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The Journal of Immunology, 2004, 173: 1085-1093.
Copyright © 2004 by The American Association of Immunologists

Dissection of the HLA-DR4 Peptide Repertoire in Endocrine Epithelial Cells: Strong Influence of Invariant Chain and HLA-DM Expression on the Nature of Ligands1

Aura Muntasell*,{dagger}, Montserrat Carrascal{ddagger}, Iñaki Alvarez*,{dagger}, Laurence Serradell*,{dagger}, Peter van Veelen§, Frank A. W. Verreck§, Frits Koning§, Joaquín Abian{ddagger} and Dolores Jaraquemada2,*,{dagger}

* Immunology Unit and {dagger} Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Campus de Bellaterra, and {ddagger} Structural and Biological Mass Spectrometry Unit, Department of Medical Bioanalysis, Institut d’Investigacions Biomèdiques de Barcelona-Consejo Superior de Investigaciones Científicas, Institut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain; and § Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Class II MHC (MHC II) expression is restricted to professional APCs and thymic epithelium but it also occurs in the epithelial cells of autoimmune organs which are the unique targets of the CD4 autoreactive T cells in endocrine autoimmune diseases. This specificity is presumably conditioned by an epithelium-specific peptide repertoire associated to MHC II at the cell surface. MHC II expression and function is dependent on the action of two main chaperones, invariant chain (Ii) and DM, whose expression is coregulated with MHC II. However, there is limited information about the in vivo expression levels of these molecules and uncoordinated expression has been demonstrated in class II-positive epithelial cells that may influence the MHC-associated peptide repertoires and the outcome of the autoimmune response. We have examined the pool of peptides associated to DR4 molecules expressed by a neuroendocrine epithelial cell and the consequences of Ii and DM coexpression. The RINm5F rat insulinoma cell line was transfected with HLA-DRB1*0401, Ii, and DM molecules in four different combinations: RIN-DR4, -DR4Ii, -DR4DM, and -DR4IiDM. The analysis of the peptide repertoire and the identification of the DR4 naturally processed ligands in each transfected cell were achieved by mass spectrometry. The results demonstrate that 1) the expression of Ii and DM affected the DR4 peptide repertoires by producing important variations in their content and in the origin of peptides; 2) these restrictions affected the stability and sequence of the peptides of each repertoire; and 3) Ii and DM had both independent and coordinate effects on these repertoires.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Class II MHC (MHC II)3 molecules are transmembrane glycoproteins which function by binding processed antigenic peptides and presenting them to CD4+ T lymphocytes to initiate or maintain a specific immune response. Constitutive MHC II expression is restricted to professional APCs including dendritic cells, B cells, and macrophages and to the epithelial cells of the thymus. MHC II expression can also be induced in other cell types and it has been described in vivo in epithelial cells from various target organs in autoimmune diseases such as autoimmune thyroiditis or insulin-dependent diabetes (1, 2, 3, 4). The relevance and specificity of Ag presentation by class II in epithelial cells is suggested by the specific destruction of these cells in endocrine autoimmunity (5) and by the T cell recognition of cryptic epitopes presented by epithelial cells and not by B cells (6, 7). Consequently, the antigenic repertoire displayed at the surface of class II-expressing epithelial cells must be relevant to the understanding of the autoreactive T cell response and its maintenance.

In a previous report, we demonstrated that the pool of peptides associated to HLA-DR4 expressed by rat insular epithelial cells was largely derived from proteins residing in internal cell compartments, including the cytosol and the secretory pathway, and some from tissue-specific proteins (8). Functional class II expression is highly dependent on the expression of two main chaperones, the invariant chain (Ii) and DM. Newly synthesized class II {alpha}- and {beta}-chains form a nonameric complex with three Ii molecules in the endoplasmic reticulum (ER) (9) where Ii blocks the peptide-binding groove (10) and guides class II dimers to the endocytic pathway where Ii is proteolytically cleaved leaving the CLIP fragment bound to class II (11). Peptide loading and editing mainly occurs in late endocytic compartments designated MHC II-containing compartments (12). HLA-DM, an endocytic chaperone, protects empty class II dimers until they bind a peptide capable to confer enough stability to the complex (13, 14). MHC II, Ii, and DM are partially coregulated (15) and their relative levels are relevant to the constitution of the final peptide pool bound to class II. In vivo, the absence of any of the two chaperones induced alterations of the peptide repertoire associated to MHC II, as inferred from the modifications of the resulting T cell pools after thymic selection in Ii and DM knockout mice (16, 17, 18, 19, 20).

