Epitope Determinants Located Within the Antibody Lymphocytes on Processing of T Cell Decarboxylase 65-Specific Autoimmune B Supp

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Suppressive Effect of Glutamic Acid Decarboxylase 65-Specific Autoimmune B Lymphocytes on Processing of T Cell Determinants Located Within the Antibody This information is current as Epitope of September 25, 2021. Juan Carlos Jaume, Sarah Louise Parry, Anne-Marie Madec, Grete Sønderstrup and Steinunn Baekkeskov J Immunol 2002; 169:665-672; ; doi: 10.4049/jimmunol.169.2.665 Downloaded from http://www.jimmunol.org/content/169/2/665

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Suppressive Effect of Glutamic Acid Decarboxylase 65-Speciﬁc Autoimmune B Lymphocytes on Processing of T Cell Determinants Located Within the Antibody Epitope1

Juan Carlos Jaume,*† Sarah Louise Parry,¤ Anne-Marie Madec,¶ Grete S¿nderstrup,¤ and Steinunn Baekkeskov2*‡

Type 1 diabetes is a T cell-mediated disease in which B cells serve critical Ag-presenting functions. In >95% of type 1 diabetic patients the B cell response to the glutamic acid decarboxylase 65 (GAD65) autoantigen is exclusively directed at conformational epitopes residing on the surface of the native molecule. We have examined how the epitope specificity of Ag-presenting autoimmune B cell lines, derived from a type 1 diabetic patient, affects the repertoire of peptides presented to DRB1*0401-restricted T cell hybridomas. The general effect of GAD65-specific B cells was to enhance Ag capture and therefore Ag presentation. The Downloaded from enhancing effect was, however, restricted to T cell determinants located outside the B cell epitope region, because processing/ presentation of T cell epitopes located within the autoimmune B cell epitope were suppressed in a dominant fashion. A similar effect was observed when soluble Abs formed immune complexes with GAD65 before uptake and processing by splenocytes. Thus, GAD65-specific B cells and the Abs they secrete appear to modulate the autoimmune T cell repertoire by down-regulating T cell epitopes in an immunodominant area while boosting epitopes in distant or cryptic regions. The Journal of Immunology, 2002,

169: 665Ð672. http://www.jimmunol.org/

ctivated B cells can take up Ag at very low concentra- play a role (see Ref. 6 for review). Yet in the nonobese diabetic tions via their Ag-specific membrane-bound Ig (mIg)3 (NOD) mouse model of type 1 diabetes, clinical signs of ␤ cell A and process it for presentation to MHC class II-restricted failure do not develop in animals carrying a mutation that results T cells. The enhanced Ag capture by B cells can lower the thresh- in B cell deficiency (7, 8). This protective effect appears to reflect old for a T cell response compared with “professional” APCs (1). a critical role of B cells as APCs for initiating and maintaining B lymphocytes can present Ag to both the Th2 and the Th1 subsets autoimmune T cell responses to pancreatic ␤ cell autoantigens (9Ð of CD4 T cells (2). The ability of B cells to present minute 11). In contrast, skewing of the B cell repertoire toward a ␤ cell by guest on September 25, 2021 amounts of Ag may be particularly important for maintaining a autoantigen promotes the development of diabetes in this model state of chronic autoimmunity and inflammation when most of the (12). While there is evidence to suggest that type 1 diabetes can target tissue has been destroyed. For example, in patients with develop in the face of a severe depletion of mature B cells in man autoimmune hypothyroidism who have no trace of remaining thy- (13), the disease process may be delayed (14). roid gland, immune responses to thyroid peroxidase, an exclusive In addition to the enhanced Ag uptake mediated by mIg of an thyroid Ag, can be detected throughout life (3, 4). In type 1 dia- Ag-presenting B cell, its Ab specificity may influence which T cell ␤ betes, autoimmune responses to the pancreatic cell autoantigen epitopes are processed and presented in the context of MHC class glutamic acid decarboxylase (GAD) 65, are often maintained for II Ags (15, 16). The endocytosed mIg-Ag complex is transported decades following the disappearance of a measurable ␤ cell func- to late endosomal compartments, where the bound Ab can pro- tion (5). Pancreatic ␤ cell destruction resulting in development of foundly influence the proteolytic processing and loading onto type 1 diabetes appears to be mediated by the Th1 subset of CD4 MHC class II Ags. It has been proposed that an enhancement of the T cells and by CD8 T cells, whereas autoantibodies do not seem to presentation of an epitope can occur when 1) the bound Ig protects it from proteolytic degradation and enhances its survival and there- *Diabetes Center, Department of Medicine, †Division of Endocrinology and Metab- fore availability for binding to MHC class II Ags; 2) the bound Ig olism, Department of Veterans Affairs Medical Center, and ‡Department of Micro- facilitates loading of the epitope into the peptide binding groove of biology and Immunology, University of California, San Francisco, CA 94143; ¤De- partment of Microbiology and Immunology, Stanford University, Palo Alto, CA MHC-class II Ags; and/or 3) the Ab suppresses presentation of 94305; and ¶Institut National de la Sante« et de la Recherche Me«dicale, Laennec other epitopes, thus increasing the availability of MHC class II Faculty of Medicine, University of Lyon, Lyon, France molecules for a specific peptide binding in the peptide binding Received for publication February 28, 2002. Accepted for publication May 10, 2002. compartment. Conversely, suppression of the presentation of an The costs of publication of this article were defrayed in part by the payment of page epitope may result from a prolonged Ag-Fab association in late charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. endosomes preventing processing into the appropriate peptide 1 This work was supported by National Institutes of Health grants (to S.B., J.C.J., and and/or from a steric blockade of the epitope (16). G.S.). J.C.J. is a recipient of the San Francisco Veterans Affairs Medical Center The general effect of soluble Abs that form immune complexes Young Investigator Award. with a protein is to enhance Ag uptake via the FcR on macro- 2 Address correspondence and reprint requests to Dr. Steinunn Baekkeskov, Diabetes phages and dendritic cells, resulting in enhanced presentation by Center, University of California, 513 Parnassus Avenue, Room HSW 1090, San Fran- cisco, CA 94143-0534. E-mail address: [email protected] such cells. Numerous studies using soluble mAbs have shown that 3 Abbreviations used in this paper: mIg, membrane-bound Ig; GAD, glutamic acid they enhance presentation of the Ag they bind to by facilitating Ag decarboxylase; NOD, nonobese diabetic. capture through the FcR (see Ref. 17 for review). Furthermore, a

