Immunology and Transplantation|August 20 2011
Thomas Delong; Rocky L. Baker; Nichole Reisdorph; Richard Reisdorph; Roger L. Powell; Michael Armstrong; Gene Barbour; Brenda Bradley; Kathryn Haskins Corresponding Author
Integrated Department of Immunology, University of Colorado Denver School of Medicine and National Jewish Health, Denver, Colorado
Diabetes 2011;60(9):2325–2330
To investigate autoantigens in β-cells, we have used a panel of pathogenic T-cell clones that were derived from the NOD mouse. Our particular focus in this study was on the identification of the target antigen for the highly diabetogenic T-cell clone BDC-5.2.9.
To purify β-cell antigens, we applied sequential size exclusion chromatography and reverse-phase high-performance liquid chromatography to membrane preparations of β-cell tumors. The presence of antigen was monitored by measuring the interferon-γ production of BDC-5.2.9 in response to chromatographic fractions in the presence of NOD antigen-presenting cells. Peak antigenic fractions were analyzed by ion-trap mass spectrometry, and candidate proteins were further investigated through peptide analysis and, where possible, testing of islet tissue from gene knockout mice.
Mass-spectrometric analysis revealed the presence of islet amyloid polypeptide (IAPP) in antigen-containing fractions. Confirmation of IAPP as the antigen target was demonstrated by the inability of islets from IAPP-deficient mice to stimulate BDC-5.2.9 in vitro and in vivo and by the existence of an IAPP-derived peptide that strongly stimulates BCD-5.2.9.
IAPP is the target antigen for the diabetogenic CD4 T-cell clone BDC-5.2.9.
CD4 T cells are major immune players in the initiation and pathogenesis of type 1 diabetes, but the identification of the β-cell antigens that drive autoreactive T-cell responses has been a long and difficult quest. Although nearly 20 different proteins have been identified as targets for T cells in the NOD mouse, and at least 12 of these also are autoantigens in human patients (1), the impact of most of these proteins on the disease process is not well understood, particularly with regard to antigens for CD4 T cells. We previously have described islet-reactive CD4 T-cell clones that are representative of memory effector CD4 T cells arising during the progression of disease in the NOD mouse (2). In response to mouse islets from a variety of strains, clones from the panel produce Th1 inflammatory cytokines, especially interferon (IFN)-γ (3), and are highly diabetogenic in vivo. The best-known T cell from this panel is BDC-2.5, the T-cell receptor of which was used to produce the BDC-2.5 T-cell receptor transgenic mouse (4), an animal used by many investigators to probe mechanisms of pathogenic T-cell activity and regulation. The BDC T-cell clones initially were selected on the basis of their ability to respond to antigens in the form of whole islet cells, presented by NOD antigen-presenting cells. We recently identified the protein antigen for BDC-2.5 and two other T-cell clones (BDC-5.10.3 and BDC-10.1) as chromogranin A (ChgA), a secretory granule protein in islet β-cells (5). Here, we report that the antigen for another diabetogenic CD4 T-cell clone, BDC-5.2.9, is islet amyloid polypeptide (IAPP), a 37–amino acid peptide hormone cosecreted with insulin.
NOD, NOD.scid, and NOD.RIP-TAg mice were bred and maintained in the Biological Resource Center at National Jewish Health. NOD.IAPP−/− mice were bred in our colony by backcrossing C57BL6.IAPP−/− mice (6) onto the NOD background; these mice have been fully backcrossed to the NOD mouse (10 generations). All experimental procedures were in accordance with the guidelines of the institutional animal care and use committee, National Jewish Health.
NOD.RIP-Tag mice (7) were used as a source of β-cell tumors for the preparation of antigen; the enrichment of membrane proteins from β-cells isolated from NOD.RIPTAg adenomas has been previously described (8). In brief, single-cell suspensions of β-cell adenomas were prepared by pressing whole-tumor tissue through a mesh screen, followed by several centrifugation steps to obtain a cellular membrane pellet; this pellet (β-Mem) is highly antigenic for the T-cell clones and serves as a positive control antigen in T-cell assays as well as the source of antigen for further purification. Membrane protein preparations were solubilized in detergent-containing buffer (20 mmol/L Tris, pH 8.0, and 1% octyl-β-glucoside), followed by centrifugation at 18,400 g (10 min at 4°C) to remove insoluble debris. Size-exclusion chromatography (SEC) was carried out on a Superdex 200 16/60 column (Amersham Biosciences) at 21°C (flow rate: 1 mL/min; fraction size: 1.25 mL; and injection volume: 2.0 mL), eluting with SEC buffer (20 mmol/L Tris, pH 8.0, and 150 mmol/L NaCl). Fractions from SEC were assayed for antigenic activity with the T-cell clones, and a peak antigenic fraction (950 μL) was acidified with 20 μL trifluoroacetic acid before adding 30 μL acetonitrile. A total of 800 μL of this mixture was then applied to a reverse-phase high-performance liquid chromatography (RP-HPLC) mRP-C18 high-recovery protein column (Agilent). A buffer gradient was used to elute proteins from the column, and a total of 36 fractions were collected between 0 and 72 min at a flow rate of 0.75 mL/min and a constant column temperature of 80°C. Antigenic peaks were again determined through assays with T-cell clones.
