H2-Mbeta 1 and H2-Mbeta 2 Heterodimers Equally Promote CLIP Removal in I-Aq Molecules from Autoimmune-prone DBA/1 Mice*

Wolfgang WalterDagger , Claudia Scheuer§, Michael LoosDagger , Torsten E. Reichert§, and Markus J. MaeurerDagger

From the Departments of Dagger  Medical Microbiology and § Oral and Maxillofacial Surgery, Johannes Gutenberg University, D-55101 Mainz, Germany

Received for publication, July 21, 2000, and in revised form, December 13, 2000



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Antigen-presenting cells degrade endocytosed antigens, e.g. collagen type II, into peptides that are bound and presented to arthritogenic CD4+ helper T cells by major histocompatibility complex (MHC) class II molecules. Efficient loading of many MHC class II alleles with peptides requires the assistance of H2-M (HLA-DM in humans), a heterodimeric MHC class II-like molecule that facilitates CLIP removal from MHC class II molecules and aids to shape the peptide repertoire presented by MHC class II to CD4+ T cells. In contrast to the HLA-DM region in humans, the beta -chain locus is duplicated in mice, with the H2-Mb1 beta-chain distal to H2-Mb2 and the H2-Ma alpha-chain gene. H2-M alleles appear to be associated with the development of autoimmune diseases. Recent data showed that Mbeta 1 and Mbeta 2 isoforms are differentially expressed in isolated macrophages and B cells, respectively. The tissue expression and functional role of these heterodimers in promoting CLIP removal and peptide selection have not been addressed. We utilized the human T2 cell line, which lacks part of chromosome 6 encompassing the MHC class II and DM genes, to construct transgenic cell lines expressing the MHC class II heterodimer I-Aq alone or in the presence of H2-Malpha beta 1 or H2-Malpha beta 2 heterodimers. Both H2-M isoforms facilitate the exchange of CLIP for cognate peptides on I-Aq molecules from arthritis-susceptible DBA/1 mice and induce a conformational change in I-Aq molecules. Moreover, I-Aq cell-surface expression is not absolutely dependent on H2-M molecules. These data suggest that I-Aq exhibits a high affinity for CLIP since virtually all I-Aq molecules on T2 cells were found to be associated with CLIP in the absence of both H2-M isoforms.



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Collagen type II (CII)1 induces a chronic polyarthritis syndrome in H2q mice that resembles some of the hallmarks of human rheumatoid arthritis. Both arthritogenic T and B cells are instrumental in initiating and perpetuating the debilitating disease. The dissociation of the capacity to induce a strong anti-CII-directed antibody response without developing arthritis or to induce an immune response ultimately leading to arthritis indicates that qualitative differences in humoral and/or cellular immune responses exist (1-8). Thus, efficient degradation, processing, and presentation of the autoantigen CII may play a role in whether an "arthritogenic" immune response ensues or not. In contrast, not only the antigen-presenting cell, but also the B and T cell repertoire influences the evolution of an autoimmune response. CD4+ T cells with an arthritogenic potential have been described and are able to initiate arthritis in DBA/1 mice (9). However, to activate, expand, and maintain these CII-specific T cells, the antigen CII has to be taken up, processed, and presented by appropriate antigen-presenting cells by I-Aq molecules.

Previous studies have indicated that substantial differences exist pertaining to the ability of different antigen-presenting cells to effectively present CII to T cell hybridomas. To stimulate CD4+ T cells, MHC class II molecules must be loaded with peptides provided by endogenous or exogenous proteins. This step is governed by the MHC class II-like H2-M molecules, which facilitate exchange of invariant chain (Ii)-derived MHC class II-associated Ii peptides (CLIP) from MHC class II for stably bound antigenic peptides (10, 11). Although dissociation of CLIP for antigenic peptides might spontaneously occur at endosome/lysosome-like pH (12), experiments using either H2-M-deficient mice or human B cell lines mutated in the HLA-DM (DM) loci demonstrated that H2-M/DM is required for that final step of peptide loading by many MHC class II alleles (13-16).

H2-M/DM appears to function as a peptide editor that serves to positively select peptides that can stably bind to MHC class II molecules (17, 18). Thus, expression of certain H2-M alleles associated with susceptibility to develop CII-induced arthritis may critically affect the peptide repertoire displayed to the T cell compartment (19).