study using GAD65 autoantibody-positive sera from type 1 dia- Ag presentation assays betic patients found an enhancement of presentation of a T cell B cells as APCs. EBV-transformed, GAD65-specific B cell lines were determinant residing in aa 274Ð286 when GAD65 immune com- incubated with graded amounts of Ag for1honiceandthen washed twice plexes rather than Ag alone were fed to peripheral blood APCs in tissue culture medium to remove unbound Ag. Ag-loaded B cells (3 ϫ 5 (18). The epitope specificity of a soluble Ab that binds to an Ag 10 ) were added to each well of a round-bottom 96-well plate containing 1.5 ϫ 105 GAD65-specific T cell hybridomas in a final volume of 150 ␮l before uptake via the FcR can also affect the processing and pre- tissue culture medium. The cells were incubated for 24 h (37¡C, 4% CO2). sentation of T cell epitopes and either suppress or boost a partic- The HLA-DRB1*0401-positive human EBV-transformed B cell line, ular epitope (15, 16). While boosting effects may be difficult to Priess (25), which does not express a GAD65-specific IgG was used as a separate from the general enhancement of uptake of immune com- negative control. IL-2 secretion was measured using a sandwich immuno- assay and a streptavidin-europium detection system (26). Experiments plexes compared with free Ag, relative suppressing effects are were repeated a minimum of three times in triplicate wells. more easily demonstrated. For instance, a mouse mAb to thyro- Splenocytes as APCs. Preformed immune complexes were generated by globulin was found to exert a relative suppression on the presen- incubating 1.5 ␮g/ml purified human mAbs and graded amounts of Ag tation of a nondominant epitope in this molecule (19). Thus, Ab (0.03Ð3.0 ␮g/ml) in a round-bottom 96-well plate for1hatroom temper- specificities are likely to modulate the T cell epitopes made avail- ature before addition of APCs. Splenocytes were prepared from HLA-DR ϩ/ϩ ϩ/ϩ ϩ/ϩ Ϫ Ϫ able by all three APCs: macrophages, dendritic cells, and B A1*0101 , B1*0401 , hCD4 , I-Ab / transgenic mice (23) and used as APCs. Splenocytes (3 ϫ 105 cells/well in 50 ␮l tissue culture lymphocytes. medium) were incubated with graded amounts of Ag alone, peptide alone, Recently, we have mapped in detail the GAD65-specific or the preformed immune complexes for 1 h before addition of T cell epitopes recognized by human mAbs derived from type 1 diabetic hybridomas (1.5 ϫ 105 cells in 100 ␮l tissue culture medium were added patients and shown that almost the entire surface of native folded per well). After 24-h incubation, the samples were processed for measure- Downloaded from ment of IL-2 secretion as described above. GAD65 is targeted by human autoimmune B cells (20). In this study we assess how the epitope specificities of four corresponding FcR blockade EBV-transformed DRB1*0401-positive B cell lines affect immu- The rat anti-mouse FcR mAb 2.4G2 (IgG2b) was used to block FcRII nodominant T cell epitopes generated from regions within and out- receptor uptake of immune complexes. The mAb was purified from super- side the B cell epitope region. Furthermore, we have studied how natants of HB197 cells (American Type Culture Collection, Manassas,

soluble Abs from the same B cell lines affect T cell epitopes pre- VA) using G4B Fast Flow (Sigma) column afﬁnity chromatography and http://www.jimmunol.org/ sented by DRB1*0401-positive splenocytes. added to splenocyte experiments (10 ␮g/ml) using preformed immune complexes as Ag.

