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International Immunology Advance Access originally published online on November 14, 2006
International Immunology 2007 19(1):31-39; doi:10.1093/intimm/dxl120
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Detection of aberrant association of DM with MHC class II subunits in the absence of invariant chain

Jürgen Neumann, Angelika König and Norbert Koch

Division of Immunobiology, Institute of Genetics, University of Bonn, Römerstrasse 164, 53117 Bonn, Germany

Correspondence to: N. Koch; E-mail: norbert.koch{at}uni-bonn.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Human HLA-DM or mouse H2-DM plays a vital role for presentation of antigenic sequences by MHC class II peptide receptors. These non-classical MHC class II molecules catalyze the release of the invariant chain (Ii) fragment CLIP from the class II cleft and facilitate acquisition of antigenic peptides by MHC class II peptide receptors. H2-DM- or Ii-deficient mice display an impaired ability of their antigen-presenting cell to present peptides to CD4+ T cells and a molecular link between the immunodeficiencies of these mouse strains may exist. We show that in transfected cells the presence of HLA-DM molecules in endocytic vesicles was strongly reduced when HLA-DM was accompanied by HLA-DR. Exclusion of HLA-DM from endocytic vesicles is explained by mixed association of HLA-DM with HLA-DR subunits and retention of the aggregates in the endoplasmic reticulum. Expression of Ii, however, impairs formation of mixed HLA-DR and HLA-DM complexes and directs matched pairing of HLA-DR and HLA-DM heterodimers. In Ii gene-deficient mice, aberrant association of H2A with H2-DM polypeptides was detected. Low expression of Ii in transgenic mice inhibits interaction of H2A with H2-DM subunits and facilitates formation of H2-DM{alpha}ß heterodimers. A role of Ii for assembly of H2-DM heterodimers partially explains the immunodeficient phenotype of Ii–/– mice.

Keywords: H2-DM, Ii-deficient mice


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
MHC class II heterodimers are peptide receptors, which present antigenic sequences on the cell surface of antigen-presenting cells (APCs) for recognition by CD4+ T cells. The non-polymorphic invariant chain (Ii) is a chaperone for assembly of MHC class II polypeptides in the endoplasmic reticulum (ER) (1). Enroute to late endosomes, Ii is sequentially degraded by acidic hydrolases and physically detached from the MHC class II molecules (2). A set of class II-associated Ii peptides (CLIP) remains bound to the HLA-DR (DR) cleft and prevents premature binding of antigenic peptides. In MIIC vesicles, which are specialized for peptide loading, HLA-DM (DM) dimers associate with the DR–CLIP complex and serve two functions: The catalytic activity of DM dislodges CLIP from the DR cleft and makes the heterodimer susceptible to capture an antigenic peptide (3). The second role of DM is to edit the repertoire of peptides yielding stable MHC class II–peptide complexes (4). APCs from H2-DM (mouse DM)-deficient mice accumulate H2A heterodimers (mouse MHC class II) associated with CLIP at the cell surface (5). APCs from H2-DM-deficient mice therefore are deficient in presenting processed antigenic peptides (6). In the absence of Ii, HLA-DM heterodimers are intracellularly transported and sorted to endosomes as demonstrated in HLA-DM transfected cell lines. Targeting to the endocytic pathway is mediated through a tyrosine-based sequence present on the cytoplasmic tail of the HLA-DMß chain (7). Ii is not essential for sorting of DM, but can rescue endosomal delivery of mutated H2-DM molecules (8). A role of Ii for DM assembly has not been reported.

Ii gene-deficient mice show reduced amounts of peptide-loaded H2A molecules on their cell surface, which impairs selection of CD4+ T cell in the thymus (9, 10). Since H2-DM facilitates acquisition of peptides to the MHC class II cleft, a dysfunction of H2-DM could contribute to the immunodeficient phenotype of Ii–/– mice.

We demonstrate here, that in the absence of Ii, HLA-DR and HLA-DM subunits form mixed complexes. Co-expression with Ii, however, promotes the formation of DR{alpha}ß and of DM{alpha}ß heterodimers and inhibits the formation of aberrant complexes. In Ii gene-deficient mice, H2-DM polypeptides associate to H2A chains and form mismatched complexes. The formation of mixed H2A–H2-DM complexes may explain the reduced half-life and the impaired function of H2-DM in APC from Ii-deficient mice.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice, antibodies and immunofluorescence
Ii–/– (9), Iilow (11) and C57BL/6 mice were from the animal breeding center at the Institute of Genetics, University of Bonn. Rabbit polyclonal antibody S35 against DRß chain and mouse mAbs anti-DR (I251), anti-DR{alpha} (1B5) and anti-DM{alpha} (5C1) have been described (12–15). HLA-DM{alpha} and HLA-DMß cDNAs were kindly provided by J. Trowsdale. HLA-DMß was mutated at amino acid residues 59–62 (NKMA) to GQIV generating an epitope for mAb 6D4 (16). mAb 6D4 was used for immunoprecipitation, western detection and immunofluorescence staining of transfected cells. mAb 2C3A to mouse H2-DM was kindly provided by L. Karlsson. The antiserum against DM{alpha} was generated by immunizing rabbits with an MBP–DM{alpha} fusion protein (16). Anti-actin mAb AC40 was purchased from Sigma (Taufkirchen, Germany).

