Autoantibodies and CD4 T cells target a ß cell retroviral envelope protein in non-obese diabetic mice

Matteo G. Levisetti1,2, Anish Suri1, Ilan Vidavsky3, Michael L. Gross3, Osami Kanagawa1 and Emil R. Unanue1

1 Department of Pathology and Immunology, and 2 Department of Medicine, Washington University School of Medicine, St Louis, MO 63110, USA 3 Department of Chemistry, Washington University, St Louis, MO 63110, USA

Correspondence to: E. R. Unanue; E-mail: unanue{at}pathbox.wustl.edu
Transmitting editor: A. Weiss


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We determined that, over a biologic time interval, from 4 to 8 weeks of age, female non-obese diabetic (NOD) mice develop antibodies against pancreatic ß-cell-surface antigens depending upon the presence of both the MHC class II susceptibility allele, I-Ag7, and other NOD background genes. We generated a mAb from a pre-diabetic NOD mouse that binds to the surface of insulinoma cells and isolated mouse ß cells, and identified the target as a retroviral envelope glycoprotein expressed on pancreatic ß cells. The cloned and expressed sequence for this protein was recognized by the mAb. The antibody as well as sera from pre-diabetic NOD mice recognized the recombinant protein. Spontaneous T cell reactivity against a peptide from the cloned protein was found in NOD mice. In conclusion, a ß cell retroviral envelope protein is a target antigen that is selected by the NOD mouse immune system early in the pathogenesis of autoimmune diabetes.

Keywords: autoimmunity, diabetes mellitus, Ig, islets of Langerhans, T lymphocyte


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Autoantibodies to ß cell antigens are detected before the onset of Type 1 diabetes in both humans and the non-obese diabetic (NOD) mouse model of the disease. Antibodies have led to the identification of multiple ß cell autoantigens and are a component of the clinical diagnosis, as well as an important prognostic factor in determining the risk of disease in relatives of individuals with Type 1 diabetes (1). These ß cell autoantibodies include islet cell antibodies, insulin autoantibodies, anti-glutamic acid decarboxylase antibodies, anti-tyrosine phosphatase antibodies as well as others (25). The prospective roles of these ß cell autoantigens in the pathogenesis of Type 1 diabetes have been extensively studied (68).

The presence of autoantibodies to surface antigens on ß cells has been recognized since the development of techniques for the isolation and purification of rodent and primate pancreatic islets (9). Previous studies in the NOD mouse documented the time course of ß cell autoantibodies against cytoplasmic antigens such as insulin and glutamic acid decarboxylase, as well as cell-surface antigens (1013). Although the NOD mouse spontaneously develops autoantibodies against ß-cell-surface antigens before the onset of disease, little is known about the nature of these antigens. A group generated anti-ß cell mAb from the NOD mouse, but their specificities have not been reported (14). In our current studies, we found that NOD mice develop antibodies to ß-cell-surface determinants between 4 and 8 weeks of age. In an effort to identify the target antigens of this spontaneous seroreactivity, we generated a B cell hybridoma from a pre-diabetic NOD mouse and have identified a new ß cell autoantigen, related to the family of retroviral envelope glycoproteins.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
The NOD mice were originally obtained from Taconic (Germantown, NY), the B6.g7 (H-2g7 congenic B6) and NOD.SCID mice from the Jackson Laboratory (Bar Harbor, Maine), and the NOD.GD (H-2g2 congenic NOD) mice were generated here. ILK3 mice expressing the protein hen egg lysozyme (HEL) under the rat insulin promoter were obtained from C. Goodnow (15) and backcrossed to B10.BR (H-2k) mice for previous studies (16).

Cell lines and antibodies
The NIT-1 cell line was a gift from Dr E. H. Leiter (Jackson Laboratory) (17), the B16 mouse melanoma tumor cell line was obtained from the ATCC (Rockville, MD) and the C3.G7 cell line was generated in our laboratory (18). The mAb AG2.42.7, which recognizes I-Ag7, was generated in our laboratory (18) and the mAb SF1.1.1, which recognizes the H-2Kd, was obtained from (PharMingen, San Diego, CA). The M562 antibody, which recognizes the mouse melanoma antigen, was a gift from M. Taniguchi.

FACS analysis of serum antibodies
Cells were incubated first with mouse sera and then with a goat anti-mouse IgG–phycoerythrin (Caltag, Burlingame, CA). FACS analysis was performed on a FACScan flow cytometer and data analysis was performed with CellQuest software (Becton Dickinson, Mountain View, CA).

