Evidence for shared recognition of a peptide ligand by a diverse panel of non-obese diabetic mice-derived, islet-specific, diabetogenic T cell clones

Kenji Yoshida1, Tracy Martin2, Ken Yamamoto3,5, Cathleen Dobbs2, Christian Münz6,9, Nobuhiro Kamikawaji4,5, Naoko Nakano7, Hans-Georg Rammensee6, Takehiko Sasazuki4,5,8, Kathryn Haskins2 and Hitoshi Kikutani1

1 Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University,3-1 Yamada-oka, Suita 565-0871, Japan 2 Barbara Davis Center for Childhood Diabetes and Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262, USA 3 Division of Molecular Population Genetics, Department of Molecular Genetics and 4 Division of Immunogenetics, Department of Immunology and Neuroscience, Medical Institute of Bioregulation, and 5 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corp., Kyushu University, Fukuoka 812-8582, Japan 6 Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen, Germany 7 Research Institute for Biological Sciences, Science University of Tokyo, Noda 278-8510, Japan 8 International Medical Center of Japan, Tokyo 162-8655, Japan 9 Present address: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021, USA 10 Present address: National Minami Fukuoka Chest Hospital, Fukuoka 811-1394, Japan

Correspondence to: H. Kikutani; E-mail: kikutani{at}ragtime.biken.osaka-u.ac.jp
Transmitting editor: K. Inaba


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
MHC class II-restricted autoreactive T cells play a major role in the development of autoimmune diabetes mellitus in both human and mouse. Two of our groups previously established panels of islet-reactive CD4+ T cell clones from prediabetic non-obese diabetic (NOD) mice. These clones express distinct sets of TCR V{alpha}, Vß, J{alpha} and Jß, and also differ in the structure of the junctional region of TCR. All of the T cell clones have been shown to cause insulitis and several induce diabetes when transferred to various recipients. The antigen specificities of these T cell clones have not been determined, but they do not react with defined islet cell antigens such as glutamic acid decarboxylase. To identify the peptide ligands recognized by these clones, we examined the reactivity of the T cell clones to peptide mixtures in which anchor residues for H2-Ag7 were fixed. Most of the clones showed similar reactivity to the peptide mixtures. To further determine the peptide ligands of the T cell clones, we synthesized several peptides based on the favored amino acid motifs and examined clone reactivity to the synthetic peptides. Some of the peptides, e.g. HLAI-RM and HIPI-RM, could stimulate most of the T cell clones tested, even though the clones expressed different TCR. The results suggest that our islet-reactive T cell clones recognize in islet ß cells a natural ligand that is similar to these peptides.

Keywords: autoantigens, combinatorial peptide libraries, islet-reactive T cells, MHC class II I-Ag7, peptide epitope


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The non-obese diabetic (NOD) mouse spontaneously develops pancreatic insulitis and diabetes, and thus has been widely used as an animal model for human insulin-dependent diabetes mellitus (IDDM) (1,2). Cumulative evidence suggests that autoimmune diabetes in NOD mice is T cell-mediated. Although very heterogeneous cell populations (including T cells, NK cells, B cells, dendritic cells and monocytes) are seen in pancreatic islets of NOD mice, the majority of infiltrating cells are T cells. One of the major diabetogenic genes, idd1, of the NOD mouse is closely linked to the MHC class II region. The fact that the molecular basis of idd1 involves the unique structure of I-Ag7 and the absence of I-E expression (310) implies that H2-Ag7-restricted autoreactive CD4+ T cells may play a critical role in pathogenesis of IDDM in the NOD mouse, although CD8+ T cells have been also shown to participate in destruction of pancreatic islets.