Materials and Methods Results EBV-B cell lines and Abs Localization and overlap of B and T cell epitopes in GAD65 The derivation of EBV-transformed, GAD65-specific, monoclonal B cell The four EBV-transformed monoclonal HLA-DRB1*0401-posi- lines DPA, DPB, DPC, and DPD from an HLA-DRB1*0401-positive type tive monoclonal B cell lines used in this study recognize four dis- 1 diabetic patient was described previously (21). The B cell lines have a tinct conformational epitopes residing on the surface of the stable production of IgG1 in tissue culture (21), and the epitopes have been GAD65 molecule (20). Fig. 1 shows the linear and spatial rela- by guest on September 25, 2021 mapped using homologue scanning mutagenesis, taking advantage of the lack of reactivity of the human mAbs with the highly homologous isoform tionships between the B cell epitopes of these four lines compared GAD67 (20). The Abs recognize four distinct conformational epitopes in with four immunodominant GAD65 T cell epitopes recognized by the N-terminal domain (DPB, DPD), middle domain (DPC), and C-termi- HLA-DRB1*0401-restricted T cell hybridomas (22, 23). DPB and nal domain (DPA) (20) (Fig. 1). Their affinity constant (Kd) for binding to DPD B cells recognize distinct conformational epitopes in the N- GAD65, determined by surface plasmon resonance on BIAcore, is 0.11 nM terminal domain of GAD65, which were mapped to aa 39Ð173 and for DPA, 9.45 nM for DPB, 0.31 nM for DPC, and 2.79 nM for DPD (A.-M. Madec, unpublished observations). The B cell lines were grown in 96Ð173, respectively (20). The determinant of the T35 T cell hy- RPMI 1640 medium (Life Technologies/BRL, Gaithersburg, MD) contain- bridoma (aa 115Ð130) overlaps or is adjacent to the DPB and DPD ing 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 epitopes. The third B cell line, DPA, recognizes a conformational ␮ ␮ g/ml streptomycin, and 50 M 2-ME (tissue culture medium). Abs were epitope residing in the C-terminal domain of GAD65, involving purified from culture supernatant by G4B fast flow (Sigma-Aldrich, St. Louis, MO) affinity chromatography. The eluate was dialyzed against PBS. residues in aa 483Ð499 and 556Ð585. In particular, H568 is critical The human mAb concentration was determined using an IgG ELISA kit for binding (20). The epitopes of two of the T cell hybridomas, (Roche, Mannheim, Germany). HY103 (aa 481Ð495) and HY81 (aa 556Ð570), overlap or are proximal to the DPA epitope. The fourth B cell line, DPC, recog- T cell hybridomas nizes a conformational epitope residing in the middle domain of GAD65 and involving aa 231Ð234 and 366Ð413. The epitope of The derivation of HLA-DRB1*0401-restricted, GAD65-specific T cell the fourth T cell hybridoma, HY91 (aa 271Ð285), localizes to the hybridomas from mice expressing HLA-DRA*0101, DRB1*0401, and human CD4 as transgenes on the I-AbϪ/Ϫ background was described pre- same domain, but does not overlap with the DPC epitope (Fig. 1). viously (22). The hybridomas 91, 103, and 81 recognizing aa 271Ð285, Thus, in contrast to the DPA, DPB, and DPD epitopes, the DPC 481Ð495, and 556Ð570 in GAD65, respectively, were used in this study. A epitope does not overlap with a T cell determinant recognized by fourth HLA-DRB1*0401-restricted, GAD65-specific T cell hybridoma, the panel of T cell hybridomas. In the three dimensional model of recognizing aa 115Ð130 (23), was donated by Dr. L. Wicker (Sharp and the middle and C-terminal regions of GAD65 (20), the HY103 and Dohme Research Laboratories, Merck, Rahway, NJ). T cell hybridomas were maintained in tissue culture medium (see above). HY81 epitopes are exposed on the surface of the GAD65 dimer, while the HY91 epitope is at the interface between the middle and C-terminal domains, which form a sandwich in each monomer GAD65 protein (Fig. 2). It is therefore buried in the native GAD65 molecule. A Human GAD65, expressed in Saccharomyces cerevisiae and purified as three-dimensional model of the N-terminal region is not available. described (24), was donated by Drs. M. Powell, B. Rees-Smith, and J. However, in the predicted secondary structure of GAD65 (20) the Furmaniak (FIRS Laboratories, Cardiff, U.K.). The purified protein was conformationally stable and was recognized by a series of human mAbs determinant of the T35 T cell hybridoma (aa 115Ð130) resides in recognizing conformational epitopes in GAD65, including the mAbs used an amphipathic ␣ helix, the hydrophilic phase of which is likely to in this study (results not shown). be exposed on the surface of native GAD65 (20). The Journal of Immunology 667 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Location of GAD65-specific epitopes recognized by autoimmune B and T cell lines. A, Location of autoimmune epitopes in the primary amino acid sequence of the N-terminal (upper panel), middle FIGURE 2. Location of T cell epitopes in a three-dimensional model (middle panel), and C-terminal (lower panel) domains of GAD65. The of the middle and C-terminal regions of the GAD65 dimer. A, A view epitopes recognized by DPA and DPC B cell lines were mapped to the of a space fill model of the GAD65 dimer showing the location of indicated regions by domain swapping and homologue-scanning mutagen- middle and C-terminal epitopes recognized by the T cell hybridomas esis, taking advantage of the high homology between GAD65 and GAD67 used in this study. The monomer on the left has the middle domain in in the middle and C-terminal domains and the fact that none of the DP Abs the front and the C-terminal domain behind it. The two monomers are recognizes GAD67 (20). Because of the low degree of homology between related by a P21 symmetry (20). Therefore, the monomer on the right GAD65 and GAD67 in the N-terminal domain, mapping of DPB and DPD has the C-terminal domain in front and the middle domain behind it. was undertaken by domain swapping and deletion mutagenesis, but was not The HY81 and HY103 epitopes in the C-terminal domain (peptides amenable to homologue-scanning mutagenesis and is therefore less de- 556Ð570 and 481Ð 495, respectively) are exposed on the surface of the tailed (20). Three of the four immunodominant T cell epitopes (HY103, dimer. In contrast, the HY91 epitope residing in the middle domain HY81, and T35) reside either in or adjacent to a B cell epitope region. (peptide 271Ð285, green) is buried in the interface between the middle Predicted ␣ helixes, which may represent potential contact points between and C-terminal domains. B, A view of the same model, where the C- complementarity-determining regions of Igs and Ag are underlined (20). B, terminal region has been removed from the monomer on the right to Location of autoimmune epitopes in a schematic representation of the reveal the location of the aa 271Ð285 epitope. three-dimensional model of GAD65. The relative location of B (ovals) and T (rectangles) cell epitopes in the N-terminal (light gray), middle (black), and C-terminal (dark gray) domains are shown. The HY91 (peptide 271Ð 285) rectangle is hatched to indicate its location on the opposite face of the recognize GAD65 epitopes, was used as a control APC. All the middle region. DP-B cell lines elicited similar IL-2 responses from the DRB1*0401-restricted, GAD65-specific T cells used in this study when synthetic GAD65 peptides were used as an Ag, indicative of a similar expression level of HLA-DRB1*0401 on their surface. Inhibition of presentation of GAD65 T cell epitopes in the mIg The Priess cell line presented synthetic GAD65 peptides to binding site of autoimmune B cells DRB1*0401-restricted GAD65-specific T cells more efficiently We evaluated the ability of the DPA, DPB, DPC, and DPD B cell than any of the DP-B cell lines (results not shown), consistent with lines to process and present GAD65 to the four T cell hybridomas a higher expression level of HLA-DRB1*0401 on its surface. used in this study (Fig. 3). The Priess B cell line, which shares the The Priess cell line has been shown to nonspecifically (pinocy- HLA-DRB1*0401 haplotype with the DP-B cells, but does not tosis) take up GAD65 at high concentrations and process it for 668 B CELL MODULATION OF AUTOIMMUNE T CELL RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. GAD65-specific B cells suppress presentation of T cell epitopes residing in their Ab binding site. IL-2 secretion by DRB1*0401- restricted mouse T cell hybridomas in response to presentation of human recombinant GAD65 protein by DRB1*0401-positive EBV-transformed B cells from a diabetic patient. Each panel shows IL-2 production by a T cell clone in response to GAD65 presentation by four GAD65-specificB cell lines and the control Priess B cell line. A, Presentation of the aa 271Ð285 epitope to the T cell hybridoma HY91. All the human B cell lines expressing GAD65-specific IgG enhance presentation of the 271Ð285 peptide compared with the Priess B cell line. IL-2 secretion by HY91 is observed at 0.1 ␮g/ml GAD65 Ag presented by DPA and DPB and at 0.2 ␮g/ml Ag presented by DPC and DPD B cells. DPC B cells induce IL-2 production by HY91 in a dose-response manner, but the maximal doses of Ag produce a lower response than with the other DP B cell lines. B, Presentation of the aa 556Ð570 epitope to the T cell hybridoma HY81. Proliferative responses of HY81 are observed at 0.1 ␮g/ml GAD65 Ag presented by DPB, DPC, and DPD B cells. In contrast, DPA B cells completely fail to present this peptide, which resides in its Ab binding site, at all concentrations (0.03Ð3 ␮g/ml). C, Presentation of the aa 481Ð 495 peptide to the T cell hybridoma HY103. Proliferative responses of HY103 are observed at 0.1 ␮g/ml Ag presented by DPB, DPC, and DPD B cells. Presentation of this peptide is suppressed by DPA compared with the other DP B cells and is only moderately enhanced compared with Priess B cells. D, Presentation of the aa 115Ð130 peptide to the T cell hybridoma T35. Proliferative responses of T35 are observed at 0.1 ␮g/ml Ag presented by DPA and DPC. Presentation of this peptide is blocked, however, by DPB cells and suppressed by DPD cells, whose epitopes reside in the same region.