Cathepsin B (CatB) was detected by immunofluorescence staining with a rabbit antiserum purchased from Calbiochem (Bad Soden, Germany) and DR with mAb I251. For immunoflourescence staining, cells were fixed in 4% PFA and permeabilized with 0.1% Triton X-100 for 5 min. Secondary antibodies were purchased from Molecular Probes (Leiden, The Netherlands). The transfected cell population was examined by western blotting for expression of the polypeptides at comparable levels. Fluorescent labeling revealed that >90% of transfected cells co-stained for the examined DR and DM subunits. The displayed stained cells are representative for the great majority of transfected cells.

Cells and transfection
COS-7 cells and the human lung fibroblast cell line IMRS were purchased from the American Type Culture Collection and the Coriell Institute (Camden, NJ, USA). Non-transfected COS-7 and IMRS cells were not reactive with antibodies, which were used in this study (data not shown). COS-7 cells were transfected with liposomal reagent DOSPER (Roche Diagnostics, Mannheim, Germany) and IMRS cells with JetPEI (Qbiogene, Heidelberg, Germany) according to instructions of the suppliers. Transfection of DR and DM cDNAs was optimized for expression of equal amounts of the polypeptides. Comparison of expression of DM and DR subunits was conducted with antibody-tagged polypeptides.

Immunoprecipitations, SDS-PAGE and immunoblotting
Cells were lysed in 0.5% NP40 (Sigma) in Tris-buffered saline pH 7.4. Lysates were pre-cleared by incubation with CL4B-Sepharose (Amersham, Freiburg, Germany). Immunocomplexes were isolated using protein A-Sepharose, separated by SDS-PAGE, and for western detection transferred to Immobilon P membrane (Millipore, Schwalbach, Germany). The membrane was blocked in PBS/Roti-Block (Roth, Karlsruhe, Germany), and probed with antibodies diluted in blocking buffer. Bands were visualized with HRP-conjugated rabbit anti-mouse (Sigma) or goat anti-rabbit (Sigma) IgG and enhanced chemiluminescent substrate (Amersham). For endoglycosidase treatment, cell lysates were digested with Endo H (2000 U) and PNgase F (1000 U) overnight at 37°C as recommended by the supplier (New England Biolabs, Frankfurt, Germany).

Immunoabsorption and micro-ultracentrifugation
Splenocytes from Ii–/– and from C57BL/6 mice were lysed at 4°C with 1% Triton X-100 in 10 mM Tris-buffered saline containing protease inhibitors at a cell concentration of 4 x 108 cells ml–1. DNA and debris were removed by centrifugation at 5000 x g for 30 min at 4°C. Ultracentrifugation was conducted with Beckman microfuge Optima TL. Cell lysates (40 µl) were centrifuged for 3 h at 10 000 and 30 000 x g or for 18 h at 100 000 x g. Thereafter, 30 µl of supernatant was mixed with 10 µl of SDS sample buffer and incubated for 5 min at 95°C. The pelleted material was mixed with 10 µl SDS sample buffer and denaturated at 95°C.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Aberrant association of HLA-DR with HLA-DM polypeptides
Combinations of DR{alpha} with DMß and of DM{alpha} with DRß were transiently expressed in COS-7 cells and examined by co-precipitation after NP40 lysis. COS-7 cells were negative for simian orthologs of DR, DM and Ii, which were examined with rabbit antisera against the respective polypeptides (data not shown). The DR and DM subunits were expressed in a ratio of ~1:1 (compare Methods). To detect inter-chain combinations of DR and DM molecules, we used antibodies that bind specifically to single DR{alpha} or DRß polypeptides. To identify the subunits, recombinant antibody tags were appended to DM and to DR chains. The antibody tags permit specific isolation and detection of free and of assembled DR and DM subunits. The ß polypeptide of the transfected subunits was immunoprecipitated and the co-precipitated {alpha} polypeptide was detected by immunoblotting (Fig. 1A). Immunoprecipitations of DMß (lane 1) or DRß (lane 4) followed by immunoblotting for DR{alpha} or DM{alpha} lead to identification of the co-precipitated heavy {alpha} chain. Expression of the polypeptides was verified by western blotting of cell lysates (lanes 2, 3, 5 and 6). (Two DM{alpha} bands, lanes 4 and 5, result from differential glycosylation (15)). The experiment in Fig. 1(A) demonstrates that DR{alpha} and DMß as well as DM{alpha} and DRß chains form mixed NP40-stable complexes. This result was confirmed by immunoprecipitation of the DR{alpha} polypeptide and detection of the DMß chain (Fig. 1B). To exclude that over-expression of DR and DM subunits in non-human COS-7 cells results in artificial aggregation, this experiment was repeated with the MHC class II and Ii-negative human lung fibroblast cell line IMRS. Again, DMß was co-isolated with DR{alpha} (Fig. 1C). Since NP40-stable, mixed complexes, such as DM{alpha}–DRß, were not detected in APC, we asked how dimer formation is controlled in the ER. To investigate binding competition between subunits, DM{alpha} and DMß were co-expressed with a single DR{alpha} or DRß chain (Fig. 1D). Immunoprecipitation of DR polypeptides was conducted against the DR{alpha} or DRß chain. Isolation of DR polypeptides and subsequent detection of DMß or DM{alpha} (lanes 1 and 5) by immunoblotting indicated their co-isolation. Expression of the individual DR and DM polypeptides was confirmed by immunoblotting of cell lysates (lanes 2–4 and 6–8). The result indicates that in the presence of single DR{alpha} or DRß chains, the DR polypeptides are engaged in NP40-stable complexes with DM chains. Co-isolation of DM and DR chains was also obtained when DR and DM heavy chains, in addition to both light chains, were co-expressed (Fig. 1E). Note that under physiological conditions dimers of DR{alpha}ß with DM{alpha}ß are unstable in NP40.