Several candidates for primary autoantigens recognized by diabetogenic T cells have been extensively studied, and include glutamic acid decarboxylase (GAD) 65, insulin and heat shock protein (HSP) 60. T cell responsiveness to GAD65 was detected in NOD mice at 3–4 weeks of age (11,12) and several islet-reactive T cell clones derived from NOD mice were shown to react with insulin B chain (13). Autoantibodies and T cell responsiveness to HSP60 were also detected in NOD mice (14). In vivo treatment by each of these candidate antigens has been demonstrated to prevent diabetes (11,12,15) and also to eliminate T cell responsiveness to other antigens (11,12). Thus, it remains to be determined whether a single dominant antigen is responsible for initiation of autoimmunity or if responses against multiple autoantigens contribute to the disease process.

Two of our groups have previously established panels of islet-reactive T cell clones and lines from pancreatic islets and spleens of prediabetic NOD mice (16,17). These T cell clones can induce overt diabetes as well as insulitis when transferred to young NOD (18,19), I-E transgenic NOD (17) or NOD-scid [(20) and Yoshida, unpublished data] mice. Production and characterization of the transgenic mouse expressing the TCR from one of these T cell clones, BDC-2.5, has clearly demonstrated that the TCR of this T cell clone is diabetogenic (21,22). However, none of these T cell clones has shown reactivity with defined islet antigens such as GAD, insulin, HSP65 and carboxypeptidase H [(17,23) and Yoshida, unpublished data]. Studies of TCR gene expression have revealed that the T cell clones express many different sets of V{alpha}, Vß, J{alpha} and Jß gene segments, which parallel the diverse TCRVß expression in T cells infiltrating pancreatic islets (17,24). Such heterogeneous TCR expression in these T cell clones might be a consequence of intra-molecular or inter-molecular epitope spreading during the disease progression as reported for T cell responses against myelin basic protein or proteolipid protein in experimental allergic encephalomyelitis (2528), and against GAD and other islet antigens in NOD mice (11,12). Alternatively, a population of autoreactive T cells, heterogeneous in terms of TCR expression, might react with a single determinant of an unknown islet antigen, a theory that might contradict the current view that the MHC–peptide complex restricts usage of TCR V gene segments and the structure of joining regions of TCR chains.

In the present study, we have analyzed reactivity of several islet-reactive T cell clones with unknown specificity against combinatorial peptide libraries. These T cell clones have been found to react with similar but not identical peptide motifs. Furthermore, some of the peptides that were synthesized on the basis of these motifs could stimulate most of the T cell clones tested. This finding raises the possibility that these T cell clones may recognize the same natural peptide ligand even though they express structurally different TCR.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and cells
Osaka
NOD/Shi colonies were maintained at the Research Institute for Microbial Diseases at Osaka University by brother–sister mating under specific pathogen-free conditions. Islet-reactive T cell clones (4-1-E2, 4-1-G4 and 4-1-L6) were established from islet-infiltrating T cells of prediabetic NOD mice as described previously (17). Cells were maintained by repeated stimulation with pancreatic islet cells in the presence of irradiated NOD spleen cells as antigen-presenting cells (APC). One T cell clone, 4-1-G4, was converted into a T cell hybridoma (G4/BW) by fusion with BW5147{alpha}ß cells.

Denver
NOD breeding mice were purchased from the Jackson Laboratory (Bar Harbor, ME) or obtained from the colony at the Barbara Davis Center (NOD/bdc). All mice were bred and maintained under pathogen-free conditions at the Center for Laboratory Animal Care at the University of Colorado Health Sciences Center. T cell clones were produced and maintained as previously described (16) by stimulation every 2 weeks with ß cell granule membrane protein obtained from ß cell tumors as a source of antigen (23), {gamma}-irradiated NOD spleen cells as APC and supernatant from EL-4 cells as a source of IL-2. Two of the T cell clones used in this report, BDC-2.5 and BDC-10.1, were in the form of T cell hybridomas (2.5/BW1-31.3 and 10.1/BW1-4.6) produced by fusions of the parent clones to the BW5147{alpha}ß fusion partner.

