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Original Article |
jblum{at}iupui.edu
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Key Words: proteasome • calpain • cathepsin • cytoplasm • protease
Peptides derived from extracellular or exogenous antigens are generated by processing reactions localized within a complex network of endosomal and lysosomal vesicles 11. Antigen denaturation by the acid pH within these organelles coupled with the action of resident endopeptidases and carboxypeptidases appears to be a key step in the generation of ligands for MHC class II complexes. Chief among these proteases are the cathepsins with acidic pH optima and a broad range of substrate specificity 12. These include the cysteine proteases cathepsins B, H, S, and L and the aspartic hydrolases cathepsins D and E. Cathepsins have been implicated in the processing of a variety of exogenous antigens for class II–restricted presentation 1314. By contrast, much less is known concerning the enzymes required for endogenous antigen processing. Nascent class II proteins are associated with an intracellular chaperone protein, the invariant chain (Ii), which must be proteolytically processed before peptide binding by these MHC antigens. Ii serves a dual purpose in protecting the ligand-binding groove of class II complexes, as well as guiding class II molecules to endocytic compartments rich in antigenic peptides. Studies of Ii processing may provide some insights into the processing environment encountered by endogenous antigens within endosomes and lysosomes, as both cysteine and aspartic cathepsins have been implicated in Ii degradation 141516. Studies using mice genetically targeted to inactivate cathepsin S suggest this protease functions at a late stage of Ii processing within professional APCs, whereas cathepsin L appears important in catalyzing this terminal reaction within thymic cells 17. Thus, endogenous antigens trafficking through endosomes may likely encounter similar proteases to those functioning in the processing of exogenous proteins. Remarkably, endogenous cytoplasmic antigens also access the class II presentation pathway, as shown by class II loading with epitopes derived from resident cytoplasmic proteins 18. It remains unclear whether acidic cathepsins or cytoplasmic proteases, optimally active at neutral pH, are required for processing cytoplasmic antigens for MHC class II–restricted presentation.
This study addresses the requirements for processing and MHC class II–restricted presentation of the autoantigen glutamate decarboxylase (GAD) derived exogenously, as well as from its natural cytoplasmic environment. GAD has been localized to the cytoplasmic face of membrane organelles in neuroendocrine cells, and this enzyme represents a key target autoantigen in several autoimmune disorders, including insulin-dependent diabetes mellitus (IDDM [19]). Our data demonstrate divergent antigen processing pathways for exogenous and cytoplasmic GAD. Although both exogenous and endogenously derived GAD access endosomal/lysosomal proteases, a distinct cytoplasmic processing pathway was identified for GAD originating in the cytosol. Thus, presentation of cytoplasmic-derived GAD required the function of a cytoplasmic Ca2+-dependent protease, calpain, and the multicatalytic proteasome. These results demonstrate that processing reactions within the cytoplasm govern the class II–restricted presentation of a subset of endogenous antigens. These studies also revealed that after translocation from the cytoplasm into membrane organelles, cytoplasmic antigens are further processed by endosomal/lysosomal proteases before presentation by class II complexes.
Inhibitors, Purified Antigen, and Synthetic Peptides.
Electroporation of Antibody into APCs.
Subcellular Fractionation and Immunoblotting.
Elution of Peptides from Surface MHC Class II.
Antigen Presentation Assays.
Introduction
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Abstract
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Materials and Methods
Results
Discussion
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MHC class II molecules selectively present peptides derived from self- and foreign antigens to CD4+ T cells, providing a physiological survey of host integrity. Biochemical studies to define the source of antigenic peptides associated with class II molecules have revealed a predominance of peptides derived from exogenous antigens, as well as epitopes from the plasma membrane and endosomal proteins 12. Such ligands would predictably intersect class II protein in transit to the cell surface via the endosomal pathway 34. By contrast, the observation that peptides derived from cytoplasmic antigens are also presented by class II proteins appears more difficult to reconcile. Epitopes from both soluble and membrane-bound proteins localized within the cytoplasm have been observed to be efficiently presented by class II complexes using human and murine APCs 56. Furthermore, studies suggest that some viral epitopes derived from cytoplasmic antigens gain access to class II proteins for presentation 78. Cytoplasmic antigens have long been established as the primary source of peptides for MHC class I molecules, although studies suggest that in specialized APCs, class I molecules may access proteins within endosomal/phagosomal compartments 910. Likewise, class II proteins may have evolved mechanisms to sample antigenic peptides within the cytoplasm, potentiating helper T cell responses to intracellular pathogens and the maintenance of self-tolerance 6.