Figure 1
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  		Fig. 1 Formation of mixed HLA-DR and HLA-DM complexes. COS-7 cells were transiently transfected with DR- and DM-encoding cDNAs, lysed with 0.5% NP40 and subjected to immunoprecipitation (IP) with chain-specific antibodies 1B5 for DR{alpha} or S35 for DRß. Immunoblotting was conducted with 1B5 (DR{alpha}), 6D4 (DMß), 5C1 (DM{alpha}) and S35 (DRß). Igheavy (H) and light (L) chains are indicated on the left. (A) DR{alpha} and DMß or DRß and DM{alpha} were co-expressed. DMß (lane 1) and DRß (lane 4) were immunoprecipitated. Immunocomplexes and cell lysates (lanes 2, 3, 5 and 6) were separated on SDS-PAGE, and western blotted as indicated. (B) Co-isolation of DMß by immunoprecipitation of DR{alpha}. DR{alpha} was immunoprecipitated (IP) from NP40 cell lysates with mAb 1B5 against DR{alpha} (lane 1). Immunoprecipitates and cell lysate were separated by SDS-PAGE and immunoblotted against DMß (mAb 6D4, lanes 1 and 2) or DR{alpha} (lane 3). (C) The human lung fibroblast cell line IMRS was transfected with DR{alpha} and DMß. Cells were lysed and DR{alpha} was immunoprecipitated. Co-isolated DMß (lane 1) was detected by western blotting. Lanes 2 and 3 show detection of the expressed polypeptides in cell lysates. (D) DM{alpha}ß co-expressed with DR{alpha} (left) or DRß (right) was examined for co-isolation. DR{alpha} and DRß immunoprecipitates were separated in lanes 1 and 5 and lysates in lanes 2–4 and 6–8. MHC class II polypeptides were detected by western blotting as indicated. (E) Co-expression of DR{alpha}ß with DM{alpha}ß. COS-7 cells were transfected with DR{alpha}ß- and with DM{alpha}ß-encoding cDNAs. The cells were lysed in 0.5% NP40 and subjected to IP against DR with mAb I251 (lane 1). Immunocomplexes and cell lysates were separated by SDS-PAGE. The proteins were transferred to a polyvinylidene difluoride membrane and detected with antibodies against DMß (mAb 6D4, lanes 1 and 2), DR{alpha} (mAb 1B5, lane 3), DRß (serum S35, lane 4) and DM{alpha} (mAb 5C1, lane 5).

