Chromogranin A is an autoantigen in type 1 diabetes

Journal name:
Nature Immunology
Year published:
Published online


Autoreactive CD4+ T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4+ T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-Ag7 in an atypical manner, occupying only the carboxy-terminal half of the I-Ag7 peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.

At a glance


  1. Purification of the antigen for the T cell clone BDC-2.5.
    Figure 1: Purification of the antigen for the T cell clone BDC-2.5.

    (a) SEC of beta-cell membrane lysate (13.8 mg). (b) IEX of pooled antigenic SEC fractions 60–62. In a,b, the protein content for each chromatographic fractionation was monitored by its absorption at 280 nm (A280). The fractions obtained were tested for the presence of antigen with the T cell clone BDC-2.5; 1 antigen unit induces the production of 10 ng/ml of IFN-γ in standard antigen assay conditions. (c) Tricine-Tris gel electrophoresis of beta-cell membrane lysate (β-Mem; 4 antigen units); pooled antigenic SEC fractions 60–62 (SEC; 4 antigen units); peak antigenic IEX fraction 21 (21 (above lane); 4 antigen units); and adjacent IEX fractions 19, 20, 22 and 23 (labels above lanes; less than 4 antigen units). Table 1 correlates to data obtained in this figure. Data are representative of at least three independent experiments.

  2. Mass spectrometry of IEX fractions.
    Figure 2: Mass spectrometry of IEX fractions.

    (a) Proteins identified (right) in the highly purified antigenic IEX fraction 21 and adjacent fractions with lower antigenic activity (fractions 19, 20, 22 and 23; Fig. 1b), which were digested with trypsin, separated by high-performance liquid chromatography and analyzed by ion-trap mass spectrometry; the resulting spectra were used to search a protein sequence database. The summarized numeric spectral intensity (middle) is indicative of the relative abundance of a specific protein in a fraction; darker colors indicate higher intensity. Tandem mass spectrometry (MS/MS) search scores (left) >20 are considered statistically significant. (b,c) Ion-trap mass spectra matching the ChgA peptides AEDQELESLSAIEAELEK (b) and SDFEEKKEEEGSAN (c). Above graphs, peptide amino acid sequence; vertical lines in the sequence correspond to b-ion series (progressing from the amino terminus to the carboxyl terminus) and the complementary y-ion series (progressing from the carboxyl terminus to the amino terminus). Data are representative of five independent experiments. (d) Complete ChgA amino acid sequence; red lettering and underlining indicates four peptides detected and matched to ChgA.

  3. Absence of the T cell antigen in islets from Chga-/- mice.
    Figure 3: Absence of the T cell antigen in islets from Chga−/− mice.

    (a,b) IFN-γ response of the BDC-2.5, BDC-10.1, BDC-5.10.3 and (insulin-reactive) PD-12.4.4 T cell clones to various concentrations of beta-cell tumor membrane proteins (a) and to various numbers of islet cells obtained from Chga−/− or (control) Chga+/+ mice (b). (c) Summary of the results in b; average concentration of antigen in islet cells from Chga−/− or Chga+/+ mice is presented as antigen units per 1 × 103 islet cells. Data are from four experiments (BDC-2.5 and PD-12.4.4; one to four replicates per experiment), two experiments (BDC-10.1; one to two replicates per experiment) or one experiment (BDC-5.10.3; single sample); error bars, s.e.m. (BDC-2.5 and PD-12.4.4).

  4. Mimotope peptide antigens for the BDC T cells suggest the region of ChgA that contains the epitope for the BDC T cells.
    Figure 4: Mimotope peptide antigens for the BDC T cells suggest the region of ChgA that contains the epitope for the BDC T cells.