Materials and Methods
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Materials and Methods
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Cell Lines.
The B lymphoblastoid cell line (B-LCL) Priess homozygous for the expression of HLA-DR4 (DRA*0101, DRB*0401) was maintained in Iscove's medium (GIBCO BRL) supplemented with 10% heat-inactivated calf serum. Retroviral transduction of the parental cell line Priess with the gene encoding the 65-kD form of human GAD resulted in a cell line constitutively expressing this endogenous antigen, termed PriessGAD. Endogenous GAD expression levels were equivalent using retroviral gene transduction or standard transfection methods with GAD expressed in the pMCFRpacGAD vector 20. Conditioned medium was generated from PriessGAD cells by culturing overnight, followed by passage of this spent medium through a 0.2-µm filter. The T cell hybridoma 33.1 is specific for the peptide GAD273–285 presented by HLA-DR4 (provided by Linda Wicker, Merck Research Laboratories, Rahway, NJ). This cell line was maintained in RPMI (GIBCO BRL) supplemented with 10% FCS, 0.1% β-mercaptoethanol, and 100 U/ml penicillin and 100 µg/ml streptomycin. Control studies with the class II–negative cell line, T2, confirmed the DR4 restriction of the 33.1 T cell. T2 is a B–T cell hybrid with a large deletion of the MHC class II locus and was maintained in the same tissue culture medium as the Priess cell lines.
Chloroquine and leupeptin were purchased from Sigma Chemical Co. and were solubilized in PBS. Cathepsin B inhibitor II, calpeptin, PD 150606, and lactacystin were purchased from Calbiochem. These inhibitors, in addition to pepstatin A and brefeldin A (BfA; Sigma Chemical Co.), were solubilized in DMSO. The recombinant 65-kD form of human GAD was produced in bacteria and purified to homogeneity using affinity chromatography, as published previously 21. Studies indicated that both a truncated form of recombinant GAD lacking the NH2-terminal domain and full-length GAD were presented equivalently by APCs. GAD273–285 (IAFTSEHSHFSLK) was synthesized using an Applied Biosystems peptide synthesizer and FMOC technology. Peptide purity and structure was confirmed by reverse phase HPLC and mass spectroscopy.
PriessGAD cells were washed twice in ice-cold PBS (GIBCO BRL) and resuspended at 2 x 107 cells/ml in ice-cold PBS. 50 µl of this cell solution was placed into 0.4-cm electroporation cuvettes (Bio-Rad) and placed on ice. 2 µl of mAb raised against calpain I or II (Calbiochem), or an IgG1 isotype control (Sigma Chemical Co.) was added (final antibody concentration of 0.16 µg/ml, 0.4 µg/ml, and 40 µg/ml, respectively). Each cuvette was gently mixed and placed on ice for 10 min, then pulsed at 0.270 kV, 0.125 uF (400 ohms resistance). Immediately afterwards, 1 ml of warm media was added, and the cells were transferred to a 48-well tissue culture plate. The cells were incubated at 37°C for 20 h and fixed in 1% paraformaldehyde, and GAD was presentation assessed.
To localize intracellular GAD, cytosolic and membrane organelle fractions were isolated from PriessGAD as described previously 22. In brief, cells were lysed using a Balch homogenizer, then differential centrifugation was performed to isolate nuclear, soluble cytoplasmic, and membrane organelle fractions. Membrane organelles were washed in a high salt buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 10 mM benzamidine, 0.1 mM p-chloromercuriphenyl sulfonic acid [p-CMPS]) to disrupt loosely bound proteins. The isolated particulate and soluble fractions were assayed by Western blot analysis. For each fraction, equal amounts of protein were boiled in reducing sample buffer and separated by 12% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane (Micron Separations, Inc.) and probed with the GAD-specific mAb GAD6 23. Bound GAD6 was visualized using goat anti–mouse horseradish peroxidase (Jackson Laboratories) followed by epichemiluminescence (Amersham Pharmacia Biotech).