Isolation and dispersion of mouse ß cells
Mouse islets were isolated as described before (19). Individual islets were selected, dispersed by trypsinization, and cultured overnight at 37°C in 5% CO2 in DMEM supplemented with 5% FCS, 10 mM HEPES, penicillin (100 U/ml), streptomycin sulfate (100 µg/ml) and glutamine (2 mM). Staining of dispersed islets from NOD and ILK3 mice was performed with mAb 5.331–biotin and mAb F10.6.6–biotin followed by staining with streptavidin–PE. Non-islet mouse tissues were dispersed through a 7-µm nylon cell strainer and stained as detailed above.

Generation of mAb
Splenocytes and peripancreatic lymph nodes were harvested from pre-diabetic NOD female mice, lipopolysaccharide activated in culture (10 µg/ml) for 72 h and fused to the Ag8 hybridoma partner cell line. Growth-positive clones were screened on NIT-1 cells by cell-surface immunofluorescence.

125I cell-surface labeling, immunoaffinity isolation and identification of mAb target
NIT-1 (2 x 107) cells were surface labeled with 125I by combining Sulfo SHPP (16 µg), chloramine-T (28 µg) and 125I (2 mCi) as Na125I (ICN, Irvine, CA) at room temperature, and then applied to the cells. The labeled cells were incubated with 5.331 (50 µg) and SF1.1.1 (50 µg) in separate aliquots, washed, and then lysed with 40 mM MEGA 8 and MEGA 9 detergents, in the presence of protease inhibitors (1 mM PMSF, 10 mM iodoacetamide and 40 µM leupeptin). The mAb–protein complexes were precipitated with Protein A–Sepharose beads (35µl) (Sigma, St Louis, MO), washed, boiled and run on a 12% SDS–PAGE gel.

For immunoaffinity isolation, NIT-1 cells (8 x 109) were lysed in PBS containing 40 mM MEGA 8 and MEGA 9 detergents, in the presence of protease inhibitors (1 mM PMSF, 10 mM iodoacetamide and 40 µM leupeptin), and incubated for 12 h at 4°C with the mAb 5.331 coupled to cyanogen bromide-activated Sepharose beads (Sigma-Aldrich, St Louis, MO). The purified proteins were eluted from the column with 5 ml of 0.01% trifluoroacetic acid (pH 1.9) and dried. The eluate was then run on a 12% SDS–PAGE gel, and the bands of interest were excised, diced into 1-mm2 pieces, extracted with 25 mM NH4HCO3/50% acetonitrile, dried to complete dryness and then gel pieces were incubated at 37°C overnight in a solution of sequencing grade modified trypsin (Promega, Madison, WI). The tryptic fragments were recovered by sonication of the digest in water (aqueous extraction) followed by sonication in 50% acetonitrile:5% formic acid (organic extraction).

Reversed-phase HPLC and MS/MS analysis
The tryptic fragments were analyzed by reversed-phase HPLC on a Zorbax C18 0.3 mm x 15 cm column (Micro-tech, Sunnyvale, CA). Solvent A: 0.6% acetic acid in water; solvent B: 0.6% acetic acid in acetonitrile. Gradient: solvent B started at 3% to 15% in 5 min and from 15 to 60% at 0.8%/min. All the column effluent was introduced into the mass spectrometer. MS and MS/MS experiments were performed on a Finnigan LCQ-deca ion trap mass spectrometer with Xcalibur 1.1 software (San Jose, CA). For MS, scan range was m/z 700–1400 in profile mode. Every three micro scans were averaged to one scan. Acquisition began 10 min after start of the chromatography run. Parent ions for MS/MS were selected automatically according to intensity by using a dependent scan mode. For each selection, the most abundant, second most abundant and third most abundant precursor ions were selected excluding isotopic peaks; scan range was m/z 700–1400 in centroid mode for the MS part, in the MS/MS scan range was from 30% of the precursor ion m/z to m/z 2000, every three micro scans were averaged to give one scan in profile mode. Precursor ions were isolated with a 2.5-m/z window and collision energy was 28% of the maximum energy (~5 eV).

Product-ion spectra from MS/MS experiments were analyzed, and peptide sequences were determined automatically using either SEQUEST software provided by the instrument manufacturer and/or MASCOT (Matrix Science, London, UK). All the automatically determined sequences were verified by interpreting manually the experimental product ion scans.

RT-PCR, PCR, molecular cloning and sequencing
RNA was isolated from NIT-1, B16 and NOD islets using the High Pure RNA isolation kit (Roche, Mannheim, Germany). RT-PCR was carried out as previously described (20). The first-strand cDNA was synthesized from 2–4 µg of total RNA by using AMV reverse transcriptase and random 9mer oligonucleotides, and then the DNAs were amplified using primer sets for retroviral envelope protein (forward 5'-CGCGGAATTcAACCATCATGGAGAGTACAA-3' backward 5'-CGCGCTCGAGTTTTATTCACGTGATTTACAAT-3') and for ß-actin (forward 5'-CATGTTTGAGACCTTCAACACCCC-3', backward: 5'-GCCATCTCCTGCTCGAAGTCTAG-3'). The PCR reaction was performed for 35 cycles under the following conditions: 94°C for 60 s, 60°C for 60 s and 72°C for 90 s. The amplified PCR products were analyzed by electrophoresis on 1% agarose and staining with ethidium bromide. The 2010-bp fragments were excised and inserted into the pBluescript cloning vector. Nucleotide sequences were determined by using the BigDye DNA sequencing kit (Applied Biosystems, Foster City, CA) under the PCR conditions recommended by the manufacturer. The nucleotide sequences of the cloned genes were compared with genes listed in the GenBank database by using the homology searching program accessible through the NCBI website.