 
It was shown that HLA-DM{alpha} and HLA-DMß chains expressed in Hela cells assemble in a transport-competent heterodimer that was localized to a pre-lysosomal/lysosomal compartment (7). We monitored intracellular localization of DM and DR in transfected COS-7 cells by co-staining with the endosomal/lysosomal marker CatB (Fig. 2A). Merging of DM and CatB staining shows that in the absence of DR, DM is largely localized in CatB-containing compartments (panel I). However, co-expression with DR, strongly impaired co-staining of DM and CatB (panel III). Independent of the presence of DM, DR is preferentially localized to CatB-free vesicles (panels II and IV). Immunostaining of DM demonstrates that co-expression with DR chains yields strongly decreased expression of DM polypeptides in lysosomal vesicles containing CatB. This result suggests that in transfected COS-7 cells, the association of DR to DM chains impacts on the presence of DM in the endosomal/lysosomal compartment. Tansfection of DM{alpha}ß, DR{alpha}ß and Ii cDNAs into COS-7 cells shows that DR, DM and Ii are co-localized with CatB (Fig. 2A, panels V, VI and VII). This experiment indicates that co-expression with Ii leads to endosomal/lysosomal localization of DM and DR in COS-7 cells. To examine export of DM subunits from the ER, cell lysates from transfected cells were treated with Endo H or PNgase F and subsequently immunoblotted for DM{alpha} chain. Figure 2(B) shows cells transfected with DM{alpha}ß-encoding cDNAs. There the DM heavy chain acquires partial Endo H resistance (lane 2, upper band). Please notice, that DM{alpha} contains two N-linked carbohydrates, of which one acquires Endo H resistance. Only small amounts of DM{alpha} are completely Endo H sensitive (lane 2, lower band). For comparison, treatment with PNgase F cleaves all the N-linked carbohydrates from DM{alpha} (lane 3). Expression of the DM{alpha} and DMß polypeptides is shown in lanes 1 and 4. Since ER export and carbohydrate modification of DM{alpha} requires assembly to DMß, this experiment confirms that DM{alpha}ß dimers are transported in transfected cells. We examined whether DR expression inhibits carbohydrate maturation of DM{alpha} chains. Cell lysates were treated with Endo H or with PNgase F (Fig. 2B, lanes 6 and 7). Samples were SDS-PAGE separated and blotted for DM{alpha}. The result shows that a substantial amount of DM{alpha} is Endo H sensitive (lane 6, lower band) and migrates in the position of PNgase F-treated DM{alpha} (lane 7). Expression of the transfected cDNAs is monitored in lanes 5, 8, 9 and 10. The result suggests that aberrantly associated DR and DM subunits are retained in the ER. By western blotting, we found that DR chains co-isolated with both, DM{alpha} and DMß chains, suggesting that the subunits are contained in larger complexes (data not shown).


Figure 2
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  		Fig. 2 Co-staining of HLA-DM or HLA-DR with the lysosomal marker CatB. (A) COS-7 cells, transfected with DM{alpha}ß (panel I), DR{alpha}ß (panel II), or DM{alpha}ß- and DR{alpha}ß-encoding cDNAs (panels III and IV) were stained for DM (red), DR (red) or endogenous CatB (green) as indicated. Transfection of COS-7 cells with DM{alpha}ß, DR{alpha}ß and Ii and staining for DM, DR and Ii of DMab, DRab and Ii transfected COS-7cells is shown in panels V, VI and VII. Co-staining of CatB with DR or DM is shown in the lower part of the figure. Controls for expression of the transfected subunits were described in Methods. (B) Cell lysates from transfected cells were treated with Endo H (EH), PNgase F (PF) or left untreated (Ø) and immunoblotted for DM{alpha} (lanes 1–3 and 5–7). Expression of DMß DR{alpha} and DRß was examined in cell lysates (lanes 4, 8, 9 and 10). (C) COS-7 cells transfected with DR{alpha}ß- and DM{alpha}ß-encoding cDNAs were lysed and immunoabsorpted for DR. Pre (lanes 1)- and post (lanes 2)-absorption lysates were SDS-PAGE separated and western blotted for DR{alpha} (upper panel) or DM{alpha} (lower panel).

 
To demonstrate that DM is contained in complexes with DR, cell lysates were immunoabsorbed for DR and subsequently blotted for DR and DM (Fig. 2C). COS-7 cells were transfected with DR{alpha}ß- and DM{alpha}ß-encoding cDNAs. Cells were lysed and immunoprecipitated for DR. Cell lysates (pre) and immunoabsorbed lysates (post) were separated by SDS-PAGE and western blotted for DR{alpha} (upper panel) or for DM{alpha} (lower panel). Absorption of DR yields reduced DR{alpha} chain band (upper panel, lane 2), when compared with non-absorbed lysate (lane 1). When blotted for DM (lower panel), the DM{alpha} band was strongly reduced (compare lane 2 with lane 1), indicating that depletion of DR also reduces the detection of DM. This result confirms that in the absence of Ii, DM physically associates to DR chains.

Ii constrains matched pairing of HLA-DR and HLA-DM polypeptides
The formation of DR inter-isotype complexes with DM chains will compete with the association of intra-isotype combinations, such as DM{alpha}ß. In APC, the formation of functional DM{alpha}ß dimers could be strongly reduced by association of DM{alpha} or DMß polypeptides with the abundantly expressed DR chains. Possibly, a chaperone-mediated rescue of DM dimers operates in APC. We asked whether Ii might serve to inhibit formation of mixed DR–DM complexes.