presentation to T cells (27) (Fig. 3). Because of the enhanced up- Table I). By contrast, the presentation of T cell epitopes residing in take of GAD65 via mIg, the DP cell lines would be expected to or adjacent to the Ab epitope was either blocked or inhibited. Thus, process and present whole GAD65 more efficiently than Priess presentation of the aa 556Ð570 peptide, which is within the Ab unless the bound Ig exerts an inhibiting effect on a particular epitope of DPA, was efficiently blocked when DPA was used for epitope. Consistent with this prediction, the DPC cell line, which Ag presentation (Fig. 3B). Similarly, presentation of another T cell binds to a conformational epitope distant from all the T cell epitope (aa 481Ð495), which resides within or adjacent to the DPA epitopes recognized by our panel of T cell hybridomas, elicited Ab epitope, was inhibited compared with presentation of this same severalfold higher IL-2 levels than Priess from the four T cell peptide by the other B cell lines and was only slightly enhanced hybridomas following incubation with whole GAD65 (Fig. 3 and compared with that of Priess (Fig. 3C). In contrast, DPA efficiently Table I). Similarly, DPA, DPB, and DPD elicited severalfold presented the N-terminal and middle domain epitopes (aa 115Ð130 higher IL-2 responses than Priess from T cell hybridomas recog- and 271Ð285, respectively) to the relevant T cells (Fig. 3, A and D, nizing T cell epitopes outside their Ab binding region (Fig. 3 and and Table I). The Journal of Immunology 669