Expression of cloned protein
The cloned 2010-bp NIT-1 cDNA was excised from pBluescript and inserted into the pCEP4 mammalian expression vector. The B cell lymphoma line, C3.G7, was transfected with the vector by electroporation. Transfected cells were sorted and selected for reactivity with the anti-I-Ag7 mAb, AG2.42.7 and 5.331, and subcloned. The cloned 2010-bp NOD cDNA was excised from pBluescript and inserted into a bacterial expression vector with the addition of a 6-His tag at the 3' end. In vitro expression was performed with the Rapid Translation System (Roche) and the protein was purified on a nickel column by standard techniques.

Serum western blot analysis of early autoantigen of diabetes 1 (EAD1) protein
The in vitro expressed EAD1 protein (1 µg/lane) was run on 12% SDS–PAGE and transferred to PVDF membranes. The membranes were incubated with mouse serum at a dilution of 1:100, then with goat anti-mouse IgG–peroxidase and developed with an ECL detection system (Amersham Biosciences, Little Chalfont, UK).

RT-PCR analysis of tissue expression of EAD1 protein
RNA was isolated from the B16 and NIT-1 cell lines and from mouse tissues using the RNeasy kit (Quiagen, Valencia, CA). First-strand cDNA was produced and PCR was performed with the following sequence-specific primers. NIT/NOD specific: 5'-TGGGCTTGTAGTACTGGACTT, 3'-TTCACGTGATTTACAATCTTCTATTA, B16 specific: 5'-TGGGCTTGTAGTAC TGGACTC, 3'-TTCACGTGATTTACAATCTCCTATTG and GAPDH: 5'-CCTGGAGAAACCTGCCAAGTATGA, 3'-GGGT GCAGCGAACTTTATTGATGG.

Peptide synthesis
A peptide library was synthesized as overlapping 20mers covering the entire 669-amino-acid sequence of the envelope glycoprotein. Peptides were synthesized at Washington University, and their identity was verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Applied Biosystems, Foster City, CA).

Peptide binding assays
Peptide binding was done as before using soluble I-Ag7 from a recombinant baculovirus system and 125I-radiolabeled reference (GKKVATTVHAGYG) peptide with increasing doses of unlabeled peptides (21).

T cell cloning, splenocyte proliferation assays and peptide immunization
Spleens from female NOD mice that were diabetic for 1 week were harvested and dispersed: 5 x 105 splenocytes were incubated with or without 10 µM of the peptides for 72 h. The cultures were pulsed on day 3 with 1 µCi of [3H]thymidine and harvested the next day. In order to isolate individual T cell clones, bulk splenocytes were cultured in DMEM with 10% FCS, 10 µM EAD61–80 peptide and IL-2 (25 U/ml). On day 10 the T cells were isolated and cultured in 96-well plates at a concentration of 0.5 cells/well in the presence of 5 x 105 irradiated NOD splenocytes, 10 µM EAD61–80 peptide and IL-2 (25 U/ml). The T cell clones were tested at a concentration of 2 x 104 cells/well with 5 x 105 irradiated NOD splenocytes and peptide. Female NOD mice, 8 weeks of age, were immunized in the hind footpads with 10 nmol of EAD61–80 peptide emulsified in complete Freund’s adjuvant (Difco, Detroit, MI). Draining lymph nodes were harvested from mice on day 7 post-immunization and single-cell suspensions were used to set-up the limiting dilution analysis (LDA) as previously described (22).

Transfer into NOD.SCID recipients
The anti-EAD61–80 T cell clones were expanded in vitro with irradiated NOD splenocytes, EAD61–80 peptide and IL-2. The cells were transferred into NOD.SCID recipients by i.v. tail vein injection of ~10 x 106 cells. The mice were followed by blood glucose determination for >1 month, and following sacrifice the pancreata were examined histologically for pathology and the spleen was assayed for reactivity with the EAD61–80 peptide.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Genetics of ß cell autoantibodies
The generation of anti-ß cell antibodies requires both the diabetes susceptibility class II MHC and background genes. The presence of anti-ß cell antibody was assayed by flow cytometric analysis of NIT-1 and dispersed mouse ß cells stained with serum from three strains of mice (Fig. 1a). NOD mice, which bear the class II susceptibility molecule I-Ag7 and disease-susceptible background genes, have antibodies that bind to the surface of the NIT-1 cell line as well as to dispersed ß cells. In our colony, 85% of female mice develop diabetes by the age of 30 weeks. In contrast, B6.G7 mice (H-2g7 congenic B6) do not produce anti-ß cell antibodies and are resistant to the development of diabetes. Likewise, NOD.GD mice (H-2g2 congenic: Kd, Db, I-Ad, I-Enull) do not have anti-ß cell antibodies and do not develop disease. This finding demonstrates that the development of anti-ß cell antibodies, and diabetes, requires both the MHC II molecule I-Ag7 and NOD background genes.