    (a) Flow cytometry of SF9 insect cells infected with an unsorted library (left), a library sorted three times (3× sorted; middle) or pS3 clonal virus (right), analyzing I-Ag7 expression and the binding of a fluorescent, oligomerized, soluble BDC-2.5 TCR used to enrich from a baculovirus library a virus encoding an I-Ag7-mimotope (pS3) that forms a strong ligand for the BDC-2.5 TCR (Online Methods). Data are from a single experiment done after sorting and cloning were complete. (b) IL-2 production by BDC T cell hybridomas stimulated in culture with immobilized H597 monoclonal antibody to the TCR β-chain constant region (Anti-TCR) or with SF9 cells expressing CD80 and ICAM-1 and infected with virus encoding pHEL–I-Ag7 or pS3–I-Ag7, assayed after 24 h. Results are representative of three experiments, each assayed in duplicate. (c) Sequences and activity of the pS3 mimotope and those identified before by other library techniques11, 12. Potency in stimulating the BDC T cell clones is presented qualitatively as follows (based on results in b for pS3 or as reported before for the other mimotopes11, 12): ++, very strong stimulation; +, modest stimulation; −, little or no stimulation; ND, not determined. Red indicates motif positions p5, p7 and p8. (d) IFN-γ production by the BDC-2.5 and BDC-10.1 T cell clones stimulated by SF9 insect cells expressing CD80 and ICAM-1 and infected with baculovirus encoding membrane-anchored I-Ag7 covalently bound to each of three peptides (pHEL, pS3 and the ChgA-derived peptide WEDKRWSRMD). Results are from a single experiment done in duplicate. (e) Flow cytometry analysis of CD69 surface expression by three BDC hybridomas with substitution of the pS3 glycine residue at position p3 (red) with other amino acids (red), including lysine, the amino acid found at this position in the ChgA peptide (pChgA); results are presented relative to those of cells activated with the unsubstituted pS3 peptide. Data are from a single experiment.

  5. The ChgA-derived peptide WE14 activates all three BDC T cells.
    Figure 5: The ChgA-derived peptide WE14 activates all three BDC T cells.

    (a) Amino acids 350–373 of ChgA; the extent of the WE14 peptide is indicated by double-headed arrow below. Putative positions in the I-Ag7 peptide-binding groove (positions 1–9) are numbered above the sequence; red indicates the motif common to the antigen peptide mimotopes. (b) IFN-γ response of the BDC-2.5, BDC-10.1, BDC-5.10.3 and PD-12.4.4 T cell clones stimulated by various concentrations of pS3, WE14, B:9-23 (Ins; SHLVEALYLVCGERG) and beta-cell tumor membrane preparation (β-Mem); peptide antigen was titrated in each assay. Data are representative of at least two separate experiments with a single measurement at each peptide concentration.

  6. Precise processing of the WE14 peptide is required for optimal presentation by I-Ag7.
    Figure 6: Precise processing of the WE14 peptide is required for optimal presentation by I-Ag7.

    (a) IFN-γ response of the BCD-2.5 T cell clone to various concentrations (5–500 μM) of ChgA-derived peptides. Data are representative of two separate experiments with a single measurement at each peptide concentration. (b) Analysis of the ability of ChgA-derivative peptides to compete with a biotinylated HEL peptide (bio-pHel) for binding to soluble I-Ag7, presented as the amount of biotinylated HEL peptide bound to I-Ag7 (percent of that bound in the absence of an inhibitor peptide) versus the concentration of inhibitor peptide. The pS3 mimotope serves as a positive control; an I-Ek-binding peptide from moth cytochrome c (pMCC) serves as a negative control. Data are the average of two experiments with similar results. (c) Multiple-regression analysis of the stimulation and inhibition curves in a and b, treated as a series of parallel polynomial curves (assessed with the program MKASSAY; available on request); results are presented as the stimulatory or inhibitory activity of the peptides relative to that of WE14.


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Author information

  1. These authors contributed equally to the work.

    • Brian D Stadinski &
    • Thomas Delong


  1. Integrated Department of Immunology, University of Colorado Denver and National Jewish Health, Denver, Colorado, USA.

    • Brian D Stadinski,
    • Thomas Delong,
    • Nichole Reisdorph,
    • Richard Reisdorph,
    • Roger L Powell,
    • Michael Armstrong,
    • Jon D Piganelli,
    • Gene Barbour,
    • Brenda Bradley,
    • Frances Crawford,
    • Philippa Marrack,
    • John W Kappler &
    • Kathryn Haskins
  2. Howard Hughes Medical Institute, National Jewish Health, University of Colorado Denver, Denver, Colorado, USA.

    • Brian D Stadinski,
    • Frances Crawford,
    • Philippa Marrack &
    • John W Kappler
  3. Department of Pediatrics, National Jewish Health, University of Colorado Denver, Denver, Colorado, USA.

    • Richard Reisdorph
  4. Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Denver, Colorado, USA.

    • Philippa Marrack
  5. Department of Medicine, University of California, San Diego and VA San Diego Healthcare System, San Diego, California, USA.

    • Sushil K Mahata
  6. Program in Biomolecular Structure, University of Colorado Denver, Denver, Colorado