Table I. Summary of the effects of autoimmune GAD65-speciﬁc B cell lines on presentation of four different T cell determinants

T Cells

B Cell T35 (115Ð130) HY91 (271Ð285) HY103 (481Ð495) HY81 (556Ð570)

Priess ϩϩ ϩ ϩ DPA (483Ð499) (556Ð586) ϩϩϩ ϩϩϩ ϩ(ϩ) Ϫ DPB (39Ð173) ϩ ϩϩϩ ϩϩϩ ϩϩϩ DPC (231Ð234) (366Ð413) ϩϩϩ ϩϩ(ϩ) ϩϩϩ ϩϩϩ DPD (96Ð173) ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ

Similar results were obtained with the DPB and DPD B cell DPA and DPC (Fig. 3D). Yet DPB was able to present the lines. DPB presented the T cell epitope 115Ð130, which resides middle and C-terminal domain epitopes efﬁciently (Fig. 3, A–C, and within or adjacent to its Ab epitope, at a similar level as Priess, Table I). Similarly, presentation of the 115Ð130 epitope by DPD was and presentation was effectively inhibited compared with that of moderately improved compared with Priess, but poor compared with Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Soluble Abs suppress presentation of T cell epitopes residing in their binding site. IL-2 secretion by DRB1*0401-restricted mouse T cell hybridomas in response to presentation of GAD65 by DRB1*0401-positive mouse splenocytes. Each panel shows the proliferation of a T cell clone in response to presentation of the GAD65 Ag alone or in an immune complex with one of the four GAD65-speciﬁc mAbs DPA, DPB, DPC, or DPD. A, Presentation of aa 271Ð285 to T cell hybridoma HY91. Incubation of splenocytes with immune complexes with each of the mAbs enhance presentation of this epitope compared with Ag alone. B, Presentation of aa 556Ð570 to the T cell hybridoma HY81. The DPB, DPC, and DPD Abs, which bind to regions distant from this T cell epitope, enhance its presentation compared with Ag alone. In comparison, presentation of the 556Ð570 determinant is suppressed by the DPA Ab, whose epitope encompasses the peptide. IL-2 secretion is only detected when Ag is in excess of the DPA Ab. C, Presentation of the aa 481Ð495 peptide to the T cell hybridoma HY103. The DPB, DPC, and DPD Abs, epitope of which are distant from this peptide, enhance its presentation compared with Ag alone. However, presentation is relatively suppressed by DPA, which binds to the same region. D, Presentation of the aa 115Ð130 peptide to the T cell hybridoma T35. The DPA and DPC Abs, the binding sites of which are distant from this peptide, enhance its presentation. In contrast, no enhancement is observed with the DPB Ab whose epitope is in the same region as the peptide. IL-2 responses are only induced in conditions of Ag excess. The DPD Ab, which binds an epitope shifted downstream compared with DPB, shows less enhancement at high Ag concentrations than DPA and DPC, but is less suppressive than DPB. 670 B CELL MODULATION OF AUTOIMMUNE T CELL RESPONSES