To test chaperone function, Ii was expressed in addition to DR{alpha}ß chains (Fig. 3, lanes 2, 4, 5, 7 and 9) and in combination with DM{alpha} (upper panel) or DMß (lower panel). Results of immunoprecipitation of DR polypeptides and subsequent immunoblotting for associated DM chains are shown in lane 1 (without Ii) and lane 2 (with Ii). Lanes 3–9 indicate expression of the individual MHC class II polypeptides and of Ii. The presence of DM polypeptides in DR immunoprecipitates (lane 1) was almost completely abolished when Ii was co-expressed (lane 2). Moreover, in the absence or presence of Ii, the DR{alpha} chain alone was co-expressed with DMß chain (Fig. 4A). Again, in the presence of Ii, co-isolation of DMß with DR{alpha} was strongly reduced (compare lanes 1 and 2). The results in Figs 3 and 4(A) show that Ii impacts on assembly of DR{alpha}ß heterodimers and inhibits formation of mixed NP40-stable DR–DM complexes. It was still unclear how Ii controls DR{alpha}ß and DM{alpha}ß pairing. Possibly, Ii inhibits formation of mixed complexes by interaction to DR subunits. We examined whether Ii expression affects formation of DM{alpha}ß heterodimers in COS-7 cells transfected with cDNAs encoding {alpha} and ß chains from DM and DR{alpha} or DRß polypeptides (Fig. 4B). Initially, DM{alpha}ß co-expressed with DR{alpha} and Ii was studied. In the presence of Ii (Fig. 4B, upper panel, lane 2) the level of DR{alpha} co-precipitated with DM was greatly reduced compared with immunoprecipitates lacking Ii (lane 1), where DR{alpha} was detected as a strong band. Ii appears to inhibit the formation of mixed complexes of DR{alpha} with DM. Detection of the DR{alpha}, DM{alpha}, DMß and Ii bands from cell lysates (lanes 3–9) indicated the level of expression of the polypeptides in transfected cells. Subsequently, combinations of DM chains with DRß and Ii polypeptides were investigated (Fig. 4B, lower panel). DM was immunoprecipitated from transfected COS-7 cells. Despite the presence of Ii, DRß was co-isolated with DM molecules (lane 2). The level of DRß co-precipitated with DM was independent of Ii expression (lanes 1 and 2). The presence of the polypeptides was monitored by immunoblotting of cell lysates (lanes 3–9). We conclude that Ii exhibits a disparate impact on binding of single DR{alpha} or DRß chains to DM subunits.


Figure 3
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  		Fig. 3 Ii inhibits aberrant association of HLA-DR to HLA-DM subunits. DR{alpha}ß and DM{alpha} or DMß polypeptides were expressed in COS-7 cells in the presence or absence of Ii. NP40 cell lysates were immunoprecipitated (IP) against DR with I251 (lanes 1 and 2). Immunoprecipitates (lanes 1 and 2) and lysates (lanes 3–9) were separated by SDS-PAGE, followed by western blotting against DM{alpha} (upper panel) (5C1, lanes 1–4); against DMß (lower panel) (6D4, lanes 1–4); against Ii (Bu43, lane 5); against DR{alpha} (1B5, lanes 6 and 7) and against DRß (S35, lanes 8 and 9).

 

Figure 4
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  		Fig. 4 Ii controls matched pairing of MHC class II subunits. (A) COS-7 cells were transfected with DR{alpha}- and DMß-encoding cDNAs, with (+) or without (–) Ii expression. Cells were lysed in 0.5% NP40 and immunoprecipitated (IP) against DMß (mAb 6D4, lanes 1 and 2). Immunocomplexes and cell lysates (lanes 3–7) were separated by SDS-PAGE, followed by western blotting against DR{alpha} (mAb 1B5, lanes 1–4), DMß (mAb 6D4, lanes 5 and 6) and Ii (mAb Bu43, lane 7). (B) COS-7 cells were transfected with DM{alpha}ß and with DR{alpha} (upper panel) or with DRß (lower panel)-encoding cDNAs, with (+) or without (–) Ii expression. Transfected COS-7 cells were lysed in 0.5% NP40 and subjected to immunoprecipitation (IP) (lanes 1 and 2). Expression of the polypeptides and co-isolation were detected by immunoblotting (lanes 3–9). DM was immunoprecipitated with 6D4 (lanes 1 and 2) and co-isolated DR{alpha} (upper panel) or DRß (lower panel) was monitored by western blotting with 1B5 (DR{alpha}) and S35 (DRß). Cell lysates were immunoblotted with 5C1 (DM{alpha}), 6D4 (DMß), Bu43 (Ii), 1B5 (DR{alpha}) and S35 (DRß). (C and D) Cell lysates expressing DM{alpha}ß and DR{alpha} ± Ii or DM{alpha}ß and DRß ± Ii were digested with Endo H (EH) or PNgase F (PF) or left untreated (Ø) and blotted for DM{alpha}. Cell lysates were examined for expression of DMß (lanes 4 and 9), DR{alpha} (C, lanes 5 and 10), DRß (D, lanes 5 and 10) and Ii (C and D, lanes 11).

 
It remains to be shown whether Ii facilitates formation of DM heterodimers and their subsequent transport. To examine assembly of DM{alpha}ß heterodimers, we monitored maturation of N-linked glycans. Endo H resistance of asparagin-bound carbohydrates indicates assembly and intracellular transport of DM{alpha}ß heterodimers. Cell lysates were treated with Endo H or PNgase F and immunoblotted for DM{alpha}. Figure 4(C) shows that DR{alpha} impacts on maturation of DM{alpha} (lane 2). Almost half of the amount of DM{alpha} remains Endo H sensitive, when DR{alpha} was co-expressed with DM{alpha}ß. However, when Ii was additionally expressed (lane 7) nearly all DM{alpha} acquires resistance to Endo H digestion. PNgase F treatment indicates the position of the DM{alpha} polypeptide with cleaved N-linked glycans (lanes 3 and 8). The expression of the expressed polypeptides is shown in lanes 1, 4, 5, 6, 9, 10 and 11.