Limited comparisons have been made of the expression levels of MHC II, Ii, and DM in epithelial cells from autoimmune target organs. Differences in expression levels of class II genes were described in Graves’ disease’s thyroid follicular cells, in multiple sclerosis patients, and in peripheral blood B cells from rheumatoid arthritis patients (21, 22). This absence of coordination could alter the repertoire presented by MHC II and consequently influence the autoimmune responses. We undertook the study of the implications of this lack of coordination in peptide presentation by class II molecules in epithelial endocrine cells, analyzing the effect of Ii and DM expression on the peptide repertoires associated to DR4 in a set of MHC II-expressing endocrine cells. The RINm5F rat insulinoma cell line was transfected with human HLA-DRB1*0401, Ii, and DM molecules in four different combinations: RIN-DR4, -DR4Ii, -DR4DM, and -DR4IiDM. The analysis of the peptide repertoires and the identification of some DR4-naturally processed ligands in each transfected cell was achieved by mass spectrometry (MS) techniques and revealed that both chaperones added restrictions to the diversity of peptides presented, demonstrating that 1) the expression of Ii and DM profoundly affected the DR4 peptide repertoires by producing strong variations in their content and in the origin of peptides; 2) these restrictions affected the stability and sequence of the peptides of each repertoire; and 3) Ii and DM had both independent and coordinate effects on those repertoires.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Abs

Four previously described (23) cell lines RINm5F-DRB1*0401(DR4),RINm5F-DRB1*0401-Ii (DR4Ii), RINm5F-DRB1*0401-HLA-DM (DR4DM),and RINm5F-DRB1*0401-Ii-HLA-DM (DR4IiDM) derived from the class II–, class I+ rat insulinoma cell line RINm5F, were used as a source of class II molecules. A DR {alpha}{beta}-specific murine mAb, L243 (Ref.24 ; American Type Culture Collection (ATCC), Rockville, MD), was used for affinity chromatography and flow cytometry in addition to the anti-DR{beta} Ab B8.11.2 (23) for immunoprecipitation experiments and the anti-DR{alpha} DA6.147 for Western blots; VICY1, a mAb specific for a Ii cytoplasmic epitope (25) and anti-Ii mAb LL1 (26) were also used, as was 5C1, a mAb anti-DM{alpha} lumenal domain. An anti-{beta}-tubulin I Ab (Sigma-Aldrich, St. Louis, MO) was used as an internal control for Western blots.

Flow cytometry

Transfected cells were fixed and permeabilized before staining with anti-Ii and anti-DM Abs. Live cells were used for surface DR. Ab binding was detected with goat anti-mouse-FITC. Samples of 10,000 cells were analyzed with a FACSCalibur using CellQuest software (both from BD Biosciences, San Jose, CA).

Bulk culture of class II-expressing cells

Transfected RINm5F cells were grown as adherent monolayers in T175 flasks using RPMI 1640 medium (Invitrogen Life Technologies, Karlsruhe, Germany) supplemented with 10% heat-inactivated FCS, 200 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin as a base. The basic medium was complemented with the corresponding selection medium as described (23) during the first expansions of each cell line in T175 flasks.

Each cell line was subsequently grown with alternate passes with and without selection until they reached 1.5 x 109 cells. DR4DM and DR4IiDM cells were further grown to 3 x 109 cells using the same method. Expression of the transfected molecules was checked every 3–4 wk by flow cytometry. For harvesting, the cells were detached using trypsin-EDTA solution. After two washes in PBS, the pellets were stored at –70°C until used.

Western blots, 125I labeling, and immunoprecipitation

Total cell extracts for Western blots were done as described (21), only 1% digitonin (Sigma-Aldrich) was used instead of Nonidet P-40. Immunoblotting was done with Abs: DA6.147, LL1, 5C1, anti-{beta}-tubulin, followed by anti-mouse IgG-HRP secondary Ab (Amersham Biosciences, Uppsala, Sweden). Blots were developed by ECL.