To remove acid-labile peptides from cell surface class II complexes, PriessGAD cells were washed twice in ice-cold 160 mM NaCl, then resuspended in 160 mM NaCl/citric acid, pH 4.0 and incubated for 30 min on ice. These cell preparations were neutralized with ice-cold medium, then washed twice with fresh tissue culture medium. No loss in cell viability was observed using these conditions.
For exogenous GAD presentation studies, Priess cells were preincubated with or without inhibitors for 30–60 min. These cells were then incubated with 20 µg/ml GAD antigen for 18 h at 37°C in the presence or absence of the inhibitors. Control cells were incubated for 16 h with or without inhibitors followed by the addition of the GAD273–285 peptide for an additional 2 h. Peptide controls were used to ensure that inhibitors affect only antigen processing and not class II maturation or cell surface expression. To monitor endogenous GAD processing and presentation, PriessGAD cells were incubated with or without inhibitors for 18 h with no visible loss of viability. For studies with the calpain inhibitor PD150606, APCs were cultured in Ca2+-free media. B-LCLs typically display a high Ca2+ influx because of the expression of the EBV-encoded latent membrane protein 1 24. In some experiments, PriessGAD cells were acid washed before incubation with inhibitors. In all cases, APCs were subsequently fixed in 1% paraformaldehyde for 10 min at room temperature and washed extensively in cold tissue culture medium. After fixation, 2 x 104 APCs were cocultured with 104 GAD-specific T hybridoma cells for 24 h at 37°C. IL-2 production was measured using the IL-2–dependent cell line, HT-2. HT-2 cells were incubated for 8–16 h with aliquots of T cell culture supernatants, followed by the addition of [3H]thymidine. After an additional 8–16 h of incubation at 37°C, HT-2 cells were harvested using a 96-well plate cell harvester (Skatron). [3H]thymidine incorporation was measured using a Wallac microplate reader. The viability of APCs was monitored in all studies with inhibitors, and the concentrations of drugs were determined to be nontoxic. The concentrations of proteases inhibitors tested did not perturb Ii proteolysis or general class II protein function, as was demonstrated in control studies monitoring Ii fragmentation and the formation of SDS-stable class II–peptide complexes using PAGE 13. All T cell assays were performed in triplicate with the mean and SD calculated for each data point. The percentage of T cell activation of inhibitor-treated APCs was calculated relative to untreated samples. All figures are representative of a minimum of three independent experiments.
Results
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Materials and Methods
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GAD Localizes to the Cytoplasmic Face of Membrane Vesicles in the B-LCL PriessGAD.
In GABA-secreting neurons, GAD associates with the cytoplasmic face of synaptic vesicles as demonstrated by microscopy and subcellular fractionation 19. Sequences within the NH2-terminal domain of GAD confer membrane binding such that the bulk of this protein is found bound to the outer face of vesicular organelles with only a small amount of soluble cytoplasmic GAD. Retroviral transduction of the B-LCL Priess with the human gad65 gene leads to a similar subcellular distribution for GAD, with the majority of this protein tightly bound to the cytosolic face of vesicular membranes (Fig. 1). The localization of GAD in the transduced B-LCL, PriessGAD, was accomplished by fractionating cells to separate cytoplasmic soluble and membrane-bound proteins, followed by Western immunoblotting with a GAD-specific mAb. Cytoplasmic GAD was predominantly found tightly bound to membrane organelles, as demonstrated by resistance to high ionic strength washes designed to disrupt weak membrane–protein interactions 22. A small but consistent fraction of GAD molecules partitioned in the cytosol of these APCs. This distribution mirrors the localization of GAD in neuroendocrine cells published previously 22.
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/β heterodimers en route to the cell surface intersect a wide variety of peptide ligands within late endosomal- and lysosomal-like compartments such as MHC class II compartment 11. By contrast, a pool of mature cell surface class II proteins transit through recycling early endosomal vesicles acquiring distinct antigenic epitopes via the process of peptide editing. The fungal product BfA has been widely used to differentiate these pathways 2728. This inhibitor of anterograde movement from the endoplasmic reticulum to the Golgi complex blocks presentation of epitopes by newly synthesized class II proteins without perturbing the display of peptides by recycling class II molecules. As expected, this inhibitor blocked exogenous GAD presentation by HLA-DR4 (Fig. 3 A). Similarly, incubation of PriessGAD cells in the presence of BfA resulted in a significant reduction in cytoplasmic GAD presentation (Fig. 3 B). Presentation of the synthetic GAD peptide was not altered by BfA, as was expected based on the binding of the peptide to preexisting cell surface class II complexes. Together, these data indicate that presentation of both cytoplasmic and exogenous GAD was dependent upon the binding of GAD epitopes to newly synthesized MHC class II /β heterodimers on route to the cell surface.
