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• W2079353381 abstract "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 β-chain locus is duplicated in mice, with theH2-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 Mβ1 and Mβ2 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-Mαβ1 or H2-Mαβ2 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. 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 β-chain locus is duplicated in mice, with theH2-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 Mβ1 and Mβ2 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-Mαβ1 or H2-Mαβ2 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. collagen type II major histocompatibility complex invariant chain invariant chain-derived MHC class II-associated Ii peptides monoclonal antibody fluorescein isothiocyanate polymerase chain reaction reverse transcriptase-polymerase chain reaction internal ribosomal entry site phosphate-buffered saline Collagen type II (CII)1induces a chronic polyarthritis syndrome in H2 q 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 (1Courtenay J.S. Dallman M.J. Dayan A.D. Martin A. Mosedale B. Nature. 1980; 283: 666-668Crossref PubMed Scopus (871) Google Scholar, 2Wooley P.H. Luthra H.S. Stuart J.M. David C.S. J. Exp. Med. 1981; 154: 688-700Crossref PubMed Scopus (584) Google Scholar, 3Kerwar S.S. Englert M.E. McReynolds R.A. Landes M.J. Lloyd J.M. Oronsky A.L. Wilson F.J. Arthritis Rheum. 1983; 26: 1120-1131Crossref PubMed Scopus (105) Google Scholar, 4Terato K. Hasty K.A. Cremer M.A. Stuart J.M. Townes A.S. Kang A.H. J. Exp. Med. 1985; 162: 637-646Crossref PubMed Scopus (120) Google Scholar, 5Holmdahl R. Klareskog L. Rubin K. Bjork J. Smedegard G. Jonsson R. Andersson M. Agents Actions. 1986; 19: 295-305Crossref PubMed Scopus (53) Google Scholar, 6Seki N. Sudo Y. Yoshioka T. Sugihara S. Fujitsu T. Sakuma S. Ogawa T. Hamaoka T. Senoh H. Fujiwara H. J. Immunol. 1988; 140: 1477-1484PubMed Google Scholar, 7Myers L.K. Terato K. Seyer J.M. Stuart J.M. Kang A.H. J. Immunol. 1992; 149: 1439-1443PubMed Google Scholar, 8Chiocchia G. Boissier M.C. Manoury B. Fournier C. Eur. J. Immunol. 1993; 23: 327-332Crossref PubMed Scopus (20) Google Scholar). 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 (9Osman G.E. Cheunsuk S. Allen S.E. Chi E. Liggitt H.D. Hood L.E. Ladiges W.C. Int. Immunol. 1998; 10: 1613-1622Crossref PubMed Scopus (34) Google Scholar). 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 (10Germain R.N. Castellino F. Han R. Reis e Sousa C. Romanoli P. Sadegh-Nasseri S. Zhong G.M. Immunol. Rev. 1996; 151: 5-30Crossref PubMed Google Scholar, 11Wolf P.R. Ploegh H.L. Annu. Rev. Cell Dev. Biol. 1995; 11: 267-306Crossref PubMed Scopus (251) Google Scholar). Although dissociation of CLIP for antigenic peptides might spontaneously occur at endosome/lysosome-like pH (12Urban R.G. Chicz R.M. Strominger J.L. J. Exp. Med. 1994; 180: 751-755Crossref PubMed Scopus (49) Google Scholar), 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 (13Morris P. Shaman J. Attaya M. Amaya M. Goodman S. Bergman C. Monaco J.J. Mellins E. Nature. 1994; 368: 551-554Crossref PubMed Scopus (346) Google Scholar, 14Fung-Leung W.P. Surh C.D. Liljedahl M. Pang J. Leturcq D. Peterson P.A. Webb S.R. Karlsson L. Science. 1996; 271: 1278-1281Crossref PubMed Scopus (242) Google Scholar, 15Stebbins C.C. Peterson M.E. Suh W.M. Sant A.J. J. Immunol. 1996; 157: 4892-4898PubMed Google Scholar, 16Wolf P.R. Tourne S. Miyazaki T. Benoist C. Mathis D. Ploegh H.L. Eur. J. Immunol. 1998; 28: 2605-2618Crossref PubMed Scopus (54) Google Scholar). 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 (17van Ham S.M. Gruneberg U. Malcherek G. Broker I. Melms A. Trowsdale J. J. Exp. Med. 1996; 184: 2019-2024Crossref PubMed Scopus (113) Google Scholar, 18Weber D.A. Evavold B.D. Jensen P.E. Science. 1996; 274: 618-620Crossref PubMed Scopus (258) Google Scholar). 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 (19Walter W. Loos M. Maeurer M.J. Immunogenetics. 