Peptide synthesis
Peptide mixtures were synthesized on a 396MPS multiple peptide synthesizer (Advanced Chem Tech, Louisville, KY), following a strategy which modified the protocol reported by Tana et al. (29). In brief, the peptide mixtures were synthesized with standard Fmoc amino acid with O-(7-azabenzotrazol-1-yl)-N,N,N',N',-tetramethyluronium hexafluorophosphate (HATU) coupling chemistry on a polystyrene resin.

HRPI-RM peptide (EKAHRPIWARMDAKK), HRPI peptide (EKAHRPIWAYNDAKK), HLAI-KM peptide (EKAHLAIWAK MDAKK), HLAI-RM peptide (EKAHLAIWARMDAKK), HVPI-RM peptide (EKAHVPIWARMDAKK), HIPI-RM peptide (EKA HIPIWARMDAKK), H2AI-RM peptide (EKAHHAIWARMD AKK), H2AI peptide (EKAHHAIWAYNDAKK), H2PI-RM peptide (EKAHHPIWARMDAKK), H2PI peptide (EKAHHPIWAYN DAKK) and ovalbumin peptide (ISQAVHAAHAEINEAGR) were obtained from Kurabo Industries (Osaka, Japan).

Peptides derived from GAD were provided by A. Quinn (La Jolla Institute for Allergy and Immunology, San Diego, CA). The insulin B9-23 peptide was synthesized by the Molecular Resource Center at the National Jewish Medical and Research Center.

Antigen responses of islet-reactive T cells
Cytokine response of islet-reactive T cell hybridoma, G4/BW
Assays were performed in 96-well plates in duplicate or triplicate by culturing 5 x 104 cells of islet-reactive T cell hybridomas with peptide and 2.5 x 105 cells of {gamma}-irradiated T cell-depleted splenocytes derived from NOD as APC for 24 h. T cell-depleted splenocytes were subjected to complement lysis by anti-Thy-1.2 antibody (F7D5) (Serotec, Oxford, UK). After 24 h, cell culture supernatants were collected and IL-2 in the supernatants was measured by CTLL-2 proliferation. In brief, 1 x 104 cells of CTLL-2 in 96-well plates were cultured with cell culture supernatants for 24 h. [3H]Thymidine was added during the last 4 h of culture. [3H]Thymidine uptake was determined by liquid scintillation analysis and is expressed as c.p.m.

Proliferative response of islet-reactive T cell clones, 4-1-E2 and 4-1-L6
Cells (1 x 105) of islet-reactive T cell clones were cultured with peptide and 2.5 x 105 cells of {gamma}-irradiated T cell-depleted splenocytes derived from NOD for 72 h. [3H]Thymidine was added during the last 24 h of culture.

Cytokine response of islet-reactive T cell hybridomas, 2.5/BW1-31 and 10.1/BW1-4.6
Assays were performed in 96-well plates with 1 x 105 T cell hybrids, 2.5 x 104 NOD peritoneal cells (PC) as APC and varying amounts of peptide antigen. IL-2 in culture supernatants was measured by preparing serial dilutions of the culture supernatants and adding HT-2 cells for 24 h. IL-2 was expressed as the reciprocal dilution of the culture supernatant.

Cytokine response of islet-reactive T cell clones, BDC-2.5, BDC-10.1, BDC-5.10.3 and BDC-6.9
Cultures were set up in 96-well plates with 2.0 x 104 T cell clones, 2.5 x 104 PC as APC and peptide antigen. After 24 h, culture supernatants were collected for measurement of IFN-{gamma} produced by ELISA.