The antigenicity of islet cells, cellular and biochemical fractions, or peptides was assessed through the IFN-γ responses of T-cell clones. In 96-well microtiter plates, assay cultures contained 2 × 10 4 responder T cells, 2.5 × 10 4 NOD peritoneal exudate cells as antigen-presenting cells, and β-cell antigen. IFN-γ production by the responder T cells was determined by ELISA (BD Biosciences). IFN-γ standards were added to the assay plates at concentrations between 0 and 100 ng/mL, but concentrations of the standard >50 ng/mL were above the assay detection range. An aliquot of β-Mem or whole NOD islet cells were used as positive controls; test wells contained SEC or RP-HPLC fractions, peptides, or islet cells. Organic solvents were removed from HPLC fractions by vacuum centrifugation prior to assay. Synthetic peptides were obtained from CHI Scientific.
The protein-identification strategy we have previously described (5) was used for mass spectrometry. Proteins were digested with trypsin, and extracted peptides were resolved by chromatography online on a C18 column and a 1200 Series HPLC system (Agilent Technologies). Analysis was carried out with a 6340 LCMS ion-trap mass spectrometer in the National Jewish Health facility. Raw data were extracted and used to search the Swiss-Prot or National Center for Biotechnology Information databases with the Spectrum Mill search engine (Rev A.03.03.038 SR1; Agilent Technologies). Data were evaluated and protein identifications were considered significant if the following confidence thresholds were met: individual peptide scores of at least 10 and scored percent intensity of at least 70%. A reverse (random) database search was simultaneously conducted, and manual inspection of spectra was used to validate the match of the spectrum to the predicted peptide fragmentation pattern.
Cell cultures of BDC-2.5 and BDC-5.2.9 were expanded in interleukin-2, and after harvesting, T cells (1 × 10 7) were injected intraperitoneally into age-matched 6- to 14-day-old NOD or NOD.IAPP−/− recipient mice. Mice were monitored on a daily basis for urine glucose using Diastix (Bayer). The presence of urine glucose was followed by blood glucose monitoring using a OneTouch Ultra glucometer (LifeScan). Mice were considered diabetic when blood glucose levels were >18 mmol/L (324 mg/dL). For disease-transfer experiments, a Wilcoxon rank sum test was used to determine statistical significance.
As previously described (5), to identify candidate antigens for the T-cell clones, we carried out biochemical fractionation of β-cell tumors and mass spectrometric analysis of peak antigenic fractions. Similar to the other clones from the panel, BDC-5.2.9 responds in vitro to pancreatic islet cells or cell extracts of β-cell adenomas presented in the context of I-A g7 (2). Our starting source of antigen was β-cell tumor tissue excised from NOD.RIPTAg mice (7) and separated by differential centrifugation to yield a highly antigenic β-cell membrane pellet (β-Mem); this preparation also served as the positive control in T-cell activity assays. After lysis, the membrane pellet was separated by sequential chromatography, first through an SEC column and then by RP-HPLC. Illustrated in Fig. 1, the presence of antigen was tracked through the IFN-γ response of the T-cell clone BDC-5.2.9 (top axis, gray lines) to protein (bottom axis, black lines) in individual fractions. Figure 1 A is the chromatogram from SEC and shows a single antigenic peak that corresponds to a low–molecular weight protein region. The antigenic fractions from SEC were further purified by RP-HPLC, yielding another single peak of antigen activity (Fig. 1 B).
FIG. 1.
Chromatographic purification of the antigen for the T-cell clone BDC-5.2.9. A: SEC of β-cell membrane lysate. B: RP-HPLC of antigenic SEC fraction no. 60. Protein elution was monitored by absorbance at 280 nm (black lines). Presence of antigen was determined by measuring the IFN-γ response (gray lines) of the T-cell clone BDC-5.2.9 to small aliquots of individual fractions in the presence of peritoneal exudate cells used as antigen-presenting cells. Data are representative of at least four separate experiments.
The highly enriched and antigenic RP-HPLC fractions were subjected to in-solution tryptic digest and were then analyzed on an ion-trap mass spectrometer. Resulting spectra were sequenced and matched to proteins by searching the Swiss-Prot protein database using the search program Spectrum Mill. We identified a total of 21 proteins distributed over HPLC fractions 11–14 (Fig. 2 A). Comparison of the antigen distribution profile with individual protein spectral-intensity values, an approximate indicator for relative protein abundances, singled out IAPP as the only candidate. Other proteins did not match the antigen distribution profile; for example, secretogranin 1 was detected only in nonantigenic fractions 11 and 14. We identified two IAPP peptides, representing 28% amino acid coverage of prepro-IAPP and 70% coverage of the 37-residue peptide hormone (Fig. 2 B and C). The peptide LANFLVR was matched with high confidence and the peptide SSNNLGPVLPPTNVGSNTY was matched with medium confidence.
FIG. 2.
Mass-spectrometric analysis of peak antigenic HPLC fractions. Antigenic
