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Editing of an Immunodominant Epitope of Glutamate Decarboxylase by HLA-DM¹

John D. Lich^2,*, Jennifer A. Jayne^3,*, Delu Zhou^*, John F. Elliott and Janice S. Blum^4,*

^* Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, and Walther Cancer Institute, Indianapolis, IN 46202; and Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada

	Abstract

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HLA-DM stabilizes peptide-receptive class II dimers and facilitates the capture of high affinity peptides, thus influencing the peptide repertoire presented by class II molecules. Variations in DM levels may therefore have a profound effect on the antigenic focus of T cell-mediated immune responses. Specifically, DM expression may influence susceptibility and resistance to autoimmune diseases. In this study the role of DM in HLA-DR4-restricted presentation of an insulin-dependent diabetes mellitus autoantigen, glutamate decarboxylase (GAD), was tested. Presentation of immunodominant GAD epitope 273–285 was regulated by endogenous DM levels in human B lymphoblasts. T cell responses to exogenous GAD as well as an endogenous cytoplasmic form of this Ag were significantly diminished with increasing cellular expression of DM. Epitope editing by DM was observed only using Ag and not small synthetic peptides, suggesting that this process occurred within endosomes. Results with cytoplasmic GAD also indicated that peptides from this compartment intersect class II proteins in endocytic vesicles where DM editing was facilitated. Changes in DM levels within APC may therefore influence the presentation of autoantigens and the development of autoimmune disorders such as type I diabetes.

	Introduction

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Activation of Ag-specific CD4⁺ Th cells requires the presentation of immunogenic peptides in the context of MHC class II proteins on the surface of APC. MHC class II heterodimers are assembled within the endoplasmic reticulum in association with the type II glycoprotein invariant chain (Ii)⁵ preceding delivery to acidic, post-Golgi vesicles (1, 2). During this early stage, MHC class II dimers are prevented from binding immunogenic epitopes becaues their peptide binding pocket is occupied by a central region of Ii, termed class II-associated Ii peptide (CLIP) (3). MHC class II maturation proceeds as acidic proteinases systematically cleave Ii, liberating the dimers (4, 5, 6). HLA-DM functions in the terminal stages of class II maturation by catalyzing the removal of CLIP and the subsequent capture of peptide epitopes by MHC class II proteins (7). Peptide binding to class II in the presence of DM is selective, such that peptides that form high stability complexes with class II proteins are displayed at the cell surface for inspection by surveying CD4⁺ T cells (8).

The function of HLA-DM was realized based on its ability to restore wild-type Ag processing and MHC class II-restricted presentation in mutant APC. While overall surface class II protein levels are not significantly reduced in these cells, the majority of surface dimers still posses CLIP within the peptide-binding groove (9). Moreover, due to their weak association with the CLIP peptide, these dimers are unstable in SDS under nonreducing conditions (10). Introduction of cDNA encoding DM and DM corrected these defects, reducing surface :CLIP and dramatically increasing the stability of isolated (11, 12). DM functionality was later confirmed in APC derived from mice defective in DM expression, which also exhibit a dramatic increase in class II-associated CLIP at the cell surface (13). Studies suggest that DM may regulate epitope immunodominance, allowing the immune response to focus upon a limited, but highly stable, set of peptides presented in the context of MHC class II molecules (14). In addition, within the thymus, T cells undergo positive and negative selection based upon the display of a repertoire of self-peptides, again influenced by the editing function of DM (15). Thus, variations in DM expression within the thymus or in the periphery may regulate class II-restricted presentation of epitopes, potentially leading to the activation of autoreactive T cells (16, 17). In this respect the importance of HLA-DM in autoimmunity remains controversial, with human genetic studies arguing both for and against a role in disease susceptibility and resistance (18, 19, 20, 21, 22, 23, 24, 25, 26).