DPA and DPC (Fig. 3D). In contrast, DPD presented the middle and presentation to levels similar to that with Ag alone (Fig. 4, B and C-terminal domains at similar levels as DPC and severalfold more C), with a more complete inhibition of epitope 556Ð570. Likewise, efficiently than Priess (Fig. 3, A–C, and Table I). loading of a complex between GAD65 and either DPB or DPD In summary, the autoimmune GAD65-specific B cell lines ap- Abs, which recognize B cell epitopes overlapping with or adjacent pear to efficiently present T cell epitopes residing outside their Ab to the 115Ð130 epitope, resulted in an inhibition of its presentation epitope region, while presentation of T cell determinants that compared with DPA and DPC Abs, with DPB Ab inhibiting pre- reside within the Ab-bound region of GAD65 is suppressed in a sentation more completely than DPD (Fig. 4D). Thus, purified dominant fashion (Table I). DPA, DPB, and DPD Abs in an immune complex with GAD65 inhibited presentation of T cell epitopes by macrophages in a sim- Ab binding and presentation of immune complexes by ilar manner as when the corresponding B cells were used for Ag macrophages and dendritic cells presentation. We next analyzed whether the inhibiting effect of the DPA, DPB, Abs enhance uptake via the FcRII on macrophages. To confirm and DPD B cell lines on processing and presentation of T cell that the stimulating effect on outlying epitopes observed in the epitopes located within their Ab-bound GAD65 regions was also experiments with soluble Abs is dependent on an enhanced uptake relevant when immune complexes were taken up, processed, and via the FcRII molecule, the experiments described above were presented by APCs like macrophages and dendritic cells. For these repeated in the presence of an FcRII Ab, which blocks uptake by experiments purified GAD65 or GAD65 in an immune complex the FcIIR, but not by pinocytosis. In the presence of the FcRII Ab, with DPA, DPB, DPC, or DPD Abs was incubated with HLA- none of the DP Abs in an immune complex with GAD65 enhanced DRB1*0401-positive splenocytes, which were then used to stim- presentation to T cell hybridomas compared with Ag alone (Fig. 5 Downloaded from ulate the T cell hybridomas. The binding of immune complexes by and results not shown), suggesting that the general boosting effect the FcRII receptor on macrophages enhances their uptake com- of soluble DPA, DPB, DPC, and DPD on T cell epitopes outside pared with Ag alone (17). the Ab binding site is the result of increased Ag capture via the Compared with incubation with Ag alone, the incubation of FcRII. splenocytes with GAD65 in an immune complex with DPA, DPB,

DPC, or DPD Abs resulted in a severalfold enhanced stimulation Discussion http://www.jimmunol.org/ of the HY91 hybridoma, recognizing the 271Ð285 peptide (Fig. The results presented here suggest that autoimmune GAD65-spe- 4A). Similarly, the presentation of peptides 115Ð130, 481Ð495, cific B cells and the Abs they secrete may play a major role in and 556Ð570 was severalfold enhanced following incubation of shaping the autoimmune T cell response by specifically suppress- splenocytes with GAD65 in an immune complex with purified DP ing presentation of T cell determinants residing within Ab-bound Abs, which do not recognize epitopes that overlap with those pep- regions. Ab effects on uptake are generally positive whether ex- tides (Fig. 4). In contrast, loading of a complex between GAD65 pressed on the surface of Ag-presenting B cells or forming soluble and DPA Ab, which recognizes an epitope overlapping with the immune complexes that are taken up by FcR on APCs. However, 481Ð495, and 556Ð570 peptides, resulted in an inhibition of their the expected increase in Ag presentation is only observed for T cell by guest on September 25, 2021