For surface labeling and immunoprecipitation, 107 cells were washed two times with cold PBS and labeled following the standard protocol with 0.5 mCi Na-125I (Amersham Biosciences) using lactoperoxidase/H2O2 and washed. Radiolabeled cells were lysed in 1 ml of lysis buffer (0.5% Nonidet-P40, 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM pefabloc, 10 mM iodoacetamide, 5 µM aprotinin, 5 µM leupeptin, 2 µM/ml pepstatin). After 30 min at 4°C, cell lysates were spun at 14,000 x g for 30 min at 4°C and then precleared for 2 h at room temperature with 75 µl of normal rabbit serum and 100 µl of protein A-Sepharose beads (Amersham Biosciences). Precleared lysates were mixed with 4 µl of B8.11.2 mAb overnight at 4°C. Ag-Ab complexes were isolated with 10 µl of protein A-Sepharose beads mixing 1 h at 4°C, washed with lysis buffer followed by washing buffer (125 mM Tris, pH 8, 150 mM NaCl), resuspended in 100 µl of sample buffer (2% SDS, 125 mM Tris, 10% glycerol, 0.1% bromphenol blue, pH 6.8), and left 30 min at room temperature. Half of the sample (boiled (B)), boiled for 3 min, and the other half (nonboiled (NB)), kept at room temperature, were electrophoresed on a SDS-12% polyacrylamide gel.

Purification of HLA-DR4 molecules from cultured cells

HLA-DR4/peptide complexes from each epithelial cell lysate were purified by affinity chromatography using L243-Sepharose beads; low m.w. peptides were eluted with 0.1% trifluoroacetic acid according to standard procedures (27). Pellets processed contained 1.5 x 109 cells from DR4 and DR4Ii transfectants and 3 x 109 cells from DR4DM and DR4IiDM transfectants.

Extraction of DR4-bound peptides and reverse phase-HPLC

Eluted class II-peptide mixtures were centrifuged through 10,000-Da cutoff ultrafiltration tubes (Centriprep 10; Millipore, Bedford, MA) and collected from the flow-through. The peptide pools were vacuum-concentrated and directly separated using a reverse-phase HPLC column (µRPC C2/C18, 2.1 x 100 mm; Amersham Biosciences) and the SMART system (Amersham Biosciences). Chromatographic analysis was monitored at a 214-nm UV wavelength. One-minute fractions were collected within the range of 5–45% acetonitrile gradient and analyzed by MS. The HPLC fractions were evaporated to dryness and redissolved in 5 µl of MeOH/H2O 1/1, 1% AcOH. Fractions were conserved at –70°C until analysis. A MALDI-TOF analysis of each fraction was performed to characterize the tentative peptide signals.

Peptide sequencing

Experiments were performed on a Finnigan LCQ ion trap mass spectrometer (ThermoQuest; Thermo Finnigan, San Jose, CA) equipped with a nanospray interface (Protana, Odense, Denmark). For analysis, 2–3 µl of each HPLC fraction were loaded in the nanospray capillary. Product ion spectra were obtained by collision-induced dissociation, mainly of the double- and triple-protonated peptide molecules using the peptide molecular mass information obtained by the MALDI-TOF spectra of the HPLC fractions as well as from the signals observed in the corresponding nanospray full MS scan.

The spray voltage applied was 0.85 kV and the capillary temperature was 110°C. The isolation window was 3 and 4 mass units wide for the MS/MS and MS (3) experiments, respectively, and the relative collision energy was 20–40%, depending on the charge state of the precursor ion. Three scan events were performed for each analysis: a full MS scan of the HPLC fraction analyzed, a high resolution zoom scan centered at the mass of the ion selected for analysis, and, finally, the sequence analysis of the tentative peptide by MS/MS.

To identify the source proteins of these peptides, the MS-Tag program (Protein Prospector; Mass Spectrometry Facility, University of California, San Francisco, CA) was used to compare the spectrum of each peptide target with the virtual spectra derived from the sequences available in public databases as protein (SwissProt, NCBInr) and DNA (dbEST) databanks. If no clear match was found, partial or total manual de novo sequencing of the peptide was conducted and the search was repeated using the MS-Edman program. After sequencing and source protein identification, an affinity peptide score was calculated for each peptide using a predictive matrix obtained by Hammer et al. (27), by peptide side chain scanning techniques, based on the DR4 peptide-binding motif (26). A value higher than 2 was considered a high score, theoretically corresponding to high affinity binding. No peptide score was assigned to peptides not fitting the DR4 motif at the P1 position.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression levels of transfected molecules; conformation of DR4 molecules in the presence or absence of Ii and DM