In the present study the role of DM in guiding HLA-DR4-restricted presentation of an immunodominant epitope from the diabetes autoantigen glutamate decarboxylase (GAD) was examined. The immunodominance of the GAD_273–285 epitope has been demonstrated in DR4 transgenic mice as well as through studies revealing the presence of CD4⁺ T cells specific for this epitope in human insulin-dependent diabetes mellitus (IDDM) patients (27, 28). HLA-DR4-restricted presentation of this ligand was severely reduced in B lymphoblastoid cell lines (B-LCL) expressing high levels of DM, while cells expressing low DM levels efficiently presented GAD_273–285. This phenomenon persisted whether the epitope was derived exogenously or originated within the cytoplasm of the APC. Prior studies have suggested that additional MHC-encoded genetic factors aside from classical HLA-DR and DQ alleles may influence the presentation of disease-linked GAD epitopes (29). The evidence offered here suggests that in humans, variations in HLA-DM expression may indeed regulate T cell responses to this diabetes autoantigen.

	Materials and Methods

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Cell lines and Ag

B-LCL Priess, JAH, and BSM are homozygous for the expression of HLA-DR4 (DRA1*0101, DRB1*0401), while B-LCL Frev and Frev.pa express HLA-DR4 and DR1 (DRA1*0101, DRB1*0401, and DRB1*0101) alleles. The line Frev.pa was isolated by subcloning and expresses lower steady state levels of intracellular DM compared with Frev (15). Retroviral transduction of the parental cell line Priess with the gene encoding the 65-kDa form of human GAD resulted in a cell line constitutively expressing this endogenous Ag, termed PriessGAD (30, 31). To obtain APC with high levels of functional DM, PriessGAD cells were transfected with cDNAs encoding DM and DM (L. Denzin, Sloan Kettering, NY). Stable DM⁺ transfectants were obtained following APC selection for DM in 0.5 µg/ml puromycin (Sigma-Aldrich, St. Louis, MO) and DM in 0.8 µg/ml hygromycin (Sigma-Aldrich). T2 is a BxT cell hybrid with a large deletion in the MHC class II locus and does not express endogenous class II proteins (32). The cell lines T2.DR4 and T2DR4.DM expressing functional class II DR4 or DR4 and DM have been described, and DR4 expression on all APC was determined by FACS using Ab 3.5.9-13F10 (32, 33). All APC were maintained in Iscove’s medium (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. For functional assays, the following T cell hybridomas were used: 33.1, specific for GAD_273–285; 17.9, specific for human serum albumin (HSA)_64–76; and 2.18, specific for Ig_188–203 (33, 34, 35). The T cell hybridomas used in this study recognize their cognate peptide presented in the context of HLA-DR4 and were maintained in RPMI (Life Technologies) supplemented with 10% FCS, 0.1% -ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. Control studies with the class II-negative cell line, T2, confirmed the DR4 restriction of these hybridomas.

The recombinant 65-kDa form of human GAD was produced in bacteria and was purified to homogeneity using affinity chromatography as previously published (36). GAD_273–285 (IAFTSEHSHFSLK) was synthesized using an ABI peptide synthesizer (PE Applied Biosystems, Foster City, CA) and F-moc technology. Peptide purity and structure were confirmed by reverse phase HPLC and mass spectroscopy.

Subcellular fractionation and immunoblotting

To localize intracellular GAD, HLA-DR, and HLA-DM, membrane organelles were isolated from APC as previously described (31, 37) and assayed by Western blot analysis. For each sample, equal amounts of protein were either boiled in reducing sample buffer or incubated at room temperature in nonreducing sample buffer and separated by 10% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane (Micron Separations, West Brook, MA) and probed with the mAbs anti-GAD65 (Sigma-Aldrich), DM.K8 recognizing DM (38) (A. Vogt, Roche Institute, Basil, Switzerland), DA6.147 specific for DR, the rabbit polyclonal anti-DM, or a rabbit polyclonal anti-DO (E. Mellins, Stanford, CA). Filter-bound Abs were visualized using goat anti-mouse HRP or goat anti-rabbit HRP (Jackson ImmunoResearch Laboratories, West Grove, PA), followed by epichemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL). Exposure times for chemiluminescence were varied to ensure accurate and quantitative signal detection.

Acid elution of peptides from surface MHC class II

APC were washed twice in ice-cold 160 mM NaCl then resuspended in 160 mM NaCl/citric acid, pH 4.0, and incubated 30 min on ice. These cell preparations were neutralized with ice-cold medium and subsequently washed twice with fresh tissue culture medium. No loss in cell viability was observed using these conditions, and APC retained their ability to activate T cells upon addition of antigenic peptides.