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MHC Class II Presents Cytoplasmic GAD Independent of Endosomal/Lysosomal Acidification.
Once resident within the endocytic pathway, newly synthesized MHC class II molecules encounter an acidic environment favoring antigen denaturation and hydrolysis 11. The low pH in late endosomes and lysosomes may also facilitate conformational alterations in MHC class II dimers, enhancing interactions with HLA-DM and peptide binding 30. However, some class II–restricted antigenic epitopes are presented independent of pH, and there is evidence that peptides may also be generated in early endosomes with only mildly acidic pH 331.
To determine whether HLA-DR4–restricted presentation of GAD epitopes was dependent upon the acidification of vesicular organelles, APCs were incubated with the lysosomotropic agent chloroquine. This drug has been shown to block the acidification of late endosomes and lysosomes, thereby inhibiting endosomal processing by resident cathepsins. Presentation of the peptide GAD273–285 derived from exogenous antigen was completely abrogated at low concentrations of chloroquine (Fig. 4 A). However, only minimal inhibition in presentation of the same epitope derived from cytoplasmic GAD was observed, with a significant portion of this endogenous processing pathway proving resistant to the action of chloroquine (Fig. 4 B). These results demonstrate that processing and presentation of endogenous GAD, in contrast with exogenous forms of the antigen, does not display absolute dependence upon endosome/lysosome acidification. The measurable endogenous GAD epitope presentation in the presence of chloroquine further suggests that, at these concentrations, this lysosomotropic agent does not abolish intracellular ligand binding to class II molecules or the transport of class II–peptide complexes to the cell surface, steps common to both endogenous and exogenous antigen presentation. Studies of exogenous peptide presentation by class II proteins were unaltered by chloroquine (Fig. 4), in agreement with control experiments (data not shown) demonstrating no change in Ii fragmentation or the formation of stable class II–peptide complexes using low drug concentrations. Thus, class II antigen function and surface expression were not grossly perturbed by the concentrations of chloroquine tested. In total, these findings suggest that presentation of exogenous GAD is absolutely dependent upon the acidic environment found within late endosomes and lysosomes. In contrast, this critical component of the classical MHC class II presentation pathway plays a less essential role in processing GAD originating in the cytoplasm of APCs.
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/β dimers and Ii proteolysis (data not shown).
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Pepstatin A is a peptide inhibitor that blocks the activity of endosomal and lysosomal aspartyl proteases and degradation of endocytosed antigens in macrophages and B cells 14. To define a role for these endosomal hydrolases in the processing of exogenous and cytoplasmic GAD, APCs were incubated with pepstatin A. This inhibitor of cathepsins D and E efficiently blocked presentation of GAD273–285 derived from the exogenous form of this antigen (Fig. 6). By contrast, presentation of cytoplasmic-derived GAD was not affected by exposure of cells to pepstatin A. Control studies confirmed that the expression and function of surface class II proteins were not altered by pepstatin A treatment of cells. These data clearly suggest that MHC class II presentation of GAD epitopes derived from both exogenous and cytoplasmic antigens follows divergent but overlapping processing pathways.
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An alternate technique to specifically interrupt the activity of intracellular enzymes is through the use of mAbs. Lukas et al. 43 have shown that mAbs specific for cyclin D1 introduced into viable cells through electroporation can arrest cell cycle progression. Two calpain isozymes have been identified in B cells: calpain I (requiring micromolar Ca2+) and calpain II (requiring millimolar Ca2+; reference 44). Remarkably, introduction of an mAb specific for calpain I into electrically permeabilized PriessGAD cells reduced cytoplasmic GAD presentation (Fig. 9). An antibody specific for calpain II displayed significantly less ability to inhibit GAD presentation. Parallel control studies with an irrelevant isotype control mAb failed to produce significant inhibition of class II restricted presentation of this cytoplasmic antigen.