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Fig. 1. The generation of antibodies against pancreatic ß cells requires both the diabetes susceptibility MHC class II and background genes. (a) The presence of anti-ß cell antibody was assayed by flow cytometric analysis of NIT-1 cells and dispersed mouse ß cells stained with serum from three strains of mice. The B6.G7 and NOD.GD mice strains do not generate anti-ß cell antibodies, and do not develop diabetes. (b) NOD mice develop anti-ß cell antibodies between 4 and 8 weeks of age. In order to characterize the time course of the development of antibodies against ß cells, serum from NOD female mice of various ages was collected and examined for the presence of antibody by staining of NIT-1 cells. The majority of animals go from seronegative to seropositive between 4 and 8 weeks of age. NOD females: 4 (three of 26), 6 (16 of 34) and 8 (35 of 44) weeks.

 
Time course of seroconversion and generation of mAb
In order to characterize the biology of anti-ß cell seroreactivity, we examined the sera of NOD mice at different ages: <10% of animals had antibodies at 4 weeks of age; however, by 8 weeks of age ~80% of animals developed antibodies against ß cells (Fig. 1b). The flow cytometric results of NIT-1 cells stained with serum from a diabetic NOD mouse demonstrated a positive shift in binding, from a mean florescence intensity of 22 in controls to 77 with NOD sera (Fig. 2a). These experiments define a biologic window during which the female NOD mouse spontaneously generates antibodies specific for antigens on the surface of the pancreatic ß cell.



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Fig. 2. FACS staining of NIT-1 cells and dispersed pancreatic islets. (a) NIT-1 cells were incubated with serum from B10.BR mice (black line), NOD mice (green line) and the mAb SF1.1.1 (red line) which recognizes H-2Kd, and then stained with a goat anti-mouse IgG. (b) NIT-1 cells stained with an isotype control IgG2b (black line), and mAb 5.331 (green line) and SF1.1.1 (red line). (c) Dispersed islets from ILK3 mice were stained with isotype control antibody (black line), mAb 5.331 (green line) and F10.6.6 (red line). (d) Dispersed NOD islets stained with isotype control (black line) and the mAb 5.331 (green line). FACS staining of dispersed NOD mouse tissue. (e) Splenocytes were incubated with an isotype control IgG2b (black line), mAb 5.331 (green line) and SF1.1.1 (red line). (f) Thymocytes. (g) Salivary gland. (h) Thyroid.

 
A B cell fusion of the spleen of a pre-diabetic NOD female mouse was performed and a mAb specific for NIT-1 cells was generated (Fig. 2b). The ß cell-specific mAb 5.331 is of isotype IgG2b and was generated from a 9-week-old normoglycemic female NOD mouse. Reactivity towards ß cells was confirmed by staining isolated and dispersed islets from NOD mice and ILK3 mice. ILK3 mice are mice of the B10.BR (H-2k) strain that express membrane-bound HEL under the rat insulin promoter. Islets from ILK3 mice were used to identify the ß cell population by co-staining with the mAb F10.6.6, which recognizes the lysozyme molecule. The mAb 5.331 stains ß cells isolated from both ILK3 mice and NOD mice, with a small, but reproducible and consistent, shift in mean florescence of 5 to 12 (Fig. 2c and d).

In order to determine the tissue specificity of 5.331, several tissues were dispersed and stained with the mAb. The mAb 5.331 was not reactive with spleen, thymus, salivary gland and thyroid (Fig. 2e–h).

Isolation and identification of mAb 5.331 target antigen
The NIT-1 cell line was cell-surface labeled with 125I and immunoprecipitation with the mAb 5.331 was performed. Three bands were identified, corresponding to proteins of 70, 50 and 20 kDa, with the latter being the most prominent (Fig. 3a). A large-scale immunoaffinity purification was then performed on NIT-1 lysate and the eluate run on a 12% SDS–PAGE gel. The Coomassie-stained gel revealed a pattern of bands that exactly recapitulated the pattern seen in the 125I-labeling experiment. Each of the bands was excised, treated with trypsin, and the extracted tryptic peptides were sequenced in part by reversed-phase HPLC and tandem MS/MS (Fig. 3b and c). In total, eight peptides were sequenced from the three immunoprecipitation products. Many of the peptides were found in each of the three bands, an indication that they derived from the same protein. Sequence analyses, using the BLAST program, revealed that these peptides were from a large family of mouse retroviral envelope glycoproteins.