Antigen responses of mononuclear cells from 2.5 TCR Tg/NOD mice
Mononuclear cells from the lymph nodes and spleens of 2.5 TCR Tg/NOD mice were incubated in the presence of peptides for 72 h. [3H]Thymidine was included during the last 16 h of culture.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reactivity of islet-reactive T cell clones from NOD mice against combinatorial peptide libraries
To prepare the peptide library, anchor motifs of H2-Ag7 binding peptides were first determined (30). Peptides were eluted from H2-Ag7 molecules purified from transfectants expressing H2-Ag7, fractionated by reverse-phase HPLC and major peaks were then sequenced. Pools of eluted peptides were also directly sequenced. Based on the sequence data of eluted peptides, our proposed anchor motif of H2-Ag7 binding peptides is shown in Fig. 1: P1, basic residues or Ser or Thr; P4, hydrophobic residues; P6, small non-charged residues such as Ala; P9, acidic residues or Ser. This is in good agreement with the optimal peptide-binding motif derived from crystallography analysis (31,32).



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Fig. 1. Proposed H2-Ag7 binding peptide motif. Position 1 (P1), position 4 (P4), position 6 (P6) and position 9 (P9) involve hydrophilic and basic amino acids, hydrophobic and aliphatic amino acids, a hydrophobic amino acid, and hydrophilic and acidic amino acids respectively.

 
To analyze peptide ligands recognized by islet-reactive T cell clones from NOD mice, we first prepared 20 peptide libraries of 15 amino acids in length, each bearing one of 20 amino acids at position 5, wherein His and Asp were fixed at positions corresponding to P1 and P9 as H2-Ag7 anchors based on the proposed motif of Fig. 1. We also added 3 amino acid residues to the end (Glu–Lys–B for the N-terminal where B denotes degenerate amino acids, B position contains a mixture of 20 amino acids; and Ala–Lys–Lys for the C-terminal) to ensure solubility of resulting peptides. Each of the peptide mixtures contained a fixed amino acid residue at P5 (a TCR contact residue) and degenerate amino acid residues at other positions. These peptide mixtures were first tested for ability to stimulate an islet-reactive T cell clone hybridoma, G4/BW, and an islet-reactive T cell clone, 4-1-E2. The T cell clones were cultured with each peptide mixture in the presence of irradiated T cell-depleted NOD spleen cells as APC. As shown in Fig. 2, both G4/BW (hybridoma) and 4-1-E2 (clone) reacted strongly to only the peptide mixture containing Trp at P5, suggesting that Trp at P5 is critical for stimulation of these T cell clones.



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Fig. 2. Different islet-reactive T cell clones react strongly with the same H2-Ag7 binding motif-oriented peptide mixture, containing Trp at P5. G4/BW (hybridoma) was cultured with peptide mixture (P5: EKBHBBBXBBBDAKK) and {gamma}-ray irradiated T cell-depleted splenocytes derived from NOD mice for 24 h. Then IL-2 in the supernatants was measured by CTLL-2 proliferation for 24 h. [3H]Thymidine was included during the last 4 h of culture (proliferation of the indicator cells). 4-1-E2 T cell clone was cultured with the peptide mixture and {gamma}-ray-irradiated T cell-depleted splenocytes derived from NOD mice for 72 h. [3H]Thymidine was included during the last 24 h of culture (direct proliferation of clone). The final concentration of each peptide mixture was ~0.5 mg/ml. Thymidine incorporation was determined by liquid scintillation analysis and is expressed as c.p.m. The data shown represent the mean ± SEM for c.p.m. determinations made on triplicate wells. B: It has degenerate amino acids. B position contains a mixture of 20 amino acids. EK and AKK: These residues are added in order to ensure solubility of the resulting peptides. H and D: They are fixed at positions corresponding to P1 and P9. X: Each peptide mixture contains a fixed amino acid residue at P5. C/G: mixtures of EKBHBBBCBBBDAKK and EKBHBBBGBBBDAKK. DDW: Double-distilled water.