FIGURE 5. Enhancement of Ag presentation by soluble Abs is blocked by an Ab to FcRII. IL-2 secretion by DRB1*0401-restricted mouse T cell hybridomas in response to presentation of GAD65 alone or GAD65 immune complexes by DRB1*0401-positive mouse splenocytes in the presence of an Ab to FcRII. Each panel shows IL-2 secretion of a T cell clone in response to presentation of GAD65 Ag alone or in an immune complex with one of the four GAD65-specific mAbs: DPA, DPB, DPC, or DPD. A, Presentation of the aa 556Ð570 peptide to T cell hybridoma HY81. The enhancement of presentation of this peptide observed with DPB, DPC, and DPD immune complexes compared with Ag alone (Fig. 4B) is eliminated when FcRII is blocked. B, Presentation of the aa 115Ð130 peptide to T cell hybridoma T35. The enhancement of presentation of this peptide observed with DPA and DPC and to a lesser degree DPD immune complexes compared with Ag alone (Fig. 4D) is eliminated when FcRII is blocked. The Journal of Immunology 671 determinants located outside the region captured by Ab. In con- Taken together, our results suggest that autoimmune GAD65- trast, T cell determinants located within or adjacent to the B cell specific B cells not only serve a critical function as APCs in type epitope are specifically suppressed, and the suppressive effect 1 diabetes, but also play a major role in shaping the epitope spec- dominates. One possible explanation for this effect is that binding ificity of the autoimmune T cell response to GAD65. Thus, auto- of a GAD65 autoantibody to its epitope is stable throughout the immune GAD65-specific B cells and the Abs they secrete appear acidic environment of Ag processing and MHC class II loading to modulate the autoimmune T cell repertoire by down-regulating compartments, resulting in masking of T cell determinants residing T cell epitopes in an immunodominant area while boosting in the region. While the binding affinities of the DP Abs varied epitopes in distant regions, providing a mechanism for autoim- 86-fold between DPA (highest) and DPB (lowest; see Materials mune T cell epitope spreading. and Methods), both mAbs and the corresponding cell lines effectively blocked the presentation of T cell determinants located within the Ab epitope. Thus, T cell epitope suppression appears to Acknowledgments occur over a wide range of Ab affinities. We are grateful to Drs. M. Powell, B. Rees-Smith, and J. Furmaniak (FIRS The findings of epitope suppression are consistent with earlier Laboratories) for donation of purified GAD65 and to Dr. L. Wicker studies in which tetanus toxin-specific mIg as well as the corre- (Merck, Sharp and Dohme) for donation of T-35. sponding soluble Abs suppressed the processing and presentation of T cell determinants within the footprint of the Ab (15, 16). In References previous studies a detailed mapping of the B cell epitopes in tet- 1. Lanzavecchia, A. 1987. Antigen uptake and accumulation in antigen-specificB anus toxin was not available. Instead, the suppressed T cell deter- cells. Immunol. Rev. 99:39. Downloaded from minants were found to be localized within the Ab footprint, i.e., the 2. Cohly, H. H., D. R. Morrison, and M. Z. Atassi. 1989. Antigen presentation by non-immune B-cell hybridoma clones: presentation of synthetic antigenic sites Ag fragment that is protected by Ab during limited proteolysis and reveals clones that exhibit no specificity and clones that present only one epitope. usually encompasses a larger area than the Ab epitope. In one case, Immunol. Invest. 18:987. the Ab footprint included both a T cell epitope that was suppressed 3. Jaume, J. C. 2000. Endocrine autoimmunity. In Basic and Clinical Endocrinol- ogy, 6th Ed. F. S. Greenspan and D. Gardner, eds. Appleton & Lange, Stamford, and an epitope that was enhanced by Ab binding (16). Based on p. 80. these results, Simitsek et al. (16) proposed a model in which the 4. Jaume, J. C., G. Costante, T. Nishikawa, D. I. W. Phillips, B. Rapoport, and http://www.jimmunol.org/ processing and MHC class II loading of a T cell epitope residing S. M. McLachlan. 1995. Thyroid peroxidase autoantibody fingerprints in hypo- thyroid and euthyroid individuals. I. Cross-sectional study in elderly women. in the actual Ab contact site (epitope) are suppressed, while the J. Clin. Endocrinol. Metab. 80:994. loading of a nearby epitope residing inside the footprint but outside 5. Christie, M. R., D. Daneman, P. Champagne, and T. L. Delovitch. 1990. Persis- the actual binding site is favored. The present study has identified tence of serum antibodies to 64,000-Mr islet cell protein after onset of type I three cases of effective suppression of immunodominant diabetes. Diabetes 39:653. 6. Baekkeskov, S., J. Kanaani, J. C. Jaume, and S. Kash. 2000. Does GAD have a DRB1*0401-restricted T cell determinants residing within or ad- unique role in triggering IDDM? J. Autoimmun. 15:279. jacent to regions constituting the epitope of human monoclonal 7. Serreze, D. V., H. D. Chapman, D. S. Varnum, M. S. Hanson, P. C. Reifsnyder, GAD65 autoantibodies arising in type 1 diabetes. We did not iden- S. D. Richard, S. A. Fleming, E. H. Leiter, and L. D. Schultz. 1996. B lympho-