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Fig. 3. The mAb 5.331 immunoprecipitates proteins of 70, 50 and 20 kDa. (a) The NIT-1 cell line was cell-surface labeled with 125I and immunoprecipitation was performed with the mAb 5.331. The eluted protein was run on a 12% acrylamide gel and exposed to film. Immunoprecipitation with SF1.1.1 was performed simultaneously as an internal control. (b) The tryptic fragments of the immunoprecipitated proteins were analyzed by reversed-phase HPLC with MS/MS. Two of the peptides identified are indicated adjacent to the peaks which contained the parent ions for the amino acid sequencing of the peptides. (c) A total of eight peptides were identified and sequenced out of the three immunoprecipitating bands.

 
Cloning, sequencing and expression of the target protein, EAD1
Based on the high degree of homology shared by all the available mouse retroviral envelope nucleotide sequences, we chose the mouse melanoma antigen (23) upon which to design primers for PCR amplification. RNA was isolated from the NIT-1 and B16 melanoma cell lines, and from NOD pancreatic ß cells, and cDNA was generated by RT-PCR. The PCR reaction performed on the cDNA, with the primers designed to cover both the start and stop codons of the mouse melanoma nucleotide sequence, yielded a 2-kb product in each sample. These cDNA were cloned and sequenced. The sequence obtained from the B16 clones was identical to the published sequence that had served as a template for the design of the primers. The NIT-1 and NOD nucleotide sequence differed from the B16 sequence by 12 and 9 nucleotides respectively, all of which encode for amino acid differences (Fig. 4). We have termed the protein reactive with the antibody 5.331 as EAD1.



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Fig. 4. Unique sequence of EAD1. The translation of the cloned and sequenced 2010-bp message recovered from the NOD ß cells is presented in alignment with the cloned B16 and NIT-1 sequences. The sequence of EAD1 differs from the B16 sequence by 9 nucleotides, which all encode for amino acid differences.

 
The differences in the amino acid sequences are summarized in Table 1.


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Table 1. Amino acid sequence differences
 
The cDNA encoding the NIT-1 EAD protein, the protein reactive with the 5.331 antibody, was inserted into a mammalian expression vector and transfected into the I-Ag7-expressing B cell lymphoma line, C3.G7. The expressed cell-surface protein on the transfected cell line, C3EAD, was recognized by the mAb 5.331, which confirms the specificity of the mAb (Fig. 5a and b). The specificity of the 5.331 antibody was further demonstrated by its lack of reactivity with the mouse melanoma antigen (Fig. 5c and d), which differs from the EAD1 protein by 12 amino acids. Reciprocally, the M562 mAb, which recognizes the mouse melanoma antigen, does not bind to the EAD1 protein (Fig. 5e and f).



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Fig. 5. The expressed EAD1 protein is recognized by the mAb 5.331. (a) The C3.G7 cell line was transfected with the EAD1 sequence with a mammalian expression vector. The non-transfected C3.G7 cell line was stained with the mAb AG2.42.7, which recognizes I-Ag7, and with the mAb 5.331. (b) The transfected cell line, C3EAD, stains positively with both mAb AG2.42.7 and mAb 5.331. Differential staining with two anti-envelope-specific antibodies. (c) The B16 melanoma cell line was stained with an isotype control (black line) and the M562 mAb which recognizes the mouse melanoma antigen (green line) with a shift in mean florescence intensity of 8 to 30. (d) The 5.331 antibody does not stain the B16 cell line (green line); isotype control (black line). (e) M562, which recognizes the mouse melanoma antigen, does not bind to the NIT-1 cell line (green line); isotype control (black line). (f) The NIT-1 cell line, which expresses EAD1, stains positively with 5.331 (green line); isotype control (black line). Analysis of tissue distribution of EAD1 mRNA expression. (g) Various tissues were probed by a sequence-specific RT-PCR technique for the presence of mRNA encoding the EAD1 sequence or the B16 sequence. The product obtained with the NOD-specific primers is shown in the top row, the product obtained with the B16 primers is shown in the middle row and the product generated with a set of GAPDH primers is displayed in the bottom row as a control. The products obtained with NIT-1 (lane 1) RNA and B16 (lane 2) RNA demonstrate the specificity of this technique, with a product generated only in the presence of the appropriate primers. Analyses of NOD islets (lane 3), kidney (lane 4), liver (lane 5), thyroid (lane 6), salivary gland (lane 7), spleen (lane 8) and thymus (lane 9) are presented. NOD mouse serum contains antibodies that are reactive with EAD1 protein by western blot. (h) The sera of control strains of mice and NOD mice of different ages was examined for the presence of antibodies reactive with the EAD1 protein by western blot analysis. The in vitro expressed protein, with a His tag at the C-terminus, was first stained with an anti-His antibody revealing both the 74- and 24-kDa fragments of the EAD1 protein. Control strains of mice, B10.BR (none of five) and CB17 (none of five), do not contain antibodies against the EAD1 protein. All NOD mice with diabetes (five of five) were positive, the majority of animals examined at 8 weeks of age were positive (four of five) and none of the NOD mice examined at 4 weeks of age were positive (none of six).