To compare the expression levels of HLA-DR4, Ii, and DM between the transfectants, a relative quantification of surface DR4 and cytoplasmic Ii and DM was performed by flow cytometry (Fig. 1A). As previously shown (23), Ii+ cells displayed higher DR4 surface staining than Ii– cells (mean fluorescence intensity (MFI) = 450 and 325 in DR4Ii and DR4IiDM, vs MFI = 218 and 183 in DR4 and DR4DM, respectively). Cytoplasmic Ii staining was higher in DR4Ii (MFI = 94) than in DR4IiDM (MFI = 77) transfectants while DM staining was higher in DR4DM (MFI = 61) than in DR4IiDM (MFI = 41) cells. The expression levels of transfected proteins were also analyzed by Western blot on total cell extracts from the transfectants (Fig. 1B). HLA-DR{alpha}, Ii, and DM expression by immunoblot was compared with the constitutively expressed protein tubulin. No significant differences were observed between the transfectants.



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  		FIGURE 1. Expression and conformation of transfected molecules. A, Flow cytometry analysis of DR4 expression (black lines) by surface staining with L243 mAb; expression of Ii (light gray lines) and HLA-DM (dark gray lines) by cytoplasmic staining using VIC-Y1 and 5C1 mAbs, respectively. The table represents the quantification of the expression of each molecule by MFI in the different cells. B, Western blot analysis of the expression of DR{alpha}, Ii, and DM{beta} in the transfected cells. Total cell extracts were blotted with DA6.147, LL1, 5C1, and anti-{beta}-tubulin mAbs, for the detection of DR{alpha}, Ii, DM{beta}, and control tubulin, respectively. C, DR molecules from 125I-labeled cell extracts were immunoprecipitated with anti-DR B8.11.2 mAb and analyzed by SDS-PAGE before (NB) or after (B) boiling, for {alpha}- and {beta}-chain dissociation. Compact dimers were mostly contained in high m.w. bands (arrow) and their presence coincided with low-density single {alpha}- and {beta}-chain bands in unboiled (NB) samples of DM-positive cells (DR4DM and DR4IiDM), which increased after sample boiling (B). Most DR4 molecules from DM-negative (DR4 and DR4Ii) transfectants were detected as individual {alpha}- and {beta}-chains in NB samples that show a low increase after boiling (B), indicating that DR dimers were mainly associated to unstably bound peptides in these cells.

 
Class II molecules stably bound to peptides form SDS-resistant complexes at room temperature (compact forms). After boiling, the complexes dissociate into {alpha}- (33 kDa) and {beta}- (29 kDa) chains. DR dimers unstably bound to peptide ligands, including CLIP, directly separate as single {alpha}- and {beta}-chains in SDS at room temperature (28). The conformation of surface DR4 molecules in the transfectants was analyzed by immunoprecipitation with B8.11.2 mAb after 125I-cell surface labeling, followed by SDS-PAGE of unboiled and boiled samples and autoradiography. The results are shown in Fig. 1C. Each two lanes correspond to SDS-PAGE of nonboiled (NB) and boiled (B) samples for each transfectant. Compact dimers were mostly contained in high m.w. bands (Fig. 1C, arrow; Ref.29) but their presence corresponded with a low density of single {alpha}- and {beta}-chain bands observed in unboiled samples of the DM+ cells and was confirmed by the increase of single {alpha}- and {beta}-chain bands after sample boiling. In contrast, most DR4 molecules from DM-negative (DR4 and DR4Ii) transfectants were detected as individual {alpha}- and {beta}-chains in NB samples, confirmed by a low increase of these bands after boiling, indicating that DR dimers were mainly associated to unstably bound peptides in these cells. These data show qualitative differences in the conformation of surface DR4 expressed by the DM+ and DM– transfected cells.