1996; 44: 19-26Crossref PubMed Scopus (17) Google Scholar). In contrast to the DM loci in humans, the H2-Mregion in the mouse contains one H2-Ma (Ma) gene, but two H2-Mb (Mb) genes termedMb1 and Mb2 (20Cho S. Attaya M. Brown M.G. Monaco J.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5197-5201Crossref PubMed Scopus (43) Google Scholar). It is still unclear whetherMb1 and Mb2 are equally expressed in vivo (15Stebbins C.C. Peterson M.E. Suh W.M. Sant A.J. J. Immunol. 1996; 157: 4892-4898PubMed Google Scholar, 21Alfonso C. Karlsson L. Annu. Rev. Immunol. 2000; 18: 113-142Crossref PubMed Scopus (135) Google Scholar), and no systematic study has been presented in which a comparison of Mα/Mβ1 and Mα/Mβ2 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-Mαβ1 (Mαβ1) or H2-Mαβ2 (Mαβ2) 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). DBA/1 mice (H2 q) and New Zealand White rabbits were purchased from Charles River Laboratories (Sulzfeld, Germany). 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 (23Salter R.D. Howell D.N. Cresswell P. Immunogenetics. 1985; 21: 235-246Crossref PubMed Scopus (508) Google Scholar). T2 cells were maintained in RPMI 1640 medium (Life Technologies, Inc., Eggenstein, Germany) supplemented with 10% heat-inactivated fetal bovine serum, 2 mml-glutamate, 100 IU/ml penicillin, and 100 μg/ml streptomycin (all from Life Technologies, Inc.) and 50 μm2-mercaptoethanol (Sigma, Deisenhofen, Germany), referred to as complete medium. 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.Mα-C.69.3, R.Mβ1/2-C.71.3, and R.hCLIP73.11 were prepared by immunizing rabbits with C-terminal peptides from Mα (amino acids 238–248) (24Cho S.G. Attaya M. Monaco J.J. Nature. 1991; 353: 573-576Crossref PubMed Scopus (110) Google Scholar) and Mβ1/Mβ2 (amino acids 228–243) (25Karlsson L. Peleraux A. Lindstedt R. Liljedahl M. Peterson P.A. Science. 1994; 266: 1569-1573Crossref PubMed Scopus (132) Google Scholar) and a CLIP peptide (amino acids 81–104 of the human p33 invariant chain isoform) (26Denzin L.K. Robbins N.F. Carboy-Newcomb C. Cresswell P. Immunity. 1994; 1: 595-606Abstract Full Text PDF PubMed Scopus (237) Google Scholar), respectively, coupled with an added amino-terminal cysteine to diphtheria toxoid (Chiron Mimotopes, Victoria, Australia). The antisera were affinity-purified usingN-hydroxysuccinimide-activated Fast Flow Sepharose 4 (Amersham Pharmacia Biotech, Freiburg, Germany) cross-linked to the respective Mα, Mβ, 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 Ma q, Mb1 q, andMb2 q. Specificity controls included Western blot analysis of T2 cells alone or T2 cells expressing I-Aq (but not H2-M genes) using anti-Mα or anti-Mβ antisera (see Fig. 4). I-Aaq (Aa q)andI-Ab q (Ab q) were amplified from a DBA/1 splenocyte cDNA library (19Walter W. Loos M. Maeurer M.J. Immunogenetics. 1996; 44: 19-26Crossref PubMed Scopus (17) Google Scholar) 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 andBamHI restriction sites (underlined) to facilitate cloning of amplified DNA. Following amplification, the 809-base pairAa q and 812-base pair Ab q PCR products were cloned into the EcoRV and BamHI sites of pBSK+ (Stratagene, Heidelberg, Germany) and sequenced (19Walter W. Loos M. Maeurer M.J. Immunogenetics. 1996; 44: 19-26Crossref PubMed Scopus (17) Google Scholar). For expression in eukaryotic cells, theAa q cDNA was subcloned (NcoI-BamHI) into the retroviral vector MFG (27Dranoff G. Jaffee E. Lazenby A. Golumbek P. Levitsky H. Brose K. Jackson V. Hamada H. Pardoll D. Mulligan R.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3539-3543Crossref PubMed Scopus (2639) Google Scholar), yielding MFG-Aaq. Subsequently, an IRES-Neocassette (28Maeurer M.J. Gollin S.M. Martin D. Swaney W. Bryant J. Castelli C. Robbins P. Parmiani G. Storkus W.J. Lotze M.T. J. Clin. Invest. 