 
Tissue distribution of EAD1 mRNA expression
The tissue distribution of EAD1 protein expression was analyzed by sequence-specific RT-PCR which differentiated between the NIT-1 and B16 sequences. NOD islets from 10- to 12-week-old mice generated a product of the expected size when probed with the NIT-1-specific primers, but not with the B16 primers (Fig. 5g, lane 3). Not shown are the results from islets from 4-week-old NOD mice, isolated from 24 animals, which were also positive for the EAD1 message. Other tissues also expressed a positive, although weak product such as liver (Fig. 5g, lane 5), thyroid (Fig. 5g, lane 6) and spleen (Fig. 5g, lane 8). It is possible that these tissues contain a sequence which is similar to the EAD sequence over the span of the primers, but is otherwise different, or that the antigenicity of the protein depends upon a conformational or post-translational modification that is unique to its expression in ß cells. Nevertheless, the EAD1 sequence is clearly recovered from NOD islets from 10- to 12-week-old animals; in addition, EAD1 sequence was recovered from NOD islets from 4-week-old animals and from NOD.SCID mice.

Western blot analysis of mouse serum reactivity against EAD1 protein
Sera from NOD mice of various ages and from other strains of mice were examined for the presence of antibodies reacting with the EAD1 protein. The presence of anti-EAD1 antibodies was specific to the NOD mouse strain B10.BR and CB17 mice were negative; this reactivity in the NOD mouse appears between 4 and 8 weeks of age, and was present in all diabetic mice examined (Fig. 5h). These results establish that a component of the anti-ß cell seroreactivity that appears in NOD mice between 4 and 8 weeks of age is targeted against the EAD1 protein.

Identification of anti-EAD1 T cell reactivity in the NOD mouse
Splenocytes from newly diabetic female NOD mice were screened for reactivity with an overlapping peptide library covering the entire 669-amino-acid sequence. Reactivity with the peptide EAD61–80 was observed in newly diabetic mice (Fig. 6a). Proliferation with this peptide was found to be ~10-fold over background. The sequence of the EAD61–80 peptide is GNHPLWTWWPDLTPDLCMLA. Based on previous studies, it is likely that the core sequence extends from Thr67 to Asp75. We previously determined that the critical amino acid in the MHC binding has an acidic side chain at P9 and Asp75 fits that requirement (18). The EAD61–80 peptide binds to soluble I-Ag7 with an IC50 of 6.6 µM (Fig. 6b). In addition to this bulk splenocyte reactivity, multiple T cell clones reactive with the EAD61–80 peptide were isolated from unmanipulated as well as immunized NOD mice (Fig. 6c and d). The sensitivity, the antigen dose that results in 50% maximal stimulation of the T cell clones derived from unmanipulated splenocytes, was 4 µM peptide while that of the T cell clones derived from the immunization was 0.6 µM. Furthermore, mice immunized with EAD61–80 demonstrated a peptide-specific primary lymph node response: the frequency of anti-peptide specific T cells was determined to be 1/8355 by limiting dilution analysis.



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Fig. 6. T cells react with peptide EAD61–80. A splenocyte proliferation assay was performed with a library of overlapping peptides covering the entire EAD1 protein. (a) Reactivity with the peptide EAD61–80 is demonstrated in the results obtained from the spleen of a 12-week-old female NOD mouse that had developed diabetes 5 days prior to sacrifice. (b) The EAD61–80 peptide binds to soluble I-Ag7 with an IC50 of 6.6 µM. Indicated is the concentration of unlabeled peptide that affects the binding of 125I reference peptide to I-Ag7 protein. (c) T cell clones reactive with EAD61–80 were isolated by subcloning out of the spleen of newly diabetic female mice and (d) following immunization with peptide. Indicated is the growth of the T cells in the presence of varying concentrations of EAD61–80.