Peptide isolation from HLA-DRB*0401 molecules expressed by transfection in epithelial cells

To analyze the DR4 peptide repertoires in the different transfectants, peptide pools were released by acid elution from affinity-purified DR4 molecules expressed by the four cell lines, following standard procedures. Reverse phase-HPLC fractionation was monitored by UV absorbance at 214 nm (Fig. 2), showing a highly heterogeneous profile of naturally processed ligands associated to DR4, which mostly eluted from 5 to 45% acetonitrile gradient range. DR4 and DR4Ii cells were analyzed in one experiment, followed by DR4DM and DR4IiDM in a second experiment, using identical protocols and RP-HPLC conditions, but doubling the amount of starting material. Analysis of HPLC fractions by MALDI-TOF confirmed the heterogeneity of samples and served to target those peptides that could be fragmented for sequencing (data not shown).



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  		FIGURE 2. Reverse-phase HPLC profiles of eluted peptides from DR4 molecules expressed by transfected cells DR4 (A), DR4Ii (B), DR4DM (C), and DR4IiDM (D). Peptides were acid eluted from affinity chromatography purified DR4 molecules and separated by reverse-phase micro-HPLC, as described in Materials and Methods. Most peptides eluted within the 5–45% acetonitrile range.

 
Identification of naturally processed peptides bound to DR4 in the transfected cells

With the exception of the two CLIP peptides sequenced by Edman degradation, all remaining sequences were obtained by tandem MS. DR4 molecules from DR4IiDM cells were associated to a more homogeneous and abundant repertoire than in the other cells (Table I). Of the 63 DR4 ligands, 4 were exogenous whereas 28, 4, 7, and 20 peptides derived from the cell surface, endocytic vesicles, secretory granules, and cytoplasmic self-proteins, respectively.


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  		Table I. Sequences of peptides associated to DR4 in DR4liDM cell transfectantsa

 
There was an important overlap between sequences identified from DR4DM and DR4IiDM eluates. Of the 28 peptides sequenced from the DR4DM eluate (Table II), nine sequences were identical to sequences of the DR4IiDM repertoire. Twelve of the remaining sequences were not identical but corresponded to nested peptides with sequences identified in the DRIiDM repertoire. One sequence corresponded to the same protein ({propto}-1-antitrypsin) but to a different epitope to the one found in the triple transfectants. Finally, only four of the peptides sequenced were unique to the DR4DM pool. These peptides derived from proteins that are not represented in the other pools, a mineralocorticoid receptor from the cytoplasm, a nuclear transcription factor ELYS, and membrane MHC and adhesion molecule cadherin.


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  		Table II. Sequences of peptides associated to DR4 in DR4DM transfectantsa

 
In addition to CLIP, 13 peptides were sequenced from the DR4Ii double transfectants (Table III): 4 exogenous peptides from fetuin, 1 single and 3 nested epitopes; 4 surface peptides, including 1 from the transfected HLA-DR{beta}-chain; 1 from a secreted protein; 1 from the ER; and 4 from the cytosol. Only 2 of the 5 cytosolic/ER sequences complied with the DR4 motif, whereas the exogenous, secreted, and surface peptides were all perfect DR4 binders. Most of the epitopes were unique to the DR4Ii repertoire.


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  		Table III. Sequences of peptides associated to DR4 in DR4li cell transfectantsa

 
The identified DR4 peptide repertoire in the absence of Ii and DM (Table IV) was entirely conformed by autologous peptides: 1 from a surface protein, 1 from a secreted molecule, and the remaining 10 from cytosolic proteins (including 1 nuclear and 1 mitochondrial) represented by single epitopes without length variants (no nested peptide sets). No affinity score could be assigned to 6 of the 12 peptides eluted from the DR4 single transfectants because their sequences did not fit with the DR4 motif. All peptides were unique to the DR4 single transfectant repertoire and, independent of their matching to the DR4 motif, all but one (9 of 10) of the cytosolic sequences corresponded to N- or C-terminal epitopes of the source proteins.


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  		Table IV. Sequences of peptides associated to DR4 in DR4 cell transfectantsa

 
In all repertoires, most peptides abundant enough to be sequenced fell within the standard class II size range. DR4 ligands derived from cell surface, endocytic, and secretory proteins were mainly grouped in nested sets of peptides with identical core sequences and ragged ends, whereas ligands from cytoplasmic resident proteins were mostly represented by a single sequence in all repertories analyzed, with few exceptions found in the DM+ cell repertoires.