In contrast to the DM loci in humans, the H2-M region in the mouse contains one H2-Ma (Ma) gene, but two H2-Mb (Mb) genes termed Mb1 and Mb2 (20). It is still unclear whether Mb1 and Mb2 are equally expressed in vivo (15, 21), and no systematic study has been presented in which a comparison of Malpha /Mbeta 1 and Malpha /Mbeta 2 heterodimers has been conducted. To address the questions of whether (i) I-Aq molecules require, for cell-surface expression, the presence of H2-M molecules and (ii) different H2-M isoforms are similar or distinct in peptide loading of MHC class II molecules, we took advantage of the T2 cell line, which lost, due a genetic defect, the MHC class II and DM molecules. Stable transfectants of the T2 cell line expressing the I-Aq allele from autoimmune-prone DBA/1 mice alone or combined with H2-Malpha beta 1 (Malpha beta 1) or H2-Malpha beta 2 (Malpha beta 2) provide the means to perform a detailed analysis of molecules involved in the MHC class II antigen presentation pathway without the possibility that freshly isolated antigen-presenting cells (e.g. macrophages) from autoimmune DBA/1 mice might be contaminated with trace amounts of different antigen-presenting cells (e.g. B cells).


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Animals-- DBA/1 mice (H2q) and New Zealand White rabbits were purchased from Charles River Laboratories (Sulzfeld, Germany).

Generation of T2 Transfectants and Culture Conditions-- The human T2 cell line (T cell × B cell hybrid, 721.174 × CEM.T2), a generous gift from Dr. R. Salter (University of Pittsburgh, Pittsburgh, PA), contains a homozygous deletion in chromosome 6p that removes the DM gene and the entire MHC class II region (23). T2 cells were maintained in RPMI 1640 medium (Life Technologies, Inc., Eggenstein, Germany) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamate, 100 IU/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies, Inc.) and 50 µM 2-mercaptoethanol (Sigma, Deisenhofen, Germany), referred to as complete medium.

Antibodies-- The hybridoma cell line N22 (anti-MHC class II) was obtained from American Type Culture Collection (Manassas, VA). Anti-I-Ab,d,q mAb YE2/36HLK was purchased from Serotec (Wiesbaden, Germany). Anti-MHC class II mAbs KH116 (anti-I-Aq) and KH118 (anti-I-Aq,b) were from Pharmingen (Hamburg, Germany). FITC-conjugated secondary staining reagents (goat anti-hamster IgG, rabbit anti-rat IgG, goat anti-mouse IgG, and goat anti-rabbit IgG) were purchased from Dianova (Hamburg), and unlabeled isotype-matched control antibodies were from Coulter-Immunotech (Hamburg). The rabbit antisera R.Malpha -C.69.3, R.Mbeta 1/2-C.71.3, and R.hCLIP73.11 were prepared by immunizing rabbits with C-terminal peptides from Malpha (amino acids 238-248) (24) and Mbeta 1/Mbeta 2 (amino acids 228-243) (25) and a CLIP peptide (amino acids 81-104 of the human p33 invariant chain isoform) (26), respectively, coupled with an added amino-terminal cysteine to diphtheria toxoid (Chiron Mimotopes, Victoria, Australia). The antisera were affinity-purified using N-hydroxysuccinimide-activated Fast Flow Sepharose 4 (Amersham Pharmacia Biotech, Freiburg, Germany) cross-linked to the respective Malpha , Mbeta , or CLIP peptide. Affinity-purified antisera were screened for specific antibody titers by enzyme-linked immunosorbent assay and Western blot analysis using T2.Aq cell lines transfected with Maq, Mb1q, and Mb2q. Specificity controls included Western blot analysis of T2 cells alone or T2 cells expressing I-Aq (but not H2-M genes) using anti-Malpha or anti-Mbeta antisera (see Fig. 4).