 
Transfer of anti-EAD T cell clones into NOD.SCID recipients
Several T cell clones reactive with the EAD61–80 peptide were expanded in vitro and injected into NOD.SCID recipients. The clones C03, C07, C09 and D07.6 were cloned out of the spleens of unmanipulated NOD mice of various ages, and all showed specific reactivity with the EAD61–80 peptide. The LDA #1 T cell line was cloned out of the popliteal lymph node of a 8-week-old NOD mouse immunized with EAD61–80 in complete Freund’s adjuvant. The recipient mice did not develop diabetes over the time that they were followed, up to 37 days post-transfer, and no pathology was detectable in the islets of the pancreas at the time of sacrifice (Table 2). Importantly, the spleens from the recipient NOD.SCID mice harbored EAD61–80-reactive T cells at sacrifice when examined in a proliferation assay; however, these cells did not appear to localize to the islets or to cause any detectable injury.


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Table 2. Transfer of anti-EAD T cell clones into NOD.SCID recipients
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We confirmed in this study the findings of many investigators that autoantibodies to ß cells develop early in the life of the NOD mouse. These autoantibodies are not only directed at intracellular antigens, as was convincingly shown by others, but also to surface exposed proteins, a finding also made by several groups (9,12,14,24). In the NOD mouse, the development of diabetes depends on the expression of the I-Ag7 molecule, as well as other proteins encoded by genes of the NOD strain (25,26). As shown here, similar requirements apply for the development of cell-surface autoantibodies. These autoantibodies develop at a time when the islet pathology is minimal, characterized by the presence of a few discrete lymphocytes around islets containing normal appearing ß cells. The purpose of our studies has been to characterize biochemically the antigens that give rise to these early antibodies. In this regard, the isolation of a mAb from the pool of autoreactive antibodies has proven effective in the identification of a ß cell autoantigen.

The autoantigen that we report here is encoded by a retroviral gene. Retroviral sequences are widely distributed in the mouse genome, but the extent of their expression among tissues remains largely undetermined. The ß cell gene identified here, with the aid of an autoantibody, differs from that encoded in the B16 and NIT-1 cell lines by several nucleotides. A sequence similar to the EAD1 sequence is present on chromosome 8 in the mouse genome database. The isolation of this autoantibody and the demonstration that a component of the anti-ß cell reactivity in NOD mouse serum is targeted against this antigen by 8 weeks of age, together with the results of others (2733), leads to an unavoidable conclusion: that retroviral gene products are a component of the spectrum of early ß cell autoreactivity in the NOD mouse. In addition, the fact that these autoantibodies are of the IgG class indicates that they evolved under T cell influence. Indeed, we showed that CD4 T cells specific for the EAD1 protein arise spontaneously or following immunization in the NOD mouse. (The autoantibody to the EAD1 protein is one of several cell-surface autoantibodies, as judged by our unpublished experiments testing the attenuation of serum staining by pre-blocking with the 5.331 mAb.)

Our results not only confirm prior findings which implicate a role for a ß cell retroviral autoantigen, but extend them along three lines: (i) to a precise molecular identification of the ß cell env protein, (ii) to the identification, for the first time, of CD4 T cells reactive to a segment of the protein, and (iii) to documenting that immune reactivity is an early, not late, event in diabetogenesis. Several findings emerge from our studies which, in turn, raise several important issues.

Specificity
The autoantibody, 5.331, appears to be specific for ß cells and insulinoma cell lines such as the NIT-1 cell line. The antibody has no reactivity with other tissues such as spleen and thymus, and does not recognize various tumor lines including the B16 melanoma line that expresses the mouse melanoma antigen, a retroviral protein that differs from EAD1 by only 9 amino acids. The specificity of the 5.331 antibody for the EAD1 protein was clearly demonstrated by showing that its expression in the B cell lymphoma line resulted in the acquisition of antibody reactivity. This result proves that the antibody is specific for the env protein that is expressed in the NOD mouse ß cell. In addition, the demonstration of NOD mouse serum reactivity against the EAD1 protein establishes that a component of the anti-ß cell reactivity which appears in the NOD mouse by 8 weeks of age is targeted against this antigen.

We are pursuing further analysis of the EAD1 gene with the intention of evaluating its pattern and regulation of expression in the ß cell. It may be that its site of integration is critically responsive to hormonal and metabolic influences specific to the ß cell. It is yet to be determined whether the protein is derived from active retroviral infection or whether it is transcribed from a stable, endogenous genomic sequence of retroviral origin. Furthermore, because the sequence for the EAD1 protein was cloned out of NOD islets by the use of primers designed to cover an envelope glycoprotein, other components (e.g. gag or pol proteins) of the retroviral structure may be present. Whether the EAD1 protein with the sequence identified is selectively expressed on other cell types, as the analysis of RNA from other tissues may suggest, requires further examination. Of note, the 5.331 antibody is not specific for NOD ß cells, but reacts with ß cells from other strains of mice such as B10.BR. The recovery of mRNA from tissues which are clearly negative for 5.331 reactivity by the use of sequence-specific primers implies that these products are different in other parts of their sequence, or that there exists a conformational or post-translational modification of EAD1 which results in the antigenicity of the expressed protein that may be unique to the ß cells of the pancreas.