Comparative analysis of the peptide repertoires. 1. Influence of DM on the peptide sequences

The predicted DR4 peptide binding motif consists of a linear core sequence of nine residues where P1, P4, P6, and P9 are the main DR4-anchor residues affecting peptide binding stability (30). Residues occupying the DR4-anchor positions of peptides were compared between DM+ and DM– cell repertoires (Fig. 3). As a first approach, all sequences, including those forming nested sets, were considered individually (n = 28 and 91 for DM– and DM+ repertoires, respectively) (Fig. 3A). The frequency of each residue in each anchor position was calculated as the percentage over the total number of sequences identified. To avoid bias from repeated nested set sequences, a second analysis was done where all peptides from nested sets were considered as one single sequence (n = 26 and 42 from DM– and DM+ repertoires, respectively) (Fig. 3B).



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  		FIGURE 3. Analysis of preferred residues occupying anchor positions of peptides from DM+ vs DM– cell repertoires. In A, data correspond to the analysis of all sequences including those forming nested sets. Numbers in the table show the frequency for each residue in each anchor position in DM+ and DM– repertoires. The last column represents the total number of sequences included in the analysis. The residue frequency was calculated as the percentage over the total number of sequences. The histograms represent the same values in DM– ({square}) and DM+ ({blacksquare}) repertoires. A second analysis is shown in B, where all peptides from each nested set were considered as one single sequence, to avoid bias from repeated nested sets sequences. Values and figures as in A.

 
In both analyses, the effect of DM was particularly evident in the constitution of the P1 anchor which was mainly occupied by a hydrophobic residue. Considering the first analysis, a high predominance of Y (51%) over F (30%) was detected for DM+ cells (Fig. 3A). The other hydrophobic amino acids, theoretically able to occupy P1 (V, L, I, W, and M), were not favored by DM expression. This was confirmed when the analysis included only one peptide representing each nested set, where the dominance of Y (36%) and F (31%) in P1 from DM+ cell peptides was maintained.

To a lesser extent, the composition of P4 and P6 were also influenced by DM expression. Residues D and E were preferred P4 anchors, and polar residues, mainly N, were more abundant as P6 anchor in DM+ cells. In contrast, DM expression was permissive for P9 anchor positions, which displayed a larger spectrum of amino acids in all sequences analyzed. In addition, a total of nine peptides from the DM-negative transfectants (DR4 and DR4Ii) did not match the DR4-affinity matrix and thus no peptide score could be assigned. Sequence alignment with ClustalW software was done to identify other possible motifs clustering these peptides, explaining their association to DR4, but no common binding motif was found.

Comparative analysis of the peptide repertoires. 2. Influence of Ii and DM in the localization of source proteins

To asses the influence of Ii and DM in the origin of peptides constituting the repertoires, the localization of the source proteins generating DR4 ligands was analyzed. Proteins were grouped according to their cellular localization and their accessibility to the class II biosynthetic pathway in four categories: exogenous proteins; cell surface and endocytic proteins were grouped together, due to their similar accessibility; secretory; and cytoplasmic (including cytosolic, nuclear, ER, and Golgi residents).

Some tendencies could be detected for each repertoire (Fig. 4). A common feature was the dominance of ligands derived from self-proteins as opposed to exogenous (serum) proteins although single transfectants were the only cells from which exogenous peptides could not be sequenced. In DR4IiDM eluates, peptides from cell surface and endocytic proteins maintained the highest proportion (44%, 17 of 39), with cytoplasmic peptides representing 36% (14 of 39), of the whole repertoire. Natural ligands eluted from DR4DM cells, all self-peptides but for two exogenous epitopes, maintained the distribution: 39% (7 of 18) from surface and endocytic proteins and 33% (6 of 18) of cytoplasmic origin. In DM–Ii+ cells (DR4Ii), non-CLIP peptides identified included exogenous peptides (9%, 1 of 11), surface and endocytic peptides (45%, 5 of 11), and a maintained proportion of cytoplasmic peptides (45%, 5 of 11) but not peptides from the secretory pathway. In the DR4 single transfectant, 83% (10 of 12) of all DR4 ligands derived from ubiquitous cytoplasmic nonendocytic proteins, including ER resident, cytosolic, mitochondrial, and nuclear enzymes. Of the remaining peptides, 8% (1 of 12) derived from cell surface molecules and one from a secretory protein (8%). No exogenous peptides were identified.