Ag presentation assays

For exogenous Ags, APC were incubated with or without Ag for 18 h at 37°C. GAD Ag was tested over a range of concentrations (1–50 µg/ml) or the fixed amount of 20 µg/ml, while HSA Ag was used at 250 µg/ml. Control samples were incubated for 16 h without Ag, then pulsed with 10 µM GAD_273–285 and incubated for an additional 2 h. The remainder of the assay was conducted as described previously (31). All T cell assays were performed in triplicate with the mean and SD calculated for each data point. All figures are representative of a minimum of three independent experiments.

	Results

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Editing of a class II-restricted epitope from the IDDM autoantigen GAD

T cells responsive to the islet cell Ag GAD are detectable in IDDM patients as well as transgenic mice expressing disease-associated DR and DQ alleles (28, 39). Studies of human T cells responsive to GAD have proven difficult, potentially due to a number of factors, including low T cell precursor frequencies, the insolubility of this protein, and genetic variability in APC. With respect to the latter, the efficiency of HLA-DR4-restricted presentation of exogenous GAD was shown to vary widely among a panel of B-LCL, suggesting that genetic differences might guide GAD epitope display in humans and contribute to disease susceptibility (29). Here, the relative importance of the MHC-encoded peptide editor, HLA-DM, on the efficiency of GAD epitope presentation by APC was assessed using functional assays (Fig. 1). Class II presentation of the epitope GAD_273–285 derived from exogenous GAD was significantly reduced in the DM-positive APC T2.DR4.DM compared with T2.DR4 cells lacking this editor (Fig. 1, A and B). The enhanced display of naturally processed GAD_273–285:class II complexes on T2.DR4 was particularly striking, as nearly 80% of the cell surface class II molecules on these cells are loaded with the Ii-derived CLIP ligand and are inaccessible to the majority of peptides generated during Ag processing (8, 9). The expression of HLA-DM in T2.DR4 cells results in the editing of unstable CLIP-class II complexes (32), suggesting that GAD_273–285 may also be susceptible to DM-mediated release from class II DR4. Differences in GAD epitope presentation were not linked to cell surface class II levels on APC^+/- DM, as flow cytometry revealed greater amounts of DR4 displayed on T2.DR4.DM (mean channel fluorescence, 149.2) compared with T2.DR4 cells (mean channel fluorescence, 77.8). To confirm that both T2.DR4 and T2.DR4.DM cells can function to activate GAD-specific T cells, these APC were incubated with the synthetic GAD_273–285 peptide (Fig. 1C). Indeed, HLA-DR4-restricted presentation of this synthetic peptide was readily detected using each APC, although T2.DR4.DM cells appear to present the synthetic GAD peptide better, possibly due to higher levels of surface DR4. The requirement for DM in generating accessible class II molecules through CLIP release has been widely documented in studies of a large number of Ags (12, 32, 40). Indeed, presentation of the exogenous Ag, HSA, was impaired in T2.DR4 cells compared with that in DM-positive cells (Fig. 1D). These results suggest that, like CLIP, an epitope derived from GAD can be released from DR4 in the presence of DM. In addition, editing of this GAD epitope was only observed following intracellular Ag processing, as the display of surface-loaded synthetic GAD_273–285 was not diminished with increasing DM levels.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. HLA-DR4-restricted presentation of exogenous GAD is diminished in the presence of DM. T2.DR4 and T2.DR4.DM cells were incubated for 18 h in the presence 20 µg/ml GAD (A), variable concentrations of GAD (B), or HSA Ag (D). C, APC were pulsed for 2 h with increasing concentrations of synthetic GAD273–285. Following these incubations, APC were plated with the appropriate T cell hybridomas (33.1 specific for GAD and 17.9 specific for HSA), and DR4-restricted presentation was determined as described in Materials and Methods. The data shown are representative of at least three individual experiments.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. HLA-DR4-restricted presentation of exogenous GAD differed among human B-LCL and was correlated with DM expression. A, APC were incubated for 18 h in the presence of GAD Ag, then cocultured with the T cell hybridoma, 33.1. HLA-DR4-restricted presentation of GAD273–285 was quantitated as described in Materials and Methods. Relative surface expression of HLA-DR4 on each APC was determined by flow cytometry: BSM, 110.39 mean channel fluorescence (mcf); JAH, 93.21 mcf; Priess, 147.59 mcf; Frev, 115.40 mcf; Frev.pa, 332.15 mcf. B–D, Quantitation of steady state levels of HLA-DM and DO. B-LCL lysates were analyzed on 10% PAGE, transferred to membranes for Western blotting, and probed with the DM-specific mAb DMK.8 (B and D) or an Ab to DO (C and D). Steady state levels of DM and DO expression were expressed as relative intensity units (RIU) for each cell line, as determined by densitometric analysis. These results are representative of three distinct experiments.