Co-expression of DM{alpha}ß with DRß and digestion with Endo H are shown in Fig. 4(D). In the presence of DRß, DM{alpha} remains largely Endo H sensitive (lane 2). A similar result was obtained when Ii was co-expressed (lane 7). In the presence of DRß the proportion of Endo H resistant DM{alpha} was not increased upon Ii expression (lanes 2 and 7). PNgase F digestion shows deglycosylated DM{alpha} chains (lanes 3 and 8). Expression of the polypeptides is demonstrated in lanes 1, 4, 5, 6, 9, 10 and 11. Figure 4(C) shows that in the presence of DR{alpha} and Ii, DM{alpha} acquires Endo H resistance, suggesting that DM{alpha}ß heterodimers are formed and exported from the ER to Golgi compartments, where the carbohydrates are modified. In contrast to DR{alpha}, interaction of DRß to DM was not inhibited by expression of Ii (Fig. 4B).

Aberrant association of H2-DM and H2A in Ii gene-deficient mice
Our data show that in the absence of Ii, mixed HLA-DR-DM complexes were identified in NP40 cell lysates. This result could suggest that in APC of Ii-null mice, assembly of H2-DM{alpha}ß dimers is impaired by binding of H2-DM chains to H2A polypeptides. To detect the interaction between H2A and H2-DM chains, spleen cells from Ii–/– mice and from wild-type mice were lysed with NP40 and immunoprecipitated against H2-DM. Immunoprecipitates were separated by SDS-PAGE and immunoblotted for the presence of H2Aß or H2A{alpha} chains. Figure 5(A and B), lanes 1, indicate co-isolation of H2Aß or H2A{alpha} in H2-DM immunoprecipitates from Ii–/– spleen cells. In contrast, spleen cells from wild-type mice exhibit no NP40-stable association between H2-DM and H2A (lanes 3). These results indicate that Ii–/– mice show aberrant association of H2-DM to H2A chains. The H2-DMß polypeptide that was detected in strongly reduced amounts in splenocytes from Ii–/– mice (17), therefore, might be largely contained in complexes with H2A subunits. Co-isolation of H2Aß and H2A{alpha} from splenocytes of Ii–/– mice by the conformation-sensitive mAb 2C3A against H2-Mß suggests that H2A and H2-M subunits are contained in larger complexes (Fig. 5A and B).


Figure 5
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  		Fig. 5 Aberrant association of H2-DM with H2A polypeptides in Ii gene-deficient mice. Spleen cells from Ii–/– mice (lane 1), from transgenic Iilow-expressing mice (Iilow, lane 2) and from wild-type mice (Iiwt, lane 3) were lysed in 0.5% NP40 followed by immunoprecipitation against H2-DM (2C3A). Immunocomplexes (lanes 1–3) and lysates (lanes–6) were separated by SDS-PAGE followed by western detection of the co-isolated H2Aß chain (A) or H2A{alpha} chain (B). Heavy (H) and light (L) chains of Ig are indicated on the left. (C) Lysates (40 µl) from splenocytes of Ii–/– and C57BL/6 mice were ultra-centrifugated for 3 h at 10 000, 30 000 x g or for 18 h at 100 000 x g. After centrifugation an upper fraction (U) of 30 µl was mixed with 10 µl SDS sample buffer. The lower fraction (L) containing the pelleted material was mixed with 10 µl SDS sample buffer. In lanes 1–4 and 7–10, 15 µl of the upper fraction and, in lanes 5, 6, 11 and 12, 10 µl of the lower fraction were separated by SDS-PAGE for western blotting. The blotted filters were developed with antisera against H2-DM{alpha} (upper panel) and H2A{alpha} (lower panel). Molecular weight markers are indicated on the right. (D) Cell lysates from 0.8, 1.6 or 2.4 million wild-type (Iiwt) or Ii–/– splenocytes were separated by SDS-PAGE and western blotted for H2-DM{alpha} (upper panel) or actin (lower panel). (E) Cell lysates from 0.8, 1.6 or 2.4 million wild-type (Iiwt) or Iilow splenocytes were separated by SDS-PAGE and western blotted for H2-DM{alpha} (upper panel) or actin (lower panel). (F) H2-DM was immunoprecipitated with mAb 2C3A from equivalent amounts of cell lysates from Ii–/–, Iilow and Iiwt splenocytes and immunoblotted with an antiserum against DM{alpha}. H and L indicate the position of the heavy and light chain of Ig used for immunoprecipitation.