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Presentation of cytoplasmic antigens by class II proteins may occur through several potential mechanisms. During infection of APCs, pathogens may enter endosomes or phagosomes before delivery into the cytoplasm, leading to processing of pathogen-derived proteins by compartmentalized acidic proteases, and presentation by class II proteins within these organelles 47. Alternatively, APCs may endocytose pathogenic antigens released during lysis of infected host cells through the process of bystander presentation 48. Finally, as demonstrated in this study, cytoplasmic antigens may be processed by proteases within the cytoplasm of the APCs, then translocated into membrane organelles containing MHC class II proteins. Evidence that peptides derived from a cytoplasmic antigen, GAD, gain direct access to newly synthesized class II proteins was obtained in EBV-transformed B-LCLs. Presentation of endogenous GAD epitopes was only marginally dependent upon key features of the classical pathway for exogenous antigen processing and display, including acidification of endosomal/lysosomal compartments and activation of cysteine proteases resident within these organelles. Rather, an absolute requirement for cytoplasmic antigen processing was observed for presentation of endogenous GAD epitopes. Thus, proteases responsible for generating peptide ligands for MHC class I molecules appear to also function in supplying epitopes for presentation by class II complexes. Evidence was obtained suggesting that both the proteasome and a calcium-activated protease calpain participate in processing antigens for class II–restricted presentation. These studies suggest a sequential mechanism whereby cytoplasmic antigens are cleaved by neutral proteases before translocation into organelles containing class II molecules. The final stages of antigen processing or trimming of these epitopes may be mediated within endosomes by proteases such as cathepsin B, before or after binding to class II complexes.
The antigen selected for this study, GAD, has been identified as a key target in several autoimmune disorders involving the neuroendocrine system 19. In insulin-dependent diabetes, autoantibodies to GAD are among the earliest hallmarks of progression to disease, with measurable T cell responses to this antigen noted in both humans and mice 19. In addition, a strong genetic link between susceptibility to IDDM and specific MHC class II alleles has been found in both humans and rodents 49, with B cells playing an essential role in progression to disease 50. The development of diabetes can be ablated in NOD mice immunized with tolerogenic levels of GAD 51. Thus, studies to examine the processing of exogenous GAD may have relevance to both the induction and prevention of this autoimmune disorder. Presentation of an immunodominant epitope derived from exogenous GAD by APCs was dependent upon acidification of endosomes and lysosomes, suggesting that processing or binding of this epitope occurs in these acidic organelles. Consistent with this result, processing of exogenous GAD by both cysteine and aspartyl proteases was a prerequisite to efficient class II–restricted presentation of this autoantigen via B cells. Disruptions in autoantigen processing can block the development of autoimmune disease, as was recently demonstrated with cathepsin S knockout mice and collagen-induced arthritis 52. Although cysteine proteases such as cathepsin S play a role in processing a variety of antigens 5354, the role of aspartyl proteases in antigen presentation appears more restricted 121415. Thus, inactivation of the aspartyl protease required for exogenous GAD processing may hold promise as a preventative treatment for IDDM.
Unexpectedly, endogenous GAD molecules produced within the cytoplasm of cells also gain access to the class II pathway for presentation 20. The processing of endogenous GAD was clearly distinct from the exogenous antigen, as determined by susceptibility to protease inhibitors. Thus, aspartyl proteases are not required for endogenous GAD presentation. Further, inhibitors of acidic cysteine proteases only partially block GAD epitope formation, suggesting that these enzymes may play a role in the final stages of epitope trimming. By contrast, at least two distinct cytoplasmic proteases, the proteasome and calpain, function in processing endogenous GAD for class II–restricted presentation.
A role for the multicatalytic proteasome in processing cytoplasmic GAD for class II–restricted presentation was demonstrated in this study using the specific inhibitor lactacystin. The proteasome rapidly catalyzes the turnover of short-lived and abnormally folded proteins within the cytoplasm 34. Yet, prior studies have shown that both long- and short-lived cytoplasmic antigens are presented by class II molecules 55. Ubiquination and processing by the proteasome also favors proteins with basic NH2-terminal residues as defined by the N-end rule pathway 56. However, generation of cytosolic antigens with basic NH2-terminal residues did not always favor class II–mediated presentation, and proteasome cleavage of substrates via the N-end rule appears to vary with cell type 8. These findings, along with the demonstration that acidification of endosomal/lysosomal compartments was sometimes important for presentation of many cytoplasmic antigens via class II molecules 755, raised questions as to the requirement for the proteasome in the class II pathway. The requirement for endosomal/lysosomal acidification is not at odds with a role for the proteasome, as proteases in these organelles may play a role in the final trimming of peptides for class II presentation. Alternatively, the acid pH within these organelles may favor peptide binding to class II molecules 30. As the proteasome can generate peptides of variable length 57, it is reasonable to hypothesize that this complex may supply ligands not only for MHC class I–restricted but also for class II–restricted presentation.