Immunogenicity
The isolation of this autoantibody, together with the results of others (2730,3235), leads to the unavoidable conclusion that retroviral gene products represent a component of the spectrum of anti-ß cell autoreactivity present in the NOD mouse. The fact that these autoantibodies are of the IgG class indicates T cell participation in their genesis. Indeed, we have shown that CD4 T cells specific for the protein arise spontaneously or following immunization in the NOD mouse. The frequency of reactive T cells as determined by limiting dilution analysis is similar to that found in response to foreign antigens. Thus it appears that the EAD1 protein is not tolerogenic in the NOD mouse. Interestingly, that segment of the EAD1 protein that appears to be the T cell epitope, EAD61–80, is conserved among many mouse env genes and interacts with I-Ag7 with moderate binding affinity.

The events that lead to the immunogenicity of the EAD1 protein or other retroviral proteins are not clear. Along these lines, neither is there an explanation for the immunogenicity for other ß cell autoantigens. Whatever the ultimate explanation, several features are important to note: (i) the expression of the EAD1 gene appears to be late in ontogeny, (ii) its appearance is not conditioned by a prior diabetogenic process mediated by T cells or inflammation, since the NOD.SCID mice expressed it, and (iii) the EAD1 protein represents an early immunological reactivity.

Pathogenicity
We need to establish whether the EAD1 antigen is critically involved in the pathogenesis of diabetes; perhaps as an initiating autoantigen or one that is secondary to an initiating event and which by itself is not pathogenic. The interpretation of defining criteria for pathogenic or non-pathogenic autoantigens is difficult because, for the most part, the ß cell antigens that trigger autoreactivity are known to a very limited extent.

The failure of the EAD-reactive T cell clones to transfer diabetes may have several explanations. First, it may be that CD8 and/or CD4 T cells targeting other epitopes of the EAD1 protein are required for pathogenic injury of the ß cells, i.e. we have yet to isolate the pathogenic effector T cell. Along these lines, we have to examine whether the present sets of isolated T cells are reactive with epitopes from the processing of the env protein: so far our indication is that the T cells react to peptides. Other work from our laboratory has indicated differences between T cell reactivity to exogenous peptides and peptides from the processing of the protein (36). Second, the levels of EAD1 protein may be below that required to induce an effector reaction at the level of the islets. This would imply that immunogenicity, i.e. the capacity of the antigen to trigger immune reactivity, is more sensitive than the effector phase, i.e. the capacity of the T cells to migrate and initiate inflammation and provoke a ß cell death program. Conceivably the EAD1 T cells may participate after some prior level of injury or inflammation which is required for them to traffic to the islets and become activated. We are evaluating this issue currently in the NOD.SCID model. Third, it is possible that the EAD1-reactive T cells may be playing an inhibitory or modulatory role in a process of ß cell injury mediated by other pathogenic T cells. This last possibility is intriguing given the mounting evidence that the development of diabetes in the NOD mouse may depend upon alterations in the balance between pathogenic and regulatory T cells (37). Some of these issues may be clarified in experimentation now in progress; isolating new CD4 T cell clones, examining for CD8 T cells, testing their reactivity with EAD1 protein and peptides, and co-transferring them with other known diabetogenic T cells.

In summary, doubts on a role for retroviral antigens up to this point come about from the lack of identification of T cells, questions on the specificity of antibodies found in serum and from the lack of well-defined ß cell retroviral antigens. Some of these doubts are resolved here, by the information obtained on the ß cell sequence, the specificity of the 5.331 antibody and the finding of spontaneously reactive T cells. The early appearance of this reactivity indicates that it is not a late product of ß cell inflammation; whether it is an initiating autoantigen, a contributing one or one that is secondary to an initiating event and which by itself is non-pathogenic needs to be defined. Our data do add EAD1 to the list of defined ß cell antigens that need to be considered in future analysis. Its role in the pathogenesis of diabetes may be revealed by future experiments involving antigen specific tolerance induction in the NOD mouse.


    Acknowledgements
 
We want to thank Kevin Clark, Thomas Esparza, Gina Filley, Kathy Frederick, Shirley Petzold and Barbara Vaupel for their excellent technical support. We thank Marco Colonna and Marina Cella for their insights and help. This work was supported by grants from the National Institutes of Health and from the Kilo Diabetes and Vascular Research Foundation (St Louis, MO). M. G. L. was supported by a clinical investigator faculty award from the Howard Hughes Medical Institute to Washington University. The mass spectrometry was supported in part by the NCRR of the NIH (grant P41RR00954).


    Abbreviations
 
EAD1—early autoantigen of diabetes 1

env—retroviral envelope protein

HEL—hen egg lysozyme

NOD—non-obese diabetic


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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