Construction of I-Aq, Maq, Mb1q, and Mb2q cDNA Expression Vectors-- I-Aaq (Aaq)and I-Abq (Abq) were amplified from a DBA/1 splenocyte cDNA library (19) by polymerase chain reaction using the ExpandTM Long Template PCR system (Roche Molecular Biochemicals, Mannheim, Germany) and standard PCR conditions. The primers used for amplification of the cDNA clones were as follows: Aaq-sense, 5'-ATACCATGGCGCGCAGCAGAGCTC-3'; Aaq-antisense, 5'-ATAGGATCCTCATAAAGGCCCTGG-3'; Abq-sense, 5'-ATACCATGGCTCTGCAGATCCC-3'; and Abq-antisense, 5'-ATAGGATCCTCACTGACGGAGCCCT-3'. The primers were designed with synthetic NcoI and BamHI restriction sites (underlined) to facilitate cloning of amplified DNA. Following amplification, the 809-base pair Aaq and 812-base pair Abq PCR products were cloned into the EcoRV and BamHI sites of pBSK+ (Stratagene, Heidelberg, Germany) and sequenced (19). For expression in eukaryotic cells, the Aaq cDNA was subcloned (NcoI-BamHI) into the retroviral vector MFG (27), yielding MFG-Aaq. Subsequently, an IRES-Neo cassette (28), consisting of an internal ribosomal entry site (IRES) sequence from the encephalomyocarditis virus and the neomycin phosphotransferase gene (Neo), was inserted into the BamHI site at the 3'-end of the Aaq cDNA in MFG-Aaq, yielding the vector DFG-Aaq-IRES-Neo. Next, an IRES-EcoRI-NcoI fragment was fused to the ATG of the Abq cDNA in pBSK+, yielding the plasmid pBSK-IRES-Abq+. After that, pBSK-IRES-Abq+ was digested with BamHI, and the IRES-Abq fragment obtained was inserted into the BamHI site at the 3'-end of the Neo cDNA of the partially digested DFG-Aaq-IRES-Neo, yielding the vector TFG-Aaq-IRES-Neo-IRES-Abq. This proviral construct, capable of coordinately expressing Aaq, Abq, and Neo, was finally termed TFG-Aq. The isolation of Maq, Mb1q, and Mb2q full-length cDNA clones has been reported previously (19). For expression in eukaryotic cells, the Abq cDNA in pBSK-IRES-Abq+ was replaced by Maq (NcoI-NotI). The IRES-Maq sequence was subcloned into the BamHI site of the eukaryotic expression vector pCEP4 (Invitrogen, Groningen, The Netherlands). Subsequently, NotI-NheI-digested Mb1q or Mb2q cDNA was inserted into the NotI-NheI sites at the 5'-end of the IRES-Maq sequence, yielding pCEP4-Mb1q-IRES-Maq and pCEP4-Mb2q-IRES-Maq, respectively.

Generation of Stable Transfectant Cell Lines-- T2 cells stably expressing I-Aq molecules from arthritis-susceptible DBA/1 mice were generated by retroviral transfection as previously described (33). Briefly, retroviral supernatant was produced by transfecting the TFG-Aq proviral construct into the psi CRIP packaging cell line (29). T2 cells (2-5 × 106) were infected with 2 ml of TFG-Aq retroviral supernatant in the presence of Polybrene (8 µg/ml) and subsequently plated in 96-well flat-bottomed plates on irradiated feeder cell layers. Stable transfectants were selected in complete medium supplemented with 1 mg/ml Geneticin (Life Technologies, Inc.). T2.Aq clones were screened for high I-Aq expression levels by flow cytometry using the I-A conformation-independent mAb N22 (30). Transfection of T2.Aq cells with the pCEP4, pCEP4-Mb1q-IRES-Maq or pCEP4-Mb2q-IRES-Maq vector was performed by electroporation as described (26). To obtain stable T2.Aq.Maq.Mb1q and T2.Aq.Maq.Mb2q cell lines, transfectants were cloned by limiting dilution in complete medium supplemented with 1 mg/ml Geneticin and 0.45 mg/ml hygromycin (Roche Molecular Biochemicals). Individual clones were screened for the presence of Maq, Mb1q, and Mb2q by RT-PCR. Clones that exhibited high Malpha beta 1 or Malpha beta 2 protein expression levels determined by Western blot analysis were used in subsequent experiments.

Template cDNA Preparation and PCR-- Total RNA isolation and cDNA synthesis have been described previously (19). PCR amplification was performed in an amplification mixture adjusted to 50 µl containing 50-100 ng of cDNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01 (w/v) gelatin, 1 mM each dNTP, 25 pmol each primer, and 2.5 units of AmpliTaq Gold polymerase (PerkinElmer Life Sciences, Weiterstadt, Germany). The RT-PCR amplification profile involved an initial denaturation step (94 °C for 10 min), followed by 35 cycles of 94 °C for 1 min, 62 °C for 1 min, and 72 °C for 1 min; the last extension was for 10 min at 72 °C. The following primers were used for PCR: Ma-sense, 5'-AAGGTATGGAGCATGAGCAGAAGT-3'; Ma-antisense, 5'-GATCAGTCACCTGAGCACGGT-3'; panMb1/2-sense, 5'-GGACCATGGCTGCACTCTGGC-3'; and panMb1/2-antisense, 5'-GCATCACGGGCTCCCTTGTGT-3'. PCR products were resolved on ethidium bromide-stained agarose gels and digitized with a Gelprint 2000i densitometer (MWG Biotech, Ebersberg, Germany).