 
We examined, whether in splenocytes of Ii–/– mice, H2-DM is contained in larger complexes. Splenocytes from Ii–/– and C57BL/6 mice were lysed and micro-centrifugated at 10 000, 30 000 or 100 000 x g. Subsequently, lysates were separated by SDS-PAGE and western blotted for H2-DM{alpha} or for H2A{alpha}. Figure 5(C) shows in lanes 1 and 7 lysates depleted of DNA and debris. Upon micro-ultracentrifugation an upper fraction of the lysates was separated in lanes 2–4 and lanes 8–10. The pelleted material was separated in lanes 3 and 4 and in lanes 11 and 12. In lane 4, it is demonstrated that H2-DM{alpha} and H2A{alpha} are depleted from the lysate of Ii–/– splenocytes by centrifugation at 100 000 x g. The amount of pelleted H2-DM{alpha} and H2A{alpha} increases from lane 5 to 6. In contrast to Ii–/– cells, splenocytes from C57BL/6 mice show no abundant reduction of H2-DM{alpha} and H2A{alpha} bands after centrifugation at 100 000 x g (lanes 7–10). The result in Fig. 5(C) suggests that in cells of Ii–/– mice H2-DM is contained in larger complexes with H2A. In addition, these data indicate strongly reduced expression of H2-DM in splenocytes from Ii–/– mice.

To monitor the level of H2-DM expression in Ii–/– and in wild-type splenocytes, cell lysates were SDS-PAGE separated for western blotting. Increasing cell equivalents were loaded onto SDS gels and immunoblotted with an antiserum against H2-DM{alpha} (Fig. 5D). The amount of the loaded cell lysates was examined by blotting for actin. Densitometric evaluation of the X-ray films revealed that Ii–/– splenocytes express steady-state levels of ~10% H2-DM{alpha} compared with wild-type splenocytes (data not shown). The reduced amount of H2-DM{alpha} is comparable to the reported decreased expression of H2-DMß in Ii-deficient mice (17).

Low amounts of Ii facilitate assembly of H2-DM heterodimers
We tested spleen cells from transgenic Ii–/+ mice [Iilowmice generated on an Ii gene-deficient background (11)] for NP40-stable H2-DM to H2A interaction. PBLs from these Iilow mice express ~17% Ii compared with PBLs from wild-type mice (11). H2-DM was immunoprecipitated from spleen cells of Iilow mice and its association with H2Aß or H2A{alpha} was monitored by immunoblotting (Fig. 5A and B, lanes 2). Weak bands corresponding to H2Aß or H2A{alpha} chains were detected in H2-DM immunoprecipitates from Iilow mice verifying that Ii controls the binding of H2A to H2-DM chains. Detection of H2Aß and H2A{alpha} in cell lysates indicated the increased mobility of H2A from Ii–/– mice compared with H2Aß and H2A{alpha} from wild-type mice, whereas H2A bands from Iilow mice exhibit an intermediate mobility consistent with a composition of mixed immature and mature H2A bands (Fig. 5A and B, lanes 4–6). Figure 5(E) demonstrates by western blotting of cell lysates that in Iilow splenocytes the level of H2-DM{alpha} gains ~30% of wild-type splenocytes, suggesting that Ii impacts on the steady-state level of H2-DM expression.

Western blotting of H2-DM immunoprecipitates in Fig. 5(F) shows that the amount of immunoprecipitated H2-DM{alpha} is strongly increased by low expression of Ii (lane 2) compared with Ii–/– splenocytes (lane 1), but highest in wild-type splenocytes (lane 3). Despite the strongly decreased level of H2-DM{alpha} in Ii–/– splenocytes, there is a large amount of H2A co-precipitated with H2-DM (compare Fig. 5A and B). Low amounts of Ii increase the level of H2-DM{alpha} in cell lysates and in immunopreciptates, but strongly reduce co-isolation of H2A with H2-DM. This result indicates that Ii inhibits aberrant association of H2A to H2-DM.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We demonstrate here that DM subunits form complexes with DR polypeptides. Association of DR to DM chains in cells lacking Ii leads to aberrant clustering of DM and DR chains. A role of Ii for assembly of DM heterodimers has not been described. It has been proposed that association of Ii prevents DM from exercising its function before arriving in the endocytic system (18). We show here that Ii inhibits aberrant association of DR{alpha} with DM subunits. Binding of single DRß chain to DM is not affected by expression of Ii. This result is consistent with detection of Ii associated to single DR{alpha}, but not to single DRß chain in NP40 cell lysates (19). We suggest that in APCs molar ratios of DR{alpha} Ii and DRß prevent quantitative association of DRß to DM subunits. DM polypeptides may benefit from successful assembly of DR polypeptides with Ii. In the absence of free DR chains DM{alpha} and DMß chains form heterodimers. Possibly, in APCs, Ii dissolves mixed DR–DM complexes and separates DM from DR heterodimers.

Marks et al. (20) showed that early after biosynthesis, MHC class II peptide receptor subunits pass through a transient phase of aggregation. Ii-free cells fail to dissociate these complexes. The MHC class II {alpha} and ß subunits associate with the ER chaperones calnexin and Bip (21). Release of DR{alpha} and DRß chains from the chaperones occurs in the presence of Ii (22).