1996; 98: 1633-1641Crossref PubMed Scopus (235) Google Scholar), consisting of an internal ribosomal entry site (IRES) sequence from the encephalomyocarditis virus and the neomycin phosphotransferase gene (Neo), was inserted into theBamHI site at the 3′-end of the Aa qcDNA in MFG-Aaq, yielding the vector DFG-Aaq-IRES-Neo. Next, an IRES-EcoRI-NcoI fragment was fused to the ATG of the Ab q cDNA in pBSK+, yielding the plasmid pBSK-IRES-Abq+. After that, pBSK-IRES-Abq+ was digested with BamHI, and the IRES-Ab q fragment obtained was inserted into theBamHI 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 Aa q,Ab q, and Neo, was finally termed TFG-Aq. The isolation of Ma q,Mb1 q, and Mb2 q full-length cDNA clones has been reported previously (19Walter W. Loos M. Maeurer M.J. Immunogenetics. 1996; 44: 19-26Crossref PubMed Scopus (17) Google Scholar). For expression in eukaryotic cells, the Ab q cDNA in pBSK-IRES-Abq+ was replaced by Ma q(NcoI-NotI). The IRES-Ma qsequence was subcloned into the BamHI site of the eukaryotic expression vector pCEP4 (Invitrogen, Groningen, The Netherlands). Subsequently, NotI-NheI-digestedMb1 q or Mb2 q cDNA was inserted into the NotI-NheI sites at the 5′-end of the IRES-Ma q sequence, yielding pCEP4-Mb1q-IRES-Maq and pCEP4-Mb2q-IRES-Maq, respectively. T2 cells stably expressing I-Aq molecules from arthritis-susceptible DBA/1 mice were generated by retroviral transfection as previously described (33Hermel E. Yuan J. Monaco J.J. Immunogenetics. 1995; 42: 136-142PubMed Google Scholar). Briefly, retroviral supernatant was produced by transfecting the TFG-Aq proviral construct into the ψCRIP packaging cell line (29Danos O. Mulligan R.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6460-6464Crossref PubMed Scopus (799) Google Scholar). 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 (30Villadangos J.A. Riese R.J. Peters C. Chapman H.A. Ploegh H.L. J. Exp. Med. 1997; 186: 549-560Crossref PubMed Scopus (179) Google Scholar). Transfection of T2.Aq cells with the pCEP4, pCEP4-Mb1q-IRES-Maq or pCEP4-Mb2q-IRES-Maq vector was performed by electroporation as described (26Denzin L.K. Robbins N.F. Carboy-Newcomb C. Cresswell P. Immunity. 1994; 1: 595-606Abstract Full Text PDF PubMed Scopus (237) Google Scholar). 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 Ma q, Mb1 q, andMb2 q by RT-PCR. Clones that exhibited high Mαβ1 or Mαβ2 protein expression levels determined by Western blot analysis were used in subsequent experiments. Total RNA isolation and cDNA synthesis have been described previously (19Walter W. Loos M. Maeurer M.J. Immunogenetics. 1996; 44: 19-26Crossref PubMed Scopus (17) Google Scholar). 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). The ratio RT-PCR assay performed in this study was based on the simultaneous amplification ofMb1 and Mb2 transcripts using panMb1/2 primers (see above) annealing within conserved regions (exons 1 and 3) of bothMb q mRNA species (19Walter W. Loos M. Maeurer M.J. Immunogenetics. 1996; 44: 19-26Crossref PubMed Scopus (17) Google Scholar). Equal amplification efficiency of both Mb transcripts was assured by comparative cycle kinetic and linear regression analysis (31Bouaboula M. Legoux P. Pessegue B. Delpech B. Dumont X. Piechaczyk M. Casellas P. Shire D. J. Biol. Chem. 1992; 267: 21830-21838Abstract Full Text PDF PubMed Google Scholar) using clonedMb1 and Mb2 full-length cDNAs (19Walter W. Loos M. Maeurer M.J. Immunogenetics. 1996; 44: 19-26Crossref PubMed Scopus (17) Google Scholar). Discrimination between co-amplified Mb transcripts was performed by restriction endonuclease analysis. We therefore took advantage of the polymorphism of Mb1 q andMb2 q genes within exon 2 (19Walter W. Loos M. Maeurer M.J. Immunogenetics. 1996; 44: 19-26Crossref PubMed Scopus (17) Google Scholar) and used the restriction enzyme HhaI, which specifically cleaves at nucleotide +321 within the Mb1 q sequence and nucleotide +264 within the Mb2 q 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 [α-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 β-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. 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 (32Kyhse-Andersen J. J. Biochem. Biophys. Methods. 1984; 10: 203-209Crossref PubMed Scopus (2145) Google Scholar). 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). 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. 