Previous studies designed to implicate the proteasome in antigen processing for class I have alluded to the potential role of other cytoplasmic proteases. In fact, it has been shown that nonproteasomal hydrolytic activity within the cytoplasm can influence class I–restricted presentation 3758. Lymphoid and myeloid cells express at least two calpain isozymes, calpain I and calpain II, with the activation of these enzymes linked to cytoplasmic calcium levels 44. In this study, MHC class II–restricted presentation of an endogenous cytoplasmic antigen was ablated after inhibition of the calcium-dependent protease calpain with the peptidyl inhibitor calpeptin. Additionally, presentation of cytoplasmic GAD was diminished in the presence of PD150606, an inhibitor of calcium-dependent calpains as well as mAbs to calpains. Remarkably, both calpain and GAD share a similar subcellular distribution within the cytoplasm, existing in both membrane and soluble forms. GAD has been localized to the cytoplasmic face of granules within neuroendocrine cells 1922, whereas calpains have been observed to bind to the cytoplasmic face of the plasma membrane, coated vesicles, and the endoplasmic reticulum 39. Whether the membrane localization of GAD and calpain play a role in favoring the translocation of GAD epitopes into organelles rich in class II proteins is currently under investigation. In contrast with cathepsins and the proteasome, calpains exhibit greater substrate selectivity and have been implicated in cleavage of I
B, the common 
chain receptor, and ZAP70 in immune cells 5960. Thus, engineering antigens for calpain cleavage and membrane translocation may hold potential as a novel methodology for vaccine delivery to the class II pathway. Calpains have been linked with several pathological conditions, including the autoimmune disorders arthritis, multiple sclerosis, and experimental allergic encephalomyelitis 4461. The importance of calpains in processing antigens for class I presentation remains unclear. Interestingly, however, several peptidyl aldehydes used to inactivate the proteasome and demonstrated to block class I presentation were originally isolated as calpain inhibitors.
MHC class II molecules bind and present peptides derived from antigens originating within the cytoplasm of the APC. Unfortunately, the mechanisms underlying this nonclassical mode of antigen presentation remain largely unknown. This study demonstrates for the first time that cytoplasmic resident proteases are involved in MHC class II presentation of endogenous antigens. The requirement for cytoplasmic antigen processing provides a key insight into this alternate pathway for antigen presentation. However, the method of translocation of cytoplasmic antigen into class II–containing vesicles remains a mystery. Delivery of intact cytoplasmic antigens into class II–rich compartments by autophagy had been speculated to be one means of facilitating the intersection of antigenic epitopes and class II molecules. Yet, the current study demonstrates a clear requirement for processing before antigen delivery into membrane organelles. Thus, a specific mechanism for peptide translocation and delivery to class II proteins appears to be necessary. Whether the transport of these epitopes is linked to transporter associated with antigen processing 762, an endosomal/lysosomal heat shock cognate protein 63, or an alternate translocation machinery requires further investigation.
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Acknowledgments |
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This work was supported with funds from the National Institute of Allergy and Infectious Diseases and the National Institute of Diabetes and Digestive and Kidney Diseases (to J.S. Blum) and from the Canadian Diabetes Association and the Juvenile Diabetes Foundation International (to J.F. Elliott). J. Lich was supported by National Institutes of Health training grant T32 DK07519.
Submitted: 10 May 1999
Revised: 17 January 2000
Accepted: 17 February 2000
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References |
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| TABLE OF CONTENTS |
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). (B) PriessGAD cells (
) were incubated with increasing concentrations of chloroquine. Control APCs (A [•] and B [
]) were pulsed with 10 µM GAD273–285 3 h before fixation in 1% paraformaldehyde. Antigen presentation was assessed as described in Materials and Methods.