Ratio RT-PCR Analysis-- The ratio RT-PCR assay performed in this study was based on the simultaneous amplification of Mb1 and Mb2 transcripts using panMb1/2 primers (see above) annealing within conserved regions (exons 1 and 3) of both Mbq mRNA species (19). Equal amplification efficiency of both Mb transcripts was assured by comparative cycle kinetic and linear regression analysis (31) using cloned Mb1 and Mb2 full-length cDNAs (19). Discrimination between co-amplified Mb transcripts was performed by restriction endonuclease analysis. We therefore took advantage of the polymorphism of Mb1q and Mb2q genes within exon 2 (19) and used the restriction enzyme HhaI, which specifically cleaves at nucleotide +321 within the Mb1q sequence and nucleotide +264 within the Mb2q sequence of the 403-base pair panMb RT-PCR product. PCR was performed using AmpliTaq Gold polymerase and standard PCR conditions. After 25 cycles, the amplified mixture was diluted 25-fold in a fresh PCR amplification mixture containing 25 nCi/µl [alpha -32P]dCTP (ICN, Eschwege, Germany), followed by two additional amplification cycles. The labeled Mb PCR products or their respective restriction fragments were separated on 6% polyacrylamide gels. To quantitate individual Mb fragments, gels were subjected to autoradiography. Corresponding bands were excised from the gel, and radioactivity was measured with a beta -counter (LS6000TA, Beckman, München, Germany) using a Cerenkov program. To calculate the ratio of Mb1 and Mb2 mRNA expression levels, their respective restriction fragments were corrected for length and cytosine and guanine (GC content) since dCTP was exclusively radioactively labeled in this assay.

Western Blot Analysis-- Cells (1 × 107/ml) were lysed in 20 mM Tris-HCl (pH 7.4) containing 1% Nonidet P-40 (Sigma), 5 mM MgCl2, 5 µg/ml chymostatin, 2.5 µg/ml leupeptin, 5 µg/ml pepstatin A, and 200 µM phenylmethylsulfonyl fluoride (all protease inhibitors were from Roche Molecular Biochemicals) for 30 min at 4 °C. Nuclei and insoluble debris were removed by centrifugation (14,000 rpm) for 30 min, and the protein concentration was determined by the BCA protein assay (Pierce). Aliquots corresponding to 10 µg of protein were mixed with Laemmli buffer, boiled for 5 min, separated on SDS-12.5% polyacrylamide gels, and then transferred onto Immobilon polyvinylidene difluoride membranes (Millipore, Eschborn, Germany) by semidry blotting as described (32). Membranes were blocked overnight with blocking reagent (Roche Molecular Biochemicals). Antibody binding was detected by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Dianova), followed by enhanced chemiluminescence using Super Signal Ultra (Pierce).

Flow Cytometry-- Cells (5 × 105/sample) were washed in PBS supplemented with 1% bovine serum albumin and incubated on ice with unlabeled primary mAb for 30 min. After washing in the PBS and bovine serum albumin, the cells were incubated with an appropriate FITC-conjugated secondary staining reagent for 30 min at 4 °C: goat anti-hamster IgG, goat anti-mouse IgG, rabbit anti-rat IgG, or goat anti-rabbit IgG. Background fluorescence was evaluated using irrelevant matched isotypes and FITC-conjugated goat anti-hamster IgG, goat anti-mouse IgG, rabbit anti-rat IgG, or goat anti-rabbit IgG. Cell-surface fluorescent labeling was visualized on an EPICS®-PROFILE II flow cytometer (Coulter-Immunotech), and data analysis was performed using EPICS®-ELITE Version 3.0 software.

Immunohistochemistry-- Transfected T2 cell lines were washed in PBS and cytocentrifuged with a Labofuge 400e (Heraeus Instruments, Hanau, Germany) at 500 × g for 5 min onto Superfrost slides (Menzel, Hannover, Germany) with 200,000 cells/dot.