 
To further analyze the fine specificity of the T cell clones, we synthesized four more sets of peptide mixtures in which His, Trp and Asp were fixed at P1, P5 and P9 as shown in Fig. 3(A). Each set consisting of 20 peptide mixtures was prepared to determine critical residues at P2, P3, P7 and P8 respectively. Figure 3(B) shows representative results with the 4-1-E2 (clone) and G4/BW (hybridoma) in which both clones showed similar but not identical reactivity patterns. In particular, only basic residues such as Arg or Lys at P7 were tolerated for both T cell clones. These clones favored non-charged residues at P2 and P3, and Met at P8. The same sets of peptide mixtures were also tested with the BDC-2.5 T cell clone in K. H.’s laboratory. A hybridoma, 2.5/BW1-31.3, made from the BDC-2.5 clone, was used to assay the peptides and IL-2 secretion from stimulated cells was measured. As shown in Fig. 3(C), although 2.5/BW1-31.3 (hybridoma) displayed a very narrow specificity for the peptide mixtures, it showed a similar reactivity pattern to clones 4-1-E2 and 4-1-G4, with strong responses to Arg at P7 and Met at P8. Two additional clones, 4-1-L6 (Osaka) and hybridoma 10.1/BW (Denver), were analyzed and the summary of specificity patterns for all five clones is shown in Table 1. There were similarities in the specificities of most of the clones and hybridomas: 4-1-E2, G4/BW, 4-1-L6 and 2.5/BW favor non-charged residues at P3, basic residues (e.g. Arg) at P7, and non-charged residues (e.g. Met) at P8.



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Fig. 3. Responses of islet-reactive T cells to H2-Ag7 binding motif-oriented peptide mixtures. (A) The design of peptide mixtures. On the basis of H2-Ag7 binding peptide motif and the preferential recognition of amino acid residue at P5 by the islet-reactive T cell clones, many sets of peptide mixtures with fixed anchor residues for H2-Ag7 and P5, one of the TCR contact residues, were synthesized. The peptide library which has Gly at P2 (see Fig. 3B,C) could not be synthesized. B: It has degenerate amino acids. B position contains a mixture of 20 amino acids. EK and AKK: These residues are added in order to ensure solubility of the resulting peptides. H, W and D: They are fixed at positions corresponding to P1, P5 and P9. X: Each peptide mixture contains a fixed amino acid residue at P2, P3, P7 or P8. (B) G4/BW (hybridoma) was cultured with the peptide mixtures and {gamma}-ray-irradiated T cell-depleted splenocytes derived from NOD mice for 24 h. Then IL-2 in the supernatants was measured by CTLL-2 proliferation (proliferation of the indicator cells). 4-1-E2 T cell clone was cultured with the peptide mixtures and {gamma}-ray-irradiated T cell-depleted splenocytes derived from NOD mice for 72 h (direct proliferation of the clone). The final concentration of each peptide mixture was ~0.5 mg/ml. The data shown represent the mean ± SEM for c.p.m. determinations made on triplicate wells. (C) 2.5BW1-31.1 (hybridoma) was cultured with peptide and NOD PC. The final concentration of each peptide mixture was ~0.5 mg/ml. Then IL-2 in the supernatants was measured by HT-2 cells (indicator cells). IL-2 was expressed as the reciprocal dilution of the supernatant.

 

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Table 1. Summary of the amino acid motifs recognized by islet-reactive T cell clones
 