Expression of DR and DM subunits in transfected cells is artificial and hardly displays the stoichiometry of the polypeptides in APCs. Therefore, the study of Ii gene-deficient mice is important to demonstrate the association of H2A with H2-DM subunits under physiological conditions.

Our data suggest that the formation of H2-DM heterodimers is diminished in Ii gene-deficient mice, caused by binding of H2-DM{alpha} and H2-DMß chains to an excess of H2A{alpha} and H2Aß chains. A molar excess at steady-state levels of 25-fold for DR to DM was reported (23). By comparison, the excess of H2A compared with the lower amount of H2-DM suggests that in Ii–/– mice H2-DM polypeptides are largely contained in complexes with H2A chains. Expression of Ii inhibits formation of mixed H2-DM–H2A complexes.

It is interesting to note that in dendritic cells (DCs) from Ii–/– mice at least 50% of H2-DM polypeptides are degraded in endosomal/lysosomal compartments (17). Inhibition of H2-DM degradation by a cysteine protease inhibitor suggests that in DC from Ii–/– mice H2-DM molecules traffic to endocytic vesicles, where proteolysis occurs. Possibly, in DC-mixed complexes of H2-DM and H2A subunits reach the proteolytic endosomal/lysosomal pathway, where they are rapidly degraded. Assembly of unusual, however, transport-competent complexes, such as HLA-DO and MHC class I heavy chains, have been detected on the surface of transfected cells (24). Hence, intracellular transport of mismatched H2A/H2-DM dimers might be possible. Many cell types can serve as APC. It is therefore possible that assembly and subsequent transport varies in APC types. As an example, MHC class II aggregates have been found in splenocytes, but not in thymic cells from Ii-mutant mice (25).

Ii gene-deficient mice show a phenotype that is similar to that of an H2-DM-deficient mouse line. Both mouse strains show reduced numbers of CD4+ T cells, caused by impaired antigen presentation, which controls selection of T cells in the thymus (26). Mice double deficient for Ii and H2-DM showed that the MHC class II phenotype of their APCs is similar, but not identical to cells from Ii–/– mice (27). In these mice, the number of CD4+ T cells is further reduced (28). MHC class II molecules on the cell membrane of APC from Ii gene-deficient mice exhibited a substantially higher ability to bind added synthetic peptide, suggesting that the MHC class II groove is empty or occupied by an easily displaced peptide (10, 29).

The CD4+ T cell-deficient phenotype of Ii–/– mice was ascribed to a role of Ii for assembly and intracellular transport of Ia molecules (29). Unexpectedly, expression of only small amounts of transgenic Ii in Ii gene-deficient mice reversed the phenotype (30). Low amounts of Ii were sufficient to retrieve positive selection of CD4+ T cells in the thymus (30). Consistent with a role of Ii for control of assembly of H2A and H2-DM polypeptides, small amounts of Ii protected against H2-DM degradation (17). Based on the increased stability of H2-DM in two Iilow isotype mouse strains compared with Ii-deficient mice, a role of Ii as a chaperone for H2-DM has been suggested (25). We found that in Iilow-transgenic mice the interaction of H2-DM with H2A polypeptides is decreased, because Ii restrains formation of aberrant H2A–H2-DM complexes. In contrast to Ii–/– mice, in APC from Iilow mice, formation of SDS stable, peptide-loaded H2A polypeptides were detected, which suggests a restored catalytic activity of H2-DM (31). H2-DM{alpha}ß heterodimers present in processing compartments from APC of Iilow mice therefore facilitate editing of high-affinity peptides for H2A binding (29). Presentation of high-affinity peptides by H2A heterodimers reconstitutes selection for CD4+ T cells in the thymus of the Iilow-expressing mice.

Ii and MHC class II are not strictly co-expressed (32, 33). In some cell types, down-regulation of Ii expression could affect the formation of DM by reducing pairing of DM{alpha}ß heterodimers. It is conceivable that aberrant aggregation of MHC class II and DM subunits occurs under reduced Ii expression in vivo. As an example, the HIV-2 protein Vpx, which interacts with Ii in infected cells, may enhance Ii degradation (34). Thus, HIV-2 infection could initiate aberrant assembly of MHC class II with DM subunits.

A physiological role of Ii could be to maintain the function of DM molecules by preventing aberrant association to MHC class II subunits.


   Acknowledgements
 
We gratefully acknowledge R. N. Germain, L. Karlsson, M. Maeurer, G. Moldenhauer and Trowsdale for providing with reagents and antibodies. The authors thank B. Dobberstein and A. McLellan for helpful comments. This work was supported by Grant Ko810/6-3 from the Deutsche Forschungsgemeinschaft.


   Abbreviations
 
APC, antigen-presenting cell
CatB, cathepsin B
CLIP, class II-associated Ii peptide
DC, dendritic cell
ER, endoplasmic reticulum
Ii, invariant chain

   Notes
 
Transmitting editor: E. Simpson

Received 24 January 2006, accepted 13 October 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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