The cytospins were air-dried overnight and afterward stored at -20 °C. Cells were fixed with 4% (w/v) paraformaldehyde (Sigma, München) for 10 min at room temperature before immunostaining. After permeabilization with 0.2% (v/v) Triton X-100 (Sigma) in PBS and blocking with serum-free protein block (Dako, Hamburg), cells were incubated with the affinity-purified rabbit anti-human primary polyclonal antibody anti-DMalpha or anti-DMbeta or the corresponding antisera (1:1000 dilution for each one). Negative controls included cells treated with PBS or normal rabbit serum. A Cy3-labeled anti-rabbit antibody (diluted 1:600; Dianova, Hamburg, Germany) was used as the second antibody. Optimal working dilutions of the antibodies were determined in titration experiments. Nuclei were counterstained with bisbenzimide (1:5000; Sigma). Slides were mounted in fluorescent mounting medium (Dako) and examined using a Leitz DM RBE fluorescence microscope (Leica, Heerbrugg, Switzerland); the red Cy3 fluorescence was detected by an N2.1 filter (wavelengths 515-560 and 590).


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Mb1 and Mb2 Are Differentially Expressed in Lymphoid and Non-lymphoid Organs-- The H2-M region contains one Ma gene, but two Mb genes termed Mb1 and Mb2 (20). Transcription of the Ma gene and both Mb genes has been observed in splenocytes of different mouse strains, including mice that carry the arthritis-susceptible H2q haplotype (19, 33). To define the H2-M gene expression pattern, we examined Ma and Mb mRNA expression in lymphoid and non-lymphoid organs or tissues from arthritis-susceptible DBA/1 (H2q) mice by RT-PCR. Constitutive Ma and Mb mRNA expression could be detected in each organ sample (data not shown). Next, we addressed the question of whether Mb1 and Mb2 are differentially expressed by ratio RT-PCR analysis (Fig. 1). The ratios of Mb1 to Mb2 mRNA given as percentages of the total Mb mRNA are listed in Table I. Mb2 represents the major Mb transcript in lymphoid organs: 60.4% in spleen; 79.5% in mesenteric lymph nodes; 82.3% in popliteal lymph nodes; and 60.2, 62.4, and 57.8% in thymus of 4-, 8-, and 12-week-old mice, respectively (Fig. 1A and Table I). Similarly, Mb2 mRNA was preferentially expressed in muscle (62.1%) and heart (57.7%). In contrast, Mb1 mRNA was found to be the dominant transcript in testis, brain, lung, liver, kidney, pancreas, and small and large intestines. With the exception of large intestine (57.1%), the relative expression of Mb1 mRNA averaged ~76% of the total Mb mRNA transcripts (Fig. 1B and Table I).



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Fig. 1.   Determination of Mb1q and Mb2q expression patterns in lymphoid and non-lymphoid organs and tissues of arthritis-susceptible DBA/1 mice (H2q). PCRs were performed with 100 ng of cDNA from the indicated lymphoid (A) and non-lymphoid (B) organs and tissues in the presence of 25 nCi/µl [alpha -32P]dCTP as described under "Experimental Procedures." Primers were chosen to co-amplify Mb1- and Mb1-specific transcripts. Co-amplified Mb isoforms were discriminated by digestion with the restriction enzyme HhaI, followed by separation on 6% polyacrylamide gels. Lanes NC show the undigested panMb1/2 PCR product; lanes H show the Mb1- and Mb2-specific fragments obtained after HhaI digestion. The lengths (in base pairs (bp)) of the undigested panMb1/2 PCR products and the restriction fragments corresponding to Mb1 or Mb2 are indicated to the right of A and B.


                              
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Table I
Mb1 and Mb2 are differentially expressed in lymphoid and non-lymphoid organs of arthritis-susceptible DBA/1 (H2q) mice
Values, presented as means ± S.D. of three determinations, indicate the relative expression of Mb1 and Mb2 mRNAs as percentages of total Mb mRNA as determined by ratio RT-PCR as described in the legend to Fig. 1.

Both Malpha beta 1 and Malpha beta 2 Can Complement for Conformation-dependent Epitopes on MHC Class II Molecules in T2 Cells-- The observation that Mb1 and Mb2 are differentially expressed in lymphoid and non-lymphoid organs (Table I) implies that both heterodimers, Malpha beta 1 and Malpha beta 2, might be functional in I-Aq/peptide assembly. To address this question, we generated T2 transfectants stably expressing either I-Aq alone (T2.Aq.pCEP4) or in combination with Malpha beta 1 (T2.Aq.Maq.Mb1q) or Malpha beta 2 (T2.Aq.Maq.Mb2q) derived from DBA/1 mice (H2q). To ensure comparable levels of I-Aq surface expression as well as Malpha beta 1 or Malpha beta 2 heterodimer expression, transgenic T2 cells were analyzed by flow cytometry (Fig. 2) and Western blotting (Fig. 3), respectively. Cell-surface staining with mAb N22, which recognizes a monomorphic determinant on Ii- or peptide-associated MHC class II alpha beta -dimers with similar efficiency (30), showed that T2.Aq.pCEP4, T2.Aq.Maq.Mb1q, and T2.Aq.Maq.Mb2q transfectants expressed comparable I-Aq levels on the cell surface (Fig. 3, left panel). Similarly, Western blot analysis of T2.Aq.Maq.Mb1q and T2.Aq.Maq.Mb2q cells demonstrated comparable Malpha beta 1 and Malpha beta 2 expression (Fig. 3).