Peptide ligands recognized by islet-reactive T cell clones
Based on the motifs of peptides favored by several islet-reactive T cell clones, we then synthesized a series of peptides with defined sequences as shown in Fig. 4(A). All of these peptides contain His, Ile, Ala and Asp at P1, P4, P6 and P9 respectively, which comprise the MHC-binding anchors and were selected as the proposed motif in Fig. 1, and Trp at P5, the first determined TCR-contacting residue. HRPI-RM contains Arg, Pro, Arg and Met at P2, P3, P7 and P8 respectively, which were selected from the motif favored by 2.5/BW. HLAI-KM was supposed to be favored by G4/BW. The other peptides are analogues of HRPI-RM and HLAI-KM, which have substitutions at P2, P3, P7 or P8. HRPI is an analogue of HRPI-RM, whose residues at P7 and P8 were replaced with those favored by 4-1-L6. HLAI-RM is an analogue of HLAI-KM, whose residue at P7 was replaced with those favored by 2.5/BW. HVPI-RM and HIPI-RM were synthesized based on the motifs favored by 4-1-E2. H2AI-RM is an analogue of HLAI-RM, whose residue at P2 was replaced with His that was favored by 4-1-L6. H2PI was synthesized based on the motifs favored by 4-1-L6. H2AI is an analogue of H2PI, in which Pro at P3 was replaced with Ala that was favored by G4/BW. H2PI-RM is an analogue of HRPI-RM, in which Arg at P2 was replaced with His that was favored by 4-1-L6. These peptides were tested for their ability to stimulate five T cell clones, whose specificities were analyzed by using the peptide library as describe above, and two additional islet-reactive T cell clones, BDC-5.10.3 and BDC-6.9. As expected from results with the peptide libraries, islet-reactive T cell clones showed similar but not identical reactivity patterns to a series of peptides with defined sequences (Fig. 4B and C). The residues at P7 and P8 were found to be very critical for stimulation of the tested T cells, whereas variations in reactivity were found with the amino acids at P2 and P3. Nevertheless, two of the peptides tested, HLAI-RM and HIPI-RM, could stimulate six of the T cell clones and half the clones were shown to give moderate to strong responses to two additional peptides, HLAI-KM and HIAI-RM. Interestingly, as indicated by the IFN-{gamma} response to the peptides, a third clone, BDC-5.10.3, from K.H.’s laboratory, was found to have a similar reactivity pattern to that of BDC-2.5, as illustrated in Fig. 4(C). One of our islet-reactive T cell clones, BDC-6.9, did not react any synthetic peptide (Fig. 4C). Taken together, our results suggest that at least six T cell clones, isolated in two different laboratories and derived from two different colonies of NOD mice, may recognize the same natural peptide ligand.



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Fig. 4. Shared recognition of the defined synthetic peptides by multiple T cell clones. (A) Amino acid sequences of synthetic peptides based on the preferential recognition of amino acid motifs by the islet-reactive T cell clones. EK and AKK: These residues are added in order to ensure solubility of the resulting peptides. (B) G4/BW (hybridoma) was cultured with 10 µM of the peptides and {gamma}-ray-irradiated T cell-depleted splenocytes derived from NOD mice for 24 h. Then IL-2 in the supernatants was measured by CTLL-2 proliferation (proliferation of the indicator cells). 4-1-E2 and 4-1-L6 T cell clones were cultured with 10 µM of the peptides and {gamma}-ray-irradiated T cell-depleted splenocytes derived from NOD mice for 72 h. Proliferation was determined by [3H]thymidine uptake (direct proliferation of the clone). Data are express as a stimulation index (mean c.p.m. of response to peptide divided by mean c.p.m. with medium only). (C) BDC-2.5, BDC-10.1, BDC-5.10.3 and BDC-6.9 T cell clone was cultured with the 10 µM of the peptides and NOD PC for 72 h. Then IFN-{gamma} in the supernatants was measured by ELISA.

 
Finally, to compare the response of one of the most widely used T cell clones in this panel, BDC-2.5, to the peptides described here and to other candidate peptide antigens from GAD and insulin, we performed assays with both the BDC-2.5 T cell clone and with T cells from the BDC-2.5 TCR transgenic mouse. Figure 5(B) illustrates the IFN-{gamma} response of the BDC-2.5 T cell clone to HRPI-RM, to insulin B9-23 and to a series of overlapping peptides through the p35 region of GAD65, identified to be an immunodominant epitope of this enzyme (12,33). The BDC-2.5 clone was previously reported as non-responsive to whole GAD (23) and, as shown here, there is no response to GAD peptides in the 523–543 portion of the molecule nor to insulin B9-23. Similarly, the transgenic T cell responses are very robust to the HRPI-RM peptide, whereas only background level responses could be measured with the GAD peptides (Fig. 5A).