Reduced presentation of the GAD epitope following processing within APC expressing abundant DM suggested that this peptide forms an unstable complex with class II DR4. The low affinity of this GAD epitope for DR4 has been established (30). Previous studies have demonstrated that unstable class II ligands such as CLIP can be displaced by HLA-DM or upon exposure of such peptide:class II complexes to low pH (8). B cells that had been preloaded with synthetic GAD peptide 273–285 and then exposed to low pH, compared with APC incubated at neutral pH, exhibited a diminished capacity to activate T cells specific for this immunodominant epitope (Fig. 3A). These results suggested that GAD_273–285, like CLIP, forms an unstable complex with MHC class II molecules. PriessGAD cells constitutively express high levels of intracellular GAD and present the immunodominant epitope GAD_273–285 in the context of HLA-DR4. A decrease in GAD-specific T cell activation was also observed following acidic pH incubation and aldehyde fixation of these APC expressing endogenous Ag (Fig. 3, A and B). Aldehyde fixation of these APC after low pH peptide stripping was required, as live PriessGAD cells within a matter of hours, generate newly formed GAD_273–285:DR4 complexes that transit to the cell surface (Fig. 3B). Acidic elution of peptides from cell surface class II molecules was also examined using an epitope known to form high affinity, stable complexes with HLA-DR4. As predicted, an epitope derived from the endogenous Ag Ig that binds to HLA-DR4 was found to be resistant to low pH dissociation (Fig. 3B). Prior studies using in vitro binding and dissociation assays have established the formation of high affinity, stable complexes of this peptide and DR that are not susceptible to DM (42). These latter studies suggest that acid treatment of cell surface class II dimers did not radically alter MHC structure or trigger indiscriminant release of ligands. Both the synthetic and naturally processed GAD_273–285 were susceptible to elution from DR4 under low pH conditions. These results support the formation of unstable or loosely bound GAD peptide:class II complexes in APC that may serve as a target for DM-mediated editing.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Instability of GAD epitope complexed with DR4 at very low pH. A, Priess or PriessGAD cells treated with the GAD273–285 synthetic peptide were incubated in ice-cold neutral or acidic pH saline buffer for 30 min, followed by washing to remove released class II ligands. The cells were then paraformaldehyde fixed and cocultured with the GAD-specific T cell hybridoma 33.1 as described in Materials and Methods. B and C, In a separate experiment, PriessGAD cells were incubated in ice-cold acidic pH saline buffer for 30 min to promote unstable peptide dissociation. Samples were divided and either paraformaldehyde-fixed or untreated before coculture with the GAD-specific T cell 33.1 (B) or the Ig-specific T cell 2.18 (C). Representative results are shown from three distinct experiments. Data are expressed as the mean of triplicate samples analyzed with the SE indicated.