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  		FIGURE 4. Comparison of the cellular localization of proteins generating DR4-associated peptides in the DR4IiDM, DR4DM, DR4Ii, and DR4 transfected cells. Bars represent the percentage of peptides from proteins of different location in the DR4 repertoires of each of the transfectants. The analysis was done on peptides derived from proteins of exogenous (), surface and endocytic ({blacksquare}), secretory (), and cytoplasmic () origin. Cell surface and endocytic proteins were grouped together because both can equally encounter DR dimers in the endocytic pathway. Cytoplasmic proteins included those from the cytosol, nucleus, ER, and GA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ectopic expression of MHC II has been described in the epithelial targets of some autoimmune diseases. The influence of this expression in in vivo autoimmune responses is not clear, but some autoepitopes are uniquely processed and presented by the endocrine cells to CD4+ T cells, what may explain their specific destruction. The relative levels of expression of Ii and DM are maintained in professional APCs (18, 31), whereas class II+ epithelial cells show variable levels of MHC II, Ii, and DM, particularly in the context of autoimmunity and tumor immunity (7, 21, 22). These variations are expected to influence the repertoire of peptides presented by the epithelial cells. In chronic inflammatory situations, this could be an important factor affecting epitope spreading and crypticity and influencing the disease development, although the actual repertoire used by class II molecules has not been analyzed.

We have studied the Ii and DM influence on the peptide repertoires associated to DR4 molecules expressed by an epithelial cell line of neuroendocrine origin. The sequences here presented are limited and may only represent a small fraction of the peptide pools associated to DR4, clearly the most abundant within the detecting capacity of our system. However, the same limitations apply to the four cells compared, so the differences we find between the cells are not expected to be due to technical bias.

HLA-DRB1*0401 is a DR allele of very low intrinsic stability, which makes it highly dependent on HLA-DM, requiring the binding of an appropriate ligand to prevent its denaturation (14). In a previous report, we showed that DM expression was enough to confer SDS resistance to DR4 in the absence of Ii, whereas in DM– cells, surface DR4 molecules were mostly SDS-sensitive (23). In this study, we demonstrate this Ii-independent editing effect of DM, showing clear similarities between the repertoires of DM+ cells in comparison to the repertoires of DM– cells. In addition, all peptides from DM+ cells fitted the DR4-binding motif and aromatic and negatively charged residues were respectively favored in P1 and P4 anchor positions, more than in peptides of the DR4-associated repertoires from DM– cells.

The class II peptide-binding groove balances between a receptive or "open" stage and a more "closed" conformation (32, 33) and DM-DR binding favors the open conformation (34). In DM+ cells, this would facilitate the occupation of P1 by aromatic residues and may expose the positively charged K in position 71 of pocket 4 (35), thus favoring the interaction with negatively charged amino acids, more abundant in DM+ pools. The importance of the P1 anchor position has been previously described by studies on peptide libraries (36) but not in studies of natural class II repertoires, and could be related to the fact that P1 is located near the DR-DM interaction site (37).

The selection of ligands by DM was also related to their availability in the relevant compartment (38). Aligning all peptide-length variants, an even more restricted pattern of P1 residue distribution was observed in DM+ ligands (Fig. 3), with dominance of Y (51%) over F (30%). This pattern could be cell type-dependent because HLA-DM editing on DR4 molecules from lymphoblastoid B cell lines appeared to select peptides having Val in P1 (30, 39) instead of the dominant Y of epithelial cells (8).

Expression of Ii in the absence of DM did not significantly limit the number of DR4 ligands but shifted the repertoire in favor of dominant CLIP sequences. Both CLIP forms identified contained the N-terminal residues 81–89, which mediate the spontaneous release of CLIP peptides (40). The non-CLIP peptides consisted of a heterogeneous pool of poorly represented peptides, some of which did not fit the DR4 motif. The surface expression of SDS-sensitive DR4 complexes in the DRIi and DR cells suggested that DR4-peptide cargo could occur at low stringency, so nonendocytic compartments such as the ER or the cell surface, cannot be ruled out (41, 42).