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Fig. 5. Activation of mononuclear cells from 2.5 TCR Tg/NOD mice and the BDC-2.5 T cell clone by GAD peptides. (A) Mononuclear cells from the lymph nodes and spleens of 2.5 TCR Tg/NOD mice were incubated in the presence of 10 µM of the indicated peptides for 72 h. [3H]Thymidine was included during the last 16 h of culture. Thymidine incorporation was determined by liquid scintillation analysis and is expressed as c.p.m. The data shown represent the mean ± SEM for c.p.m. determinations made on triplicate wells. (B) BDC-2.5 T cell clones was incubated in the presence of 7 µM of the indicated peptides or NOD islet cells (10,000/well) for 72 h. The amount of IFN-{gamma} in cell-free supernatants was determined by ELISA and is expressed as ng/ml. The data shown represent the mean ± SEM for IFN-{gamma} determinations made on triplicate wells.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study using synthetic combinatorial peptide libraries, we attempted to identify peptide ligands recognized by islet-reactive T cell clones derived from two independent laboratories. As previously reported (1620), these T cell clones were derived from islets or spleens of prediabetic NOD mice and could transfer diabetes or insulitis in young NOD, irradiated NOD, I-E transgenic NOD or NOD-scid recipients. Our results show that T cell clones from the two panels display very similar, but not identical, reactivity patterns to peptide libraries. Furthermore, they could react with some of the peptides designed from their reactivity patterns, suggesting that they may recognize the same natural peptide ligand. On the other hand, these T cell clones are quite diverse in their TCR expression (17,24). The peptides, which could stimulate them, contain a basic residue, Arg or Lys, at position 7, implying that reactive T cell clones may have an acidic residue in CDR3 loops of their TCR. However, their CDR loops did not share particular structural features including acidic residues.

We used not only T cell clones but also T cell hybridomas to screen peptide libraries. There may be some differences in responses to peptides between islet-reactive T cell clones and their hybridoma counterparts with regard to density of TCR, CD4, co-stimulatory molecules and adhesion molecules. However, in our hands, hybridomas exhibited reactivities very similar to those of their parent T cell clones as long as we selected hybridoma cells expressing TCR brightly (data not shown). On the other hand, the sensitivity of T cell clones varied depending on their culture conditions, particularly when large-scale assays were performed. Therefore, islet-reactive hybridomas may be suitable for screening combinatorial peptide libraries. For some T cell clones, IFN-{gamma} production was investigated because this was the most sensitive assay for these clones. Although IFN-{gamma} is known to be produced as a consequence of partial agonism as well as full agonism, almost comparable reactive patterns were also obtained in proliferation assays (data not shown).

Two characteristic features have been described for generation of autoreactive T cells in animal autoimmune models. First, T cells responding to dominant determinants of autoantigens have been reported to exhibit restricted TCR repertoires. In the case of experimental autoimmune encephalomyelitis (EAE), when B10.PL and PL mice were immunized with the N-terminal peptide, myelin basic protein (MBP 1–11), the encephalitogenic T cell clones induced were found to largely utilize the Vß8 gene segment for the TCR ß chain (34,35). It has been also reported that the majority of NOD-derived insulin-reactive T cell clones preferentially use V{alpha}13 coupled with one of two homologous J{alpha} segments (36). The second feature of autoreactive T cells is epitope spreading. Immunoreactivity to non-dominant determinants has been observed in the course of chronic EAE in mice immunized exclusively with immunodominant peptides of autoantigens and takes place not only in an intra-molecular manner but also in an inter-molecular manner (2528). Intra- and inter-molecular epitope spreading has been also reported for NOD mice. For example, T cells reactive to GAD65 are observed within 4 weeks of age in NOD mice, and are subsequently followed by T cell responses to other antigens such as carboxypeptidase H, insulin and HSP65 (11,12). Determinant spreading within GAD65 has also been reported during the course of disease in NOD mice (12). Thus, the diverse TCR repertoire within islet-infiltrating T cells has been thought to be a consequence of epitope spreading. However, assuming that the T cell clones employed in this study are representative of islet-reactive T cells in pancreatic islets and spleens of prediabetic NOD mice, an assumption that gains support from the existence of T cells that can be stained by tetramers of H2-Ag7 and one of the peptides identified in this study in islets and spleens of prediabetic NOD (Stratmann et al., unpublished data), our findings raise the alternative possibility that a large proportion of heterogeneous islet-reactive T cells may share a few unknown dominant self-determinants rather than diverse determinants as a consequence of epitope spreading. This interpretation is further suggested by our observation that two of the clones tested in this study, each isolated from different NOD donors, have almost identical peptide reactivity patterns.