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-DMα or anti-DMβ 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). The H2-M region contains one Ma gene, but two Mb genes termedMb1 and Mb2 (20Cho S. Attaya M. Brown M.G. Monaco J.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5197-5201Crossref PubMed Scopus (43) Google Scholar). Transcription of theMa gene and both Mb genes has been observed in splenocytes of different mouse strains, including mice that carry the arthritis-susceptible H2 q haplotype (19Walter W. Loos M. Maeurer M.J. Immunogenetics. 1996; 44: 19-26Crossref PubMed Scopus (17) Google Scholar, 33Hermel E. Yuan J. Monaco J.J. Immunogenetics. 1995; 42: 136-142PubMed Google Scholar). To define the H2-M gene expression pattern, we examinedMa and Mb mRNA expression in lymphoid and non-lymphoid organs or tissues from arthritis-susceptible DBA/1 (H2 q) mice by RT-PCR. Constitutive Ma andMb mRNA expression could be detected in each organ sample (data not shown). Next, we addressed the question of whetherMb1 and Mb2 are differentially expressed by ratio RT-PCR analysis (Fig. 1). The ratios ofMb1 to Mb2 mRNA given as percentages of the total Mb mRNA are listed in TableI. Mb2 represents the majorMb 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. 1 A 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.1 B and Table I).Table IMb1 and Mb2 are differentially expressed in lymphoid and non-lymphoid organs of arthritis-susceptible DBA/1 (H2q) miceOrgan/tissueMb1Mb2Thymus4 weeks39.9 ± 0.860.1 ± 0.98 weeks37.6 ± 2.962.4 ± 1.412 weeks42.2 ± 3.857.8 ± 2.3Spleen39.6 ± 1.460.4 ± 3.2Mesenteric lymph node20.5 ± 1.679.5 ± 2.1Popliteal lymph node17.7 ± 1.782.3 ± 1.6Small intestine81.4 ± 2.118.6 ± 1.5Large intestine57.1 ± 1.142.9 ± 0.9Muscle37.9 ± 1.262.1 ± 1.0Heart42.3 ± 2.157.7 ± 1.9Testis75.1 ± 1.524.9 ± 1.1Brain82.5 ± 1.817.5 ± 1.3Lung79.7 ± 2.320.3 ± 2.5Liver71.2 ± 0.928.8 ± 1.4Kidney80.4 ± 1.619.6 ± 1.8Pancreas59.6 ± 2.440.4 ± 2.8Values, presented as means ± S.D. of three determinations, indicate the relative expression of Mb1 and Mb2mRNAs as percentages of total Mb mRNA as determined by ratio RT-PCR as described in the legend to Fig. 1. Open table in a new tab Values, presented as means ± S.D. of three determinations, indicate the relative expression of Mb1 and Mb2mRNAs as percentages of total Mb mRNA as determined by ratio RT-PCR as described in the legend to Fig. 1. The observation that Mb1 andMb2 are differentially expressed in lymphoid and non-lymphoid organs (Table I) implies that both heterodimers, Mαβ1 and Mαβ2, 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 Mαβ1 (T2.Aq.Maq.Mb1q) or Mαβ2 (T2.Aq.Maq.Mb2q) derived from DBA/1 mice (H2 q). To ensure comparable levels of I-Aq surface expression as well as Mαβ1 or Mαβ2 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 αβ-dimers with similar efficiency (30Villadangos J.A. Riese R.J. Peters C. Chapman H.A. Ploegh H.L. J. Exp. Med. 1997; 186: 549-560Crossref PubMed Scopus (179) Google Scholar), 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 Mαβ1 and Mαβ2 expression (Fig. 3).Figure 3Comparison of Mαβ1 and Mαβ2 expression in T2.Aq transfectants. MHC class II antigen-processing mutant T2 cells were stably transfected with I-Aq α- and β-chain genes and then super-transfected with Ma andMb1 (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 Mα, or Mβ1, and Mβ2 monomers as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 inDM mutant cell lines (13Morris P. Shaman J. Attaya M. Amaya M. Goodman S. Bergman C. Monaco J.J. Mellins E. Nature. 1994; 368: 551-554Crossref PubMed Scopus (346) Google Scholar, 26Denzin L.K. Robbins N.F. Carboy-Newcomb C. Cresswell P. Immunity. 1994; 1: 595-606Abstract Full Text PDF PubMed Scopus (237) Google Scholar) or H2-M-deficient mice (14Fung-Leung W.P. Surh C.D. Liljedahl M. Pang J. Leturcq D. Peterson P.A. Webb S.R. Karlsson L. Science. 1996; 271: 1278-1281Crossref PubMed Scopus (242) Google Scholar), 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 firstand second lanefrom left) and polymorphic determinants (Fig. 2, thirdand fourth lanefrom left). Staining with mAb KH118," @default.
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