Class II MHC proteins have the capacity to present peptide ligands derived from both exogenous and endogenous Ags (43, 44). While DM functions to enhance class II accessibility for most Ags, select endogenous proteins are presented even in the absence of DM (33). Epitopes derived from these endogenous Ags may intersect peptide-receptive class II complexes within specialized sites in the endo/lysosomal pathway (45). Little is known about the mechanisms regulating cytoplasmic peptide presentation by class II molecules, although studies have demonstrated that cytoplasmic processing is a prerequisite for the display of these antigenic epitopes (31). To determine whether complexes of cytoplasmic peptides and class II intersect DM and undergo selective editing, the presentation of epitopes derived from cytoplasmic GAD was examined in cells expressing high or low levels of DM. PriessGAD cells expressing cytoplasmic GAD were transfected with HLA-DM and subclones isolated with increased DM levels. Western blot analysis of the cells PriessGAD and PriessGAD.DM confirmed the enhanced expression of DM and DM in the latter cell line (Fig. 4A). Notably, the low expression of DM in the parental PriessGAD cell line was functionally demonstrated by an abundance of class II dimers exhibiting instability under nonreducing SDS-PAGE (Fig. 4B). Enhanced expression of DM resulted in an increase in SDS-stable dimers in the PriessGAD.DM cells (Fig. 4C). The ability of HLA-DM to enhance the formation of stable peptide:class II complexes in cells has long been shown to correlate with the appearance of SDS-stable class II dimers on PAGE (10, 12). Remarkably, the enhanced expression of DM also correlated with a significant reduction in HLA-DR4-restricted presentation of cytoplasmic-derived GAD_273–285 (Fig. 5). This reduction occurred even though intracellular GAD protein levels remained equivalent in the two cell lines (Fig. 4D). The addition of exogenous GAD peptide to these cells resulted in similar levels of T cell activation, in line with flow cytometric data indicating similar levels of DR4 expression by these cells (data not shown). Together these data suggest that DM encounters and edits DR4 molecules complexed with GAD_273–285 regardless of the route of Ag delivery. Furthermore, these results suggest that cytoplasmic-derived epitopes may access class II molecules in the absence of DM, yet the resulting peptide: complexes later can undergo DM-meditated editing before display on the cell surface.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Overexpression of HLA-DM in APC enhances the formation of SDS-stable class II:peptide complexes. Western blot analysis was used to evaluate relative HLA-DM, DR, and GAD levels in PriessGAD and PriessGAD.DM cells. Membrane fractions from PriessGAD and PriessGAD.DM were solubilized, and equal amounts of protein from each were fractionated by SDS-PAGE followed by Western immunoblotting. A, C, and D, Samples prepared in reducing SDS sample buffer and boiled for 5 min were probed for DM and DM, DR, or GAD levels. B, Samples prepared in nonreducing SDS sample buffer and incubated for 15 min at room temperature were probed for SDS-stable class II dimers and dissociated DR. The Ab used here, DA6.147, preferentially recognizes free DR compared with dimerized DR, thus accounting for the ready detection of dissociated DR in cells lacking DM. In all cases identical amounts of cell proteins were loaded in each lane, separated on 10% PAGE, and transferred to membranes, and Ab were probed. Each blot is representative of at least three independent experiments.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. DM-mediated inhibition of cytoplasmic GAD presentation by HLA-DR4. PriessGAD or PriessGAD.DM cells were cultured in fresh medium with or without the GAD273–285 peptide for 2 h before washing and were cocultured with the GAD-specific T cell hybridoma, 33.1. Ag-specific activation of T cell hybridoma lines was measured as described in Materials and Methods. Representative results are shown from three separate experiments, with the data expressed as the mean of triplicate samples and the SE indicated.

	Discussion

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Analysis of peptides eluted from APC with and without DM indicates that this editor alters the repertoire of self-peptides presented by class II proteins (7, 52). The expression of certain class II alleles in combination with variable DM levels may therefore influence the development and progression of autoimmune disease. T cells reactive to epitopes containing the core sequence GAD_273–285 have been isolated from individuals with IDDM (27) with differential presentation of this and other GAD epitopes reported using APC from healthy individuals and diabetes patients (16, 29). The results presented here strongly suggest that the steady state levels of DM within APC may be a key factor regulating the presentation of this autoantigen. HLA-DM is constitutively expressed by B cells, although lymphocyte activation can result in reduced levels of this MHC heterodimer (53). Interestingly, B cells derived from rheumatoid arthritis patients have recently been shown to express reduced amounts of DM compared with cells derived from nonautoimmune arthritis patients (20). However, conflicting reports argue for and against a linkage between specific HLA-DM alleles and susceptibility to rheumatoid arthritis (20, 21, 22, 23, 24, 25, 26) and other autoimmune disorders. For instance, HLA-DM expression has been reported to be up-regulated in Graves’ disease (54). Far less is known concerning the role of DM in type I diabetes, although studies suggest genetic association due to DM allelic polymorphisms (18, 19). DM has also been shown to edit an epitope from myelin basic protein, a target autoantigen in multiple sclerosis (55), although analysis of patients has not revealed a genetic correlation (56). Thus, further studies to evaluate how DM expression and polymorphisms influence autoimmune disease in humans are needed.