As our T cell clones do not react with known autoantigens such as GAD and insulin, one of the important questions still to be answered is the identity of the natural autoantigen recognized by these clones. At present, we have not identified through a database search proteins containing the HLAI-RM, HIPI-RM or HRPI-RM sequences. This may be due to the fact that peptides employed in this study contain particular residues at MHC anchors selected to ensure strong MHC binding. A natural ligand recognized by these T cell clones might be a weak or intermediate MHC binder and could differ from peptides such as HLAI-RM at the MHC anchors. In preliminary experiments, a T cell line that reacted with the HIPI-RM peptide, established from NOD mice immunized with HIPI-RM peptide, responded only very weakly to islet cells in vitro although the line showed a very strong proliferative response to the HIPI-RM peptide. This observation also supports the notion that these peptides are still divergent from a natural ligand expressed in pancreatic ß cells, although we cannot rule out the possibility that peptide immunization may induce T cells reactive to the flanking sequences rather than the core sequence.

Finally, we have addressed the recent speculation that the natural ligand for one of our T cell clones, BDC-2.5, may be derived from GAD65. Using T cells from the BDC-2.5 TCR transgenic mouse, Judkowski et al. screened a combinatorial peptide library and found >100 decapeptides that stimulated transgenic T cells (37). Notably, peptides with the highest stimulatory activity for the 2.5 TCR transgenic T cells contained W at P6 (P5 in our peptide configuration), and RM at P8 and P9 (corresponding to our P7 and P8). In addition, some of these peptides included sequences very similar to those found within the 528–539 fragment of GAD65. Our data (Fig. 5) obtained from testing the original BDC-2.5 T cell clone, as well as T cells from two transgenic mice, indicate that overlapping 15-amino-acid GAD sequences within the GAD65 521–543 region, or with the entire 521–543 sequence (data not shown), have no antigenic activity for BDC-2.5 T cells. Furthermore, previous work had established that the BDC-2.5 clone had no reactivity with NOD APC and whole GAD (23). We therefore think that it is unlikely that GAD is the source of the natural peptide ligand for BDC-2.5, although it is intriguing to discover that limited substitution of amino acids in peptides from GAD65 can lead to peptides with agonistic activity for this T cell clone.


    Acknowledgements
 
We thank Shouta Takenaka, Kazunori Inoue and Taigo Kato for technical assistance, Miki Sonoda for peptide synthesis, and Kyoko Kubota for secretary assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to H. K., from the Japan Science and Technology Corp. to T. S., and JDFI research award 1-2000-322 and NIH grant NIDDK 50561 to K. H.


    Abbreviations
 
APC—antigen-presenting cell

EAE—experimental autoimmune encephalomyelitis

GAD—glutamic acid decarboxylase

HSP—heat shock protein

IDDM—insulin-dependent diabetes mellitus

NOD—non-obese diabetic

PC—peritoneal cells


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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