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Fig. 2.   Expression of Malpha beta 1 or Malpha beta 2 in T2.Aq restores the expression of the KH116 and YE2/36HLK epitopes. MHC class II and DM mutant T2 cells expressing I-Aq were generated by retroviral transfection as described under "Experimental Procedures." To express Malpha beta 1 or Malpha beta 2 heterodimers, T2.Aq cells were super-transfected with Maq in combination with Mb1q or Mb2q. Transfection with the pCEP4 vector alone served as a control. I-Aq surface expression was measured by flow cytometry using MHC class II conformation-independent mAbs N22 (43) and KH118 (22) and MHC class II conformation-sensitive mAbs KH116 (22) and YE2/36HLK (48). Data are plotted as fluorescence intensity (mean fluorescence channel) versus cell number. Closed histograms show binding of specific antibodies, whereas open histograms show isotype-matched control antibodies. Antibody names are listed across the top, and cell lines are indicated to the right.



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Fig. 3.   Comparison of Malpha beta 1 and Malpha beta 2 expression in T2.Aq transfectants. MHC class II antigen-processing mutant T2 cells were stably transfected with I-Aq alpha - and beta -chain genes and then super-transfected with Ma and Mb1 (T2.Aq.Maq.Mb1q), Mb2 (T2.Aq.Maq.Mb1q), or pCEP4 control vector (T2.Aq.pCEP4). Laemmli buffer-solubilized cell lysates (10 µg) derived from the indicated cell lines were separated on denaturing 12.5% SDS-polyacrylamide gels and analyzed on Western immunoblots by staining with affinity-purified rabbit antisera to Malpha , or Mbeta 1, and Mbeta 2 monomers as described under "Experimental Procedures."

In view of the well documented observation that absent or reduced binding of certain mAbs recognizing conformation-dependent epitopes on MHC class II molecules directly correlates with a failure to exchange CLIP for other peptides on MHC class II molecules in DM mutant cell lines (13, 26) or H2-M-deficient mice (14), we analyzed the I-Aq surface phenotype of the T2.Aq.pCEP4, T2.Aq.Maq.Mb1q, and T2.Aq.Maq.Mb2q cell lines by flow cytometry using a panel of mAbs to monomorphic (Fig. 2 first and second lane from left) and polymorphic determinants (Fig. 2, third and fourth lane from left). Staining with mAb KH118, which also recognizes a monomorphic determinant on I-Aq molecules (34), confirmed the results obtained with mAb N22, demonstrating that individual transfectants express I-Aq molecules at equal levels on the cell surface. In contrast, differential staining was obtained with mAbs KH116 and YE2/36HLK, both recognizing polymorphic determinants on I-Aq (34, 35). Both mAbs equally stained T2.Aq.Maq.Mb1q and T2.Aq.Maq.Mb2q cells, but stained T2.Aq.pCEP4 cells with a reduced intensity. These findings suggest that the I-Aq conformation on T2.Aq cells expressing either Malpha beta 1 or Malpha beta 2 differs from that on the H2-M-negative T2.Aq.pCEP4 cells. However, expression of the KH116 and YE2/36HLK mAb epitopes in T2.Aq cells requires the expression of both Maq and either Mb1q or Mb2q since transfectants expressing each gene individually remained KH116- and YE2/36HLK-negative.2

To evaluate whether the expression of the KH116 and YE2/36HLK epitopes in T2.Aq.Maq.Mb1q or T2.Aq.Maq.Mb2q cells might result from CLIP exchange for cognate peptides on I-Aq molecules, cell-surface levels of CLIP were determined by flow cytometry using the antibody R.hCLIP73.11, which recognizes a peptide derived from the CLIP region (amino acids 81-104 of the human p33 Ii isoform). As expected, T2.Aq.pCEP4 cells exhibited high levels of anti-CLIP staining compared with T2.Aq.Maq.Mb1q and T2.Aq.Maq.Mb2q cells (Fig. 4). These data show that (i) I-Aq molecules are occupied with CLIP in the absence of Malpha beta 1 or Malpha beta 2 heterodimers; (ii) I-Aq assembly, transport, and ultimately cell-surface expression do not require H2-M molecules; and (iii) either Malpha beta 1 or Malpha beta 2 heterodimers are sufficient to exchange CLIP for cognate peptides displayed by the I-Aq molecules.