DM’s function has been shown in part to be regulated by the MHC-encoded molecule, HLA-DO. Unlike DM, DO exhibits a more limited tissue distribution as it is expressed by B cells and thymic epithelial cells and has been localized to some of the same intracellular compartments as DM (57). Inhibition of DM editing by DO occurs at neutral or mildly acidic pH, and only the presentation of some Ag was altered in mice lacking DO (58). This has led to the suggestion that in compartments such as MIIC, DM may function independently of DO. In this study DO expression was variable, but did not correlate with GAD epitope presentation. Exogenous GAD presentation by Frev and Frev.pa cells, which express nearly identical levels of DO, was linked only to changes in steady state DM. Similarly, PriessGAD and the DM-overexpressing PriessGAD.DM cells differentially present GAD epitopes, yet have similar levels of intracellular DO (data not shown). Together these results suggest that GAD epitope 273–285 binds class II molecules, and these complexes intersect DM in compartments where DO may not be functional. These results are also consistent with our previously published work demonstrating that exogenous GAD is processed in mature acidic endosomal or lysosomal compartments (31).

MHC class II-restricted presentation of cytoplasmic Ags is well established (33, 59, 60). However, the precise processing pathways followed by the majority of these endogenous Ags remain a mystery. In the B lymphoblastoid cell line PriessGAD, which constitutively expresses endogenous glutamate decarboxylase, the naturally processed epitope GAD_273–285 is efficiently presented by HLA-DR4 class II proteins to Ag-specific T cells (30, 31). Endogenous GAD produced by these cells is processed by cytoplasmic proteases such as calpains and the proteosome before epitope presentation in the context of class II molecules (31). Inhibitor studies also suggested that fragments of cytoplasmic GAD may be cleaved by acidic cysteine proteases; however, it remained unclear whether this occurred in conventional endosomal/lysosomal compartments before or after peptide binding to HLA class II molecules. Here the observation that DR4-restricted presentation was ablated when DM levels were increased in APC suggested that, like exogenous GAD, peptides derived from a cytoplasmic Ag bind class II DR and transit to DM-containing endosomes. DM is known to accumulate predominantly within a compartment termed the MIIC (61) where the majority of peptide binding to conventional class II molecules takes place (60, 62). In addition, DM has been found to function at the cell surface (55). However, equivalent presentation of synthetic peptides pulsed onto T2.DR4 cells in the presence or the absence of high levels of DM suggest that DM does not edit this epitope at the cell surface. A requirement for DM in the class II-restricted presentation of cytoplasmic Ags has been previously reported (15). Yet, this effect was most likely due to DM’s role in CLIP release and the formation of peptide-receptive class II molecules. The results presented here show directly that cytoplasmic-derived peptides upon binding class II proteins undergo DM-editing reactions identical with those of epitopes from exogenous Ag-processing pathways.

	Acknowledgments

 
We thank Heather A. Bruns, Alicia Cecil, Sheila Jackson, Ping Li, M. Azizul Haque, Patrick O’Donnell, and James Walsh for their help and encouragement.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI33418 and AI49589 (to J.S.B.), T32DK07519 (to J.D.L.), and the Indiana University Diabetes Center (to J.A.J. and D.Z.).

² Current address: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599.

³ Current address: Department of Biology, Southern Utah University, Science Center 105, Cedar City, UT 84720.

⁴ Address correspondence and reprint requests to Dr. Janice S. Blum, Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, MS 420, Indianapolis, IN 46202. E-mail address: jblum{at}iupui.edu

⁵ Abbreviations used in this paper: Ii, invariant chain; B-LCL, B lymphoblastoid cell line; CLIP, class II-associated Ii peptide; GAD, glutamate decarboxylase; HSA, human serum albumin; IDDM, insulin-dependent diabetes mellitus.

Received for publication August 1, 2002. Accepted for publication May 15, 2003.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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