cytes are essential for the initiation of T cell-mediated autoimmune diabetes: by guest on September 25, 2021 tify a T cell epitope whose processing/presentation were relatively analysis of a new “speed congenic” stock of NOD.Igmnull mice. J. Exp. Med. enhanced by Ab binding to the epitope area, but the possibility is 184:2049. not excluded by this limited set of B cell lines and T cell 8. Akashi, T., S. Nagafuchi, K. Anzai, S. Kondo, D. Kitamura, S. Wakana, J. Ono, M. Kikuchi, Y. Niho, and T. Watanabe. 1997. Direct evidence for the contribu- hybridomas. tion of B cells to the progression of insulitis and the development of diabetes in Both immunization of DRB1*0401-positive transgenic mice non-obese diabetic mice. Int. Immunol. 9:1159. (22, 23) and derivation of human T cell lines from human patients 9. Serreze, D. V., S. A. Fleming, H. D. Chapman, S. D. Richard, E. H. Leiter, and R. M. Tisch. 1998. B lymphocytes are critical antigen-presenting cells for the (28) have identified the aa 271Ð285 determinant as perhaps the initiation of T cell-mediated autoimmune diabetes in nonobese diabetic mice. most immunodominant DRB1*0401-restricted T cell epitope in J. Immunol. 161:3912. the GAD65 molecule. In the three-dimensional model of the mid- 10. Falcone, M., J. Lee, G. Patstone, B. Yeung, and N. Sarvetnick. 1998. B lymphocytes are crucial antigen-presenting cells in the pathogenic autoimmune response dle and C-terminal regions of GAD65, the 271Ð285 epitope is to GAD65 antigen in nonobese diabetic mice. J. Immunol. 161:1163. buried in the native folded molecule. This is in contrast to both the 11. Noorchashm, H., Y. K. Lieu, N. Noorchashm, S. Y. Rostami, S. A. Greeley, aa 481Ð495 and 556Ð570 T cell epitopes, which are exposed on A. Schlachterman, H. K. Song, L. E. Noto, A. M. Jevnikar, C. F. Barker, et al. 1999. I-Ag7-mediated antigen presentation by B lymphocytes is critical in over- the surface. More than 95% of type 1 diabetic patients exclusively coming a checkpoint in T cell tolerance to islet ␤ cells of nonobese diabetic mice. make autoantibodies to conformational epitopes on the surface of J. Immunol. 163:743. the native GAD65 molecule (Ref. 20 and references therein). The 12. Hulbert, C., B. Riseili, M. Rojas, and J. W. Thomas. 2001. B cell specificity contributes to the outcome of diabetes in nonobese diabetic mice. J. Immunol. aa 271Ð285 T cell epitope does not, therefore, appear to reside in 167:5535. the Ab binding site of a typical autoimmune B cell in type 1 di- 13. Martin, S., D. Wolf-Eichbaum, G. Duinkerken, W. A. Scherbaum, H. Kolb, abetes. For example, while the epitopes of two other human mAbs J. G. Noordzij, and B. O. Roep. 2001. Development of type 1 diabetes despite derived from diabetic patients, MICA 6 and MICA 10, have been severe hereditary B-lymphocyte deficiency. N. Engl. J. Med. 345:1036. 14. McDevitt, H. 2001. Closing in on type 1 diabetes. N. Engl. J. Med. 345:1060. mapped close to the 271Ð285 T cell epitope (242Ð282 region), both 15. Watts, C., and A. Lanzavecchia. 1993. Suppressive effect of antibody on pro- Abs bind the surface of native intact GAD65, suggesting that the cessing of T cell epitopes. J. Exp. Med. 178:1459. buried 271Ð285 residues are not part of the epitope. Furthermore, 16. Simitsek, P. D., D. G. Campbell, A. Lanzavecchia, N. Fairweather, and C. Watts. 1995. Modulation of antigen processing by bound antibodies can boost or sup- in experiments using splenocytes as APCs, MICA 10 in an im- press class II major histocompatibility complex presentation of different T cell mune complex with GAD65 does not suppress presentation of the determinants. J. Exp. Med. 181:1957. 271Ð285 determinant (W. Richter, J. C. Jaume, S. L. Parry, G. 17. Ravetch, J. V., and S. Bolland. 2001. IgG Fc receptors. Annu. Rev. Immunol. S¿nderstrup, and S. Baekkeskov, unpublished observations). We 19:275. 18. Reijonen, H., T. L. Daniels, A. Lernmark, and G. T. Nepom. 2000. GAD65- hypothesize that the absence of B cell reactivity to the aa 271Ð285 specific autoantibodies enhance the presentation of an immunodominant T-cell region, and the consequent absence of suppression of processing epitope from GAD65. Diabetes 49:1621. and presentation of T cell determinants in this region by B cells 19. Dai, Y., K. A. Carayanniotis, P. Eliades, P. Lymberi, P. Shepherd, Y. M. Kong, and G. Carayanniotis. 1999. Enhancing or suppressive effects of antibodies on and APCs may contribute to the immunodominance of this processing of a pathogenic T cell epitope in thyroglobulin. J. Immunol. 162:6987. epitope. 20. Schwartz, H. L., J. M. Chandonia, S. Kash, J. Kanaani, E. Tunnell, A. Domingo, 672 B CELL MODULATION OF AUTOIMMUNE T CELL RESPONSES

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