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Fig. 4.   CLIP persistence in the absence of Malpha beta 1 or Malpha beta 2. T2.Aq cells transfected with the pCEP4 control vector alone and with Maq and either Mb1q or Mb2q were stained with R.hCLIP73.11 (anti-CLIP-(81-104)) and analyzed by flow cytometry. Cell line designations are indicated across the top.

Cell-surface Expression of H2-M-- T2, T2.Aq.pCEP4, T2.Aq.Maq.Mb1q, and T2.Aq.Maq.Mb2q cells were examined for Malpha and Mbeta protein expression (Fig. 5). Both transgenic T2 cell lines expressing Malpha q/Mbeta 1q or Malpha q/Mbeta 2q heterodimers exhibited a strong cytoplasmic and cellular membrane staining pattern.



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Fig. 5.   Cell-surface expression of H2-M in transgenic cell lines. T2 cells (a and e), T2.Aq cells (b and f), T2.Aq Malpha beta 1 transfectants (c and g), and T2.Aq Malpha beta 2 transfectants (d and h) were evaluated for expression of Malpha (a-d) and Mbeta (e-h). T2 cells and T2.Aq cells were negative for Malpha and Mbeta . In contrast, the transfectants showed strong staining of the cytoplasm and the cell membrane. Magnification × 1000.



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REFERENCES

H2-M molecules not only facilitate CLIP removal, but also select high affinity binding peptides for MHC class II (17, 18). Presumably, low affinity binding peptides would not be loaded onto MHC class II molecules if the MHC class II·H2-M complex exhibits a higher affinity compared with a potential MHC class II·peptide complex. However, the role of different Malpha beta 1 and Malpha beta 2 heterodimers in mediating these functions has not been addressed. Previous studies have shown that Ma, Mb1, and Mb2 are coexpressed in splenocytes of mice carrying different haplotypes (19, 33, 36), indicating that Malpha beta 1 and Malpha beta 2 heterodimers might be implemented in antigen presentation by many MHC class II alleles/isotypes. As shown in this study, Mb2 is predominantly expressed in lymph nodes and spleen. In contrast, Mb1 represents the predominant H2-M transcript in solid organs in DBA/1 mice (Fig. 1 and Table I). A similar distribution of Mb1/Mb2 expression has also been observed in Balb/c (H2d) and C57BL/6 (H2b) mice.2 Additionally, we have recently been able to demonstrate that Mb1 is predominantly expressed in cells of epithelial and mesenchymal origin. Mb genes at almost equal levels are expressed in splenic dendritic cells, whereas Mb2 is expressed in B cells (37-39). Of note, highly purified peritoneal macrophages constitutively express Mb2, which can be switched to Mb1 expression upon interferon-gamma treatment (37). Thus, the differential pattern of Ma/Mb1/Mb2 expression may reflect either the composition of immune cells present in lymphoid organs (reviewed in Ref. 40) or the local cytokine milieu, which impacts on Mb1/Mb2 mRNA expression significantly (37, 38). Thus, Malpha beta 1/Malpha beta 2 could potentially exert different functions concerning (i) efficiency in removing CLIP from MHC class II heterodimers or (ii) the selection of the peptide repertoire loaded onto MHC class II molecules. Here, we show that both isoforms are able (i) to effectively remove CLIP from MHC class II molecules (Fig. 4) and (ii) to promote peptide loading of MHC class II molecules as detected with antibodies that define conformational epitopes in MHC class II molecules, indicating MHC class II occupancy with peptides except CLIP (Figs. 2 and 4).

To this end, the efficacy of Malpha /Mbeta 1 or Malpha /Mbeta 2 heterodimers in CLIP removal has not been compared. Here, we show that both Mbeta 1/Mbeta 2 isoforms are able to release CLIP from Aq molecules. This results in a conformational change of the Aq molecule, a finding that has been observed in H2-M-/- mice. In these animals, MHC class II cell-surface expression appears normal, but the conformational shape of the class II molecule (H2-Ab) appears to be altered, probably due to the occupancy by CLIP (14, 41, 42). A similar situation was found to be true for Aq cell-surface molecules: the class II epitope defined by mAbs KH116 and YE2/36HLK is not accessible if Aq is occupied by CLIP (Figs. 2 and 4). Examination of peptides harvested from T2.Aq cells expressing either the Malpha beta 1 or Malpha beta 2 heterodimer will ultimately reveal if the peptide repertoire presented to CD4+ T cells is qualitatively or quantitatively different.

Additionally, we have been able to demonstrate that I-Aq does not necessarily require H2-M to be transported and expressed on the cell surface (Fig. 2). These MHC class II cell-surface molecules may be occupied by CLIP since H2-M is not present in these cells to exchange CLIP for antigenic peptides. Not mutually exclusive, peptides may also occupy the MHC class II heterodimer, which may exhibit a high affinity for I-Aq, leading to CLIP replacement. The apparent lack or need of certain human or mouse MHC class II alleles to require H2-M/DM for CLIP removal in vivo, e.g. the murine alleles H2b and H2d (14, 15), has been attributed to different affinities of these MHC class II alleles for CLIP as determined by in vitro binding studies (43). Although the affinity of I-Aq for CLIP has yet to be defined, we propose that I-Aq exhibits a high affinity in T2 transfectants at least for human CLIP since virtually all I-Aq molecules on T2 cells appeared to be associated with CLIP in the absence of both H2-M isoforms (Fig. 4). In support of this hypothesis, the I-Ad allele, which exhibits a high affinity for both human and mouse CLIP in vitro (43), requires H2-M/DM for CLIP release and acquisition of peptides in H2-M/DM-deficient T2/Ltk-transfected cells. In contrast, I-Ak, although showing a low affinity for CLIP in vitro, is also capable of displacing CLIP without the assistance of H2-M/DM (15).

The observation that both Malpha /Mbeta 1 and Malpha /Mbeta 2, although different in exon 2 (19, 33), are equally effective in CLIP removal is of particular interest in view of previous observations that either peritoneal macrophages or unsorted spleen cells are able to present naive or denatured CII to T cells compared with purified splenic dendritic cells, Langerhans cells, and primed or unprimed B cells (expressing exclusively Mb2 (37)), which cannot present CII appropriately (44, 45).

The obvious difference pertaining to the role of antigen-presenting cells (macrophage versus B cell) in activation of MHC class II-restricted and CII-specific CD4+ T cells (46) may stem from the fact that B cells utilized for CD4+ T cell stimulation are derived from arthritis-susceptible H2g7 mice, but not from mice with the H2q haplotype (43). Of note, the Mb2 allele, which represents the dominant H2-M transcript in B cells (37), differs in H2g7 mouse strains (33) compared with the Mb2 allele in H2q mice (19). Hence, the different capacity of B cells obtained from H2g7 and H2q mice to select for arthritogenic peptides provided by CII may stem not only from differences in the MHC class II binding cleft, but also from a different peptide repertoire selected by alternate Mb2 alleles.

It is noteworthy that transgenic expression of either Malpha beta 1 or Malpha beta 2 heterodimers results not only in a cytoplasmic staining pattern, but also in cell membrane staining (Fig. 5). Earlier studies showed that H2-M/DM molecules reside in the endosomal/lysosomal system (MHC class II compartments, MIICs) (25, 47, 48), but recent studies suggested that H2-M/DM may also be present at the plasma membrane (21, 22). The physiology of H2-M membrane expression, either alone or in association with MHC class II molecules, has to be addressed in future studies.

In summary, the results of this study provide further evidence that the expression of genes involved in MHC class II antigen processing is tissue-dependent. Yet, our findings established for the first time that Malpha beta 1 and Malpha beta 2 can select for MHC class II/peptide assembly utilizing the I-Aq heterodimer from autoimmune-prone mice. Identification of functional differences between H2-M isoforms appears to be of particular interest for a better understanding as to why co-adaptation of two alternative Mbeta chains has been evolved for immune surveillance in mice without apparently being required in other species analyzed thus far.


    ACKNOWLEDGEMENTS

We thank Kirsten Freitag and Claudia Neukirch for excellent technical assistance.


    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 311/A16.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-6131-393-3645; Fax: 49-6131-393-5580; E-mail: maeurer@mail.uni-mainz.de.

Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M006521200

2 W. Walter, unpublished observation.


    ABBREVIATIONS

The abbreviations used are: CII, collagen type II; MHC, major histocompatibility complex; Ii, invariant chain; CLIP, invariant chain-derived MHC class II-associated Ii peptides; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; IRES, internal ribosomal entry site; PBS, phosphate-buffered saline.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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