DR4 molecules expressed alone were associated to a very unconventional peptide repertoire with single peptides derived from nonendocytic cytoplasmic proteins corresponding mostly to N- or C-terminal sequences that did not respect the DR4-binding motif or had low theoretical affinity to DR4 (27). Only one peptide from this repertoire belonged to a surface protein, that could be able to bind recycling DR4 in the endocytic pathway, independently of Ii and DM expression (43, 44, 45). However, the absolute majority (83%) of the peptides derived from proteins of cytoplasmic origin that most likely do not use the endolysosomal pathway for their degradation. This suggests that these peptides may be generated or loaded in compartments other than MHC II-containing compartments where distinct chaperones and a different physicochemical milieu may influence ligand availability, binding-groove conformation, and sequence requirements. Accumulation of DR4 molecules in ER-Golgi shown in these cells by confocal microscopy (Ref.23 and data not shown) point to this compartment as a likely site for cytosolic peptide loading in the absence of Ii and DM. To meet DR4 dimers, peptides would need to be translocated either to the ER by cytosol-ER peptide channels such as TAP or Sec61, or to the endocytic pathway by autophagy or selective protein-import channels (46). Furthermore, many of the sequences corresponded to C or N termini of the source protein which could not be related to any known endocytic or cytosolic protease activity. Protection of protein termini before and during proteasome cleavage by chaperones could be an explanation for the survival of such peptides.

Peptides from different compartments of the cytoplasm were found in all repertoires, independent of the expression of chaperones as a specific feature of epithelial cells, compared with constitutively expressing DR4+ cells (47). Ii and DM expression also influenced the origin of the peptides constituting the DR4 pools. In their absence, DR4 was mostly associated to peptides from cytosolic proteins (83%). The expression of Ii alone forced the association of CLIP to DR4 but also of some non-CLIP peptides derived from exogenous, membrane, or cytosolic proteins (DR4Ii vs DR4 repertoires). This agrees with a partial switch of the peptide loading location from the ER to the endocytic pathway accessible to exogenous peptides and with the reported DM-independent loading in early endosomes that occurs after receptor-mediated endocytosis of some Ags (43, 44). DM expression alone resulted in a very similar composition of the pool with respect to peptide origin, cell surface and endocytic, nonendocytic cytoplasmic peptides, exogenous peptides, and a number of peptides from the secretory pathway. However, the sequences from the DR4DM cell pool were very different from the other transfectants, with the exception of peptides from the triple transfectants, again indicating a role for DM in sequence edition. All repertoires, except the CLIP-dominated DR4Ii pool, had a similar proportion of peptides from tissue-restricted secreted proteins that are cell type-specific. This is important in the view that most described autoantigens in organ-specific autoimmunity are tissue-specific molecules from the secretory pathway (i.e., glutamic acid decarboxylase, insulin, and IA-2 in type-1 diabetes; thyroid peroxidase in thyroid-specific autoimmunity). The reported variability in the relative expression of MHC II, Ii, and DM (21) in autoimmune tissues has been related to the maintenance of autoimmune responses (22). Our data suggest that low or absent DM expression can prevent the presentation of dominant epitopes, and lead to the display of heterogeneous repertoires, presumably able to stimulate nontolerant T cell pools. Low stringency in DM-peptide editing due to low DM expression may allow new or cryptic endogenous epitopes to be presented at the cell surface of endocrine cells.

In summary, the relative expression of class II, Ii, and DM determine the composition of the natural peptide repertoires associated to class II in endocrine epithelial cells. Therefore, a more accurate knowledge of their expression in target organs of autoimmune diseases is crucial 1) to identify epithelial cell-specific peptides, 2) to understand the generation and maintenance of these autoepitopes, and 3) to provide new ways of developing effective cellular vaccines in autoimmunity.


   Acknowledgments
 
We thank Drs. P. Benaroch, P. Cresswell, G. Hammerling, P. Jensen, W. Knapp, C. Navarrete, P. Morton, P. Roche, and J. Trowsdale for their generous gifts of Abs and Dr. J. López de Castro for critically reading the manuscript.


   Footnotes
 
1 This work was supported by Comision Interministerial de Ciencia y Tecnologia Grant SAF00-0131-C02-01 from the Spanish Science and Technology Ministry and in part by Fundació La Marató de TV3 Grant 00-4832-3. I.A. is a fellow of the Training of Researchers Program of the Basque Government. Back

2 Address correspondence and reprint requests to Dr. Dolores Jaraquemada, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. E-mail address: Dolores.Jaraquemada{at}uab.es Back

3 Abbreviations used in this paper: MHC II, class II MHC; Ii, invariant chain; ER, endoplasmic reticulum; MS, mass spectometry; MFI, mean fluorescence intensity. Back

Received for publication September 22, 2003. Accepted for publication May 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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