II molecule H2-M is targeted to an endosomal compartment by a tyrosine-based targeting motif. Immunity 3:561.[Medline]
  22. Marks, M. S., P. A. Roche, E. van Donselaar, L. Woodruff, P. J. Peters, J. S. Bonifacino. 1995. A lysosomal targeting signal in the cytoplasmic tail of the ß chain directs HLA-DM to MHC class II compartments. J. Cell Biol. 131:351.[Abstract/Free Full Text]
  23. Bikoff, E. K., L.-Y. Huang, V. Episkopou, J. van Meerwijk, R. N. Germain, E. J. Robertson. 1993. Defective major histocompatibility complex class II assembly, transport, peptide acquisition, and CD4+ T cell selection in mice lacking invariant chain expression. J. Exp. Med. 177:1699.[Abstract/Free Full Text]
  24. Viville, S., J. Neefjes, V. Lotteau, A. Dierich, M. Lemeur, H. Ploegh, C. Benoist, D. Mathis. 1993. Mice lacking the MHC class II-associated invariant chain. Cell 72:635.[Medline]
  25. Elliott, E. A., J. R. Drake, S. Amigorena, J. Elsemore, P. Webster, I. Mellman, R. A. Flavell. 1994. The invariant chain is required for intracellular transport and function of major histocompatibility complex class II molecules. J. Exp. Med. 179:681.[Abstract/Free Full Text]
  26. Martin, W. D., G. G. Hicks, S. K. Mendiratta, H. I. Leva, H. E. Ruley, L. Van Kaer. 1996. H2-M mutant mice are defective in the peptide loading of class II molecules antigen presentation, and T cell repertoire selection. Cell 84:543.[Medline]
  27. Miyazaki, T., P. Wolf, S. Tourne, C. Waltziner, A. Dierich, N. Barois, H. Ploegh, C. Benoist, D. Mathis. 1996. Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway. Cell 84:531.[Medline]
  28. Fung-Leung, W. P., C. D. Surh, M. Liljedahl, J. Pang, D. Leturcq, P. A. Peterson, S. R. Webb, L. Karlsson. 1996. Antigen presentation and T cell development in H2-M-deficient mice. Science 271:1278.[Abstract]
  29. Van Kaer, L., P. G. Ashton-Rickardt, H. L. Ploegh, S. Tonegawa. 1992. TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4-8+ T cells. Cell 71:1205.[Medline]
  30. Kontgen, F., G. Suss, C. Stewart, M. Steinmetz, H. Bluethmann. 1993. Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int. Immunol. 5:957.[Abstract/Free Full Text]
  31. Jr Janeway, C. A., P. J. Conrad, E. A. Lerner, J. Babich, P. Wettstein, D. B. Murphy. 1984. Monoclonal antibodies specific for Ia glycoproteins raised by immunization with activated T cells: possible role of T cellbound Ia antigens as targets of immunoregulatory T cells. J. Immunol. 132:662.[Abstract]
  32. Murphy, D. B., D. Lo, S. Rath, R. L. Brinster, R. A. Flavell, A. Slanetz, Jr C. A. Janeway. 1989. A novel MHC class II epitope expressed in thymic medulla but not cortex. Nature 338:765.[Medline]
  33. Eastman, S., M. Deftos, P. C. DeRoos, D.-H. Hsu, L. Teyton, N. S. Braunstein, C. J. Hackett, A. Rudensky. 1996. A study of complexes of class II invariant chain peptide: major histocompatibility complex class II molecules using a new complex-specific monoclonal antibody. Eur. J. Immunol. 26:385.[Medline]
  34. Symington, F. W., J. Sprent. 1981. A monoclonal antibody detecting an Ia specificity mapping in the I-A or I-E subregion. Immunogenetics 14:53.[Medline]
  35. Bhattacharya, A., M. E. Dorf, T. A. Springer. 1981. A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication. J. Immunol. 127:2488.[Abstract]
  36. Germain, R. N., L. R. Hendrix. 1991. MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding. Nature 353:134.[Medline]
  37. Hugo, P., J. W. Kappler, D. I. Godfrey, P. C. Marrack. 1992. A cell line that can induce thymocyte positive selection. Nature 360:679.[Medline]
  38. Rudensky, A. Y., P. Preston-Hurlburt, B. K. Al-Ramadi, J. Rothbard, Jr C. A. Janeway. 1992. Truncation variants of peptides isolated from MHC class II molecules suggest sequence motifs. Nature 359:429.[Medline]
  39. Shachar, I., R. A. Flavell. 1996. Requirement for invariant chain in B cell maturation and function. Science 274:106.[Abstract/Free Full Text]
  40. Rao, M., W. T. Lee, D. H. Conrad. 1987. Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE. J. Immunol. 138:1845.[Abstract]
  41. Waldschmidt, T. J., D. H. Conrad, R. G. Lynch. 1988. The expression of B cell surface receptors. I. The ontogeny and distribution of the murine B cell IgE Fc receptor. J. Immunol. 140:2148.[Abstract]
  42. Dornmair, K., H. M. McConnell. 1990. Refolding and reassembly of separate {alpha} and ß chains of class II molecules of the major histocompatibility complex leads to increased peptide-binding capacity. Proc. Natl. Acad. Sci. USA 87:4134.[Abstract/Free Full Text]
  43. Stern, L. J., D. C. Wiley. 1992. The human class II MHC protein HLA-DR1 assembles as empty {alpha}ß heterodimers in the absence of antigenic peptide. Cell 68:465.[Medline]
  44. Germain, R. N., Jr A. G. Rinker. 1993. Peptide binding inhibits protein aggregation of invariant-chain free class II dimers and promotes surface expression of occupied molecules. Nature 363:725.[Medline]
  45. Zhong, G., F. Castellino, P. Romagnoli, R. N. Germain. 1996. Evidence that binding site occupancy is necessary and sufficient for effective major histocompatibility complex MHC class II transport through the secretory pathway redefines the primary function of class II-associated invariant chain peptides CLIP. J. Exp. Med. 184:2061.[Abstract/Free Full Text]
  46. Androlewicz, M. J., P. Cresswell. 1996. How selective is the transporter associated with antigen processing?. Immunity 5:1.[Medline]
  47. Momburg, F., J. Roelse, G. J. Hammerling, J. J. Neefjes. 1994. Peptide size selection by the major histocompatibility complex-encoded peptide transporter. J. Exp. Med. 179:1613.[Abstract/Free Full Text]
  48. Tourne, S., H. M. van Santen, M. van Roon, A. Berns, C. Benoist, D. Mathis, H. Ploegh. 1996. Biosynthesis of major histocompatibility complex molecules and generation of T cells in Ii TAP1 double-mutant mice. Proc. Natl. Acad. Sci. USA 93:1464.[Abstract/Free Full Text]
  49. Henderson, R. A., H. Michel, K. Sakaguchi, J. Shabanowitz, E. Appella, D. F. Hunt, V. H. Engelhard. 1992. HLA-A2.1-associated peptides from a mutant cell line: a second pathway of antigen presentation. Science 255:1264.[Abstract/Free Full Text]
  50. Wei, M. L., P. Cresswell. 1992. HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides. Nature 356:443.[Medline]
  51. Busch, R., I. Y. Vturina, J. Drexler, F. Momburg, G. J. Hammerling. 1995. Poor loading of major histocompatibility complex class II molecules with endogenously synthesized short peptides in the absence of invariant chain. Eur. J. Immunol. 25:48.[Medline]
  52. Busch, R., I. Cloutier, R.-P. Sekaly, G. J. Hammerling. 1996. Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum. EMBO J. 15:418.[Medline]
  53. Zhong, G., P. Romagnoli, R. N. Germain. 1997. Related leucine-based cytoplasmic targeting signals in invariant chain and major histocompatibility complex class II molecules control endocytic presentation of distinct determinants in a single protein. J. Exp. Med. 185:429.[Abstract/Free Full Text]
  54. Kearney, J. F., M. D. Cooper, J. Klein, E. R. Abney, R. M. E. Parkhouse, A. R. Lawton. 1977. Ontogeny of Ia and IgD on IgM-bearing B lymphocytes in mice. J. Exp. Med. 146:297.[Abstract/Free Full Text]
  55. Mond, J. J., E. Seghal, J. Kung, F. D. Finkelman. 1981. Increased expression of I-region-associated antigen Ia on B cells after cross-linking of surface immunoglobulin. J. Immunol. 127:881.[Abstract]
  56. Davidson, H. W., P. A. Reid, A. Lanzavecchia, C. Watts. 1991. Processed antigen binds to newly synthesized MHC class II molecules in antigen-specific B lymphocytes. Cell 67:105.[Medline]
  57. Bonnerot, C., D. Lankar, D. Hanau, D. Spehner, J. Davoust, J. Salamero, W. H. Fridman. 1995. Role of B cell receptor Ig{alpha} and Igß subunits in MHC class II-restricted antigen presentation. Immunity 3:335.[Medline]
  58. Hayakawa, K., D. Tarlinton, R. R. Hardy. 1994. Absence of MHC class II expression distinguishes fetal from adult B lymphopoiesis in mice. J. Immunol. 152:4801.[Abstract]
  59. Lam, K.-P., A. M. Stall. 1994. Major histocompatibility complex class II expression distinguishes two distinct B cell developmental pathways during ontogeny. J. Exp. Med. 180:507.[Abstract/Free Full Text]
  60. Cosgrove, D., D. Gray, A. Dierich, J. Kaufman, M. Lemeur, C. Benoist, D. Mathis. 1991. Mice lacking MHC class II molecules. Cell 66:1051.[Medline]
  61. Grusby, M. J., R. S. Johnson, V. E. Papaioannou, L. H. Glimcher. 1991. Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice. Science 253:1417.[Abstract/Free Full Text]
  62. Markowitz, J. S., P. R. Rogers, M. J. Grusby, D. C. Parker, L. H. Glimcher. 1993. B lymphocyte development and activation independent of MHC class II expression. J. Immunol. 150:1223.[Abstract]
  63. Nadimi, F., J. Moreno, F. Momburg, A. Heuser, S. Fuchs, L. Adorini, G. Hammerling. 1991. Antigen presentation of hen egg-white lysozyme but not of ribonuclease A is augmented by the major histocompatibility complex class II-associated invariant chain. Eur. J. Immunol. 21:1255.[Medline]
  64. Bikoff, E. K.. 1992. Formation of complexes between self peptides and MHC class II molecules in cells defective for presentation of exogenous protein antigens. J. Immunol. 149:1.[Abstract]
  65. Peterson, M., J. Miller. 1992. Antigen presentation enhanced by the alternatively spliced invariant chain gene product p41. Nature 357:596.[Medline]
  66. Bodmer, H., S. Viville, C. Benoist, D. Mathis. 1994. Diversity of endogenous epitopes bound to MHC class II molecules limited by invariant chain. Science 263:1284.[Abstract/Free Full Text]
  67. Pinet, V., M. S. Malnati, E. O. Long. 1994. Two processing pathways for the MHC class II-restricted presentation of exogenous influenza virus antigen. J. Immunol. 152:4852.[Abstract]
  68. Pinet, V., M. Vergelli, R. Martin, O. Bakke, E. O. Long. 1995. Antigen presentation mediated by recycling of surface HLA-DR molecules. Nature 375:603.[Medline]
  69. Lindner, R., E. R. Unanue. 1996. Distinct antigen MHC class II complexes generated by separate processing pathways. EMBO J. 15:6910.[Medline]
  70. Brooks, A. G., P. L. Campbell, P. Reynolds, A. M. Gautam, J. McCluskey. 1994. Antigen presentation and assembly by mouse I-Ak class II molecules in human APC containing deleted or mutated HLA DM genes. J. Immunol. 153:5382.[Abstract]
  71. Stebbins, C. C., Jr G. E. Loss, C. G. Elias, A. Chervonsky, A. J. Sant. 1995. The requirement for DM in class II-restricted antigen presentation and SDS-stable dimer formation is allele and species dependent. J. Exp. Med. 181:223.[Abstract/Free Full Text]
  72. Bijlmakers, M. J. E., P. Benaroch, H. L. Ploegh. 1994. Assembly of HLA DR1 molecules translated in vitro: binding of peptide in the endoplasmic reticulum precludes association with invariant chain. EMBO J. 13:2699.[Medline]
  73. Hedley, M. L., R. G. Urban, J. L. Strominger. 1994. Assembly and peptide binding of major histocompatibility complex class II heterodimers in an in vitro translation system. Proc. Natl. Acad. Sci. USA 91:10479.[Abstract/Free Full Text]
  74. Roche, P. A., P. Cresswell. 1990. Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding. Nature 345:615.[Medline]
  75. Teyton, L., D. O. O’Sullivan, P. W. Dickson, V. Lotteau, A. Sette, P. Fink, P. A. Peterson. 1990. Invariant chain distinguishes between the exogenous and endogenous antigen presentation pathways. Nature 348:39.[Medline]
  76. Roche, P. A., C. L. Teletski, D. R. Karp, V. Pinet, O. Bakke, E. O. Long. 1992. Stable surface expression of invariant chain prevents peptide presentation by HLA-DR. EMBO J. 11:2841.[Medline]
  77. Katz, J. F., C. Stebbins, E. Appella, A. J. Sant. 1996. Invariant chain and DM edit self-peptide presentation by major histocompatibility complex MHC class II molecules. J. Exp. Med. 184:1747.[Abstract/Free Full Text]
  78. Lightstone, L., R. Hargreaves, G. Bobek, M. Peterson, G. Aichinger, G. Lombardi, R. Lechler. 1997. In the absence of the invariant chain, HLA-DR molecules display a distinct array of peptides which is influenced by the presence or absence of HLA-DM. Proc. Natl. Acad. Sci. USA 94:5772.[Abstract/Free Full Text]
  79. Simonsen, A., F. Momburg, J. Drexler, G. Hammerling, O. Bakke. 1993. Intracellular distribution of the MHC class II molecules and the associated invariant chain Ii in different cell lines. Int. Immunol. 5:903.[Abstract/Free Full Text]
  80. Schneider, F.-J., B. Opel, W. Ballhausen, W. Henkes, P. Steinlein, K. Reske. 1987. Synthesis and expression of MHC class II molecules in the absence of attached invariant chains by recombinant interferon-{gamma}-activated bone marrow-derived macrophages. Eur. J. Immunol. 17:1235.[Medline]
  81. Momburg, F., P. Moller. 1988. Non-co-ordinate expression of HLA-DR antigens and invariant chain. Immunology 63:551.[Medline]
  82. Momburg, F., K. Koretz, A. Von Herbay, P. Moller. 1988. Nonimmune human cells can express MHC class II antigens in the absence of invariant chain: an immunohistological study on normal and chronically inflamed small intestine. Clin. Exp. Immunol. 72:367.[Medline]
  83. Vidal, K., C. Samarut, J.-P. Magaud, J.-P. Revillard, D. Kaiserlian. 1993. Unexpected lack of reactivity of allogeneic anti-Ia monoclonal antibodies with MHC class II molecules expressed by mouse intestinal epthelial cells. J. Immunol. 151:4642.[Abstract]
  84. Tourne, S., T. Miyazaki, P. Wolf, H. Ploegh, C. Benoist, D. Mathis. 1997. Functionality of major histocompatibility complex class II molecules in mice doubly deficient for invariant chain and H-zM complexes. Proc. Natl. Acad. Sci. USA 94:9255.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Int ImmunolHome page
Y. Kuwano, C. M. Prazma, N. Yazawa, R. Watanabe, N. Ishiura, A. Kumanogoh, H. Okochi, K. Tamaki, M. Fujimoto, and T. F. Tedder
CD83 influences cell-surface MHC class II expression on B cells and other antigen-presenting cells
Int. Immunol., August 1, 2007; 19(8): 977 - 992.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
J. Neumann, A. Konig, and N. Koch
Detection of aberrant association of DM with MHC class II subunits in the absence of invariant chain
Int. Immunol., January 1, 2007; 19(1): 31 - 39.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Ohmura-Hoshino, Y. Matsuki, M. Aoki, E. Goto, M. Mito, M. Uematsu, T. Kakiuchi, H. Hotta, and S. Ishido
Inhibition of MHC Class II Expression and Immune Responses by c-MIR
J. Immunol., July 1, 2006; 177(1): 341 - 354.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. K. Nanda and E. K. Bikoff
DM Peptide-Editing Function Leads to Immunodominance in CD4 T Cell Responses In Vivo
J. Immunol., November 15, 2005; 175(10): 6473 - 6480.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. H. Koonce and E. K. Bikoff
Dissecting MHC Class II Export, B Cell Maturation, and DM Stability Defects in Invariant Chain Mutant Mice
J. Immunol., September 1, 2004; 173(5): 3271 - 3280.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Muntasell, M. Carrascal, I. Alvarez, L. Serradell, P. van Veelen, F. A. W. Verreck, F. Koning, J. Abian, and D. Jaraquemada
Dissection of the HLA-DR4 Peptide Repertoire in Endocrine Epithelial Cells: Strong Influence of Invariant Chain and HLA-DM Expression on the Nature of Ligands
J. Immunol., July 15, 2004; 173(2): 1085 - 1093.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. Benlagha, S.-H. Park, R. Guinamard, C. Forestier, L. Karlsson, C.-H. Chang, and A. Bendelac
Mechanisms Governing B Cell Developmental Defects in Invariant Chain-Deficient Mice
J. Immunol., February 15, 2004; 172(4): 2076 - 2083.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. Rajagopalan, M. K. Smart, C. J. Krco, and C. S. David
Expression and Function of Transgenic HLA-DQ Molecules and Lymphocyte Development in Mice Lacking Invariant Chain
J. Immunol., August 15, 2002; 169(4): 1774 - 1783.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. M. Tompkins, J. Padilla, M. C. Dal Canto, J. P.-Y. Ting, L. Van Kaer, and S. D. Miller
De Novo Central Nervous System Processing of Myelin Antigen Is Required for the Initiation of Experimental Autoimmune Encephalomyelitis
J. Immunol., April 15, 2002; 168(8): 4173 - 4183.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
D. Matza, F. Lantner, Y. Bogoch, L. Flaishon, R. Hershkoviz, and I. Shachar
Invariant chain induces B cell maturation in a process that is independent of its chaperonic activity
PNAS, February 20, 2002; (2002) 52703299.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
P. Kraj, R. Pacholczyk, H. Ignatowicz, P. Kisielow, P. Jensen, and L. Ignatowicz
Positive Selection of CD4+ T Cells Is Induced In Vivo by Agonist and Inhibited by Antagonist Peptides
J. Exp. Med., August 13, 2001; 194(4): 407 - 416.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. K. Bikoff, G. Wutz, G. A. Kenty, C. H. Koonce, and E. J. Robertson
Relaxed DM Requirements During Class II Peptide Loading and CD4+ T Cell Maturation in BALB/c Mice
J. Immunol., April 15, 2001; 166(8): 5087 - 5098.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Kraj, R. Pacholczyk, and L. Ignatowicz
{{alpha}}{{beta}}TCRs Differ in the Degree of Their Specificity for the Positively Selecting MHC/Peptide Ligand
J. Immunol., February 15, 2001; 166(4): 2251 - 2259.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
L. Flaishon, R. Hershkoviz, F. Lantner, O. Lider, R. Alon, Y. Levo, R. A. Flavell, and I. Shachar
Autocrine Secretion of Interferon {gamma} Negatively Regulates Homing of Immature B Cells
J. Exp. Med., November 6, 2000; 192(9): 1381 - 1388.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. Kenty and E. K. Bikoff
BALB/c Invariant Chain Mutant Mice Display Relatively Efficient Maturation of CD4+ T Cells in the Periphery and Secondary Proliferative Responses Elicited upon Peptide Challenge
J. Immunol., July 1, 1999; 163(1): 232 - 241.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Matza, O. Wolstein, R. Dikstein, and I. Shachar
Invariant Chain Induces B Cell Maturation by Activating a TAFII105-NF-kappa B-dependent Transcription Program
J. Biol. Chem., July 13, 2001; 276(29): 27203 - 27206.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
D. Matza, F. Lantner, Y. Bogoch, L. Flaishon, R. Hershkoviz, and I. Shachar
Invariant chain induces B cell maturation in a process that is independent of its chaperonic activity
PNAS, March 5, 2002; 99(5): 3018 - 3023.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kenty, G.
Right arrow Articles by Bikoff, E. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kenty, G.
Right arrow Articles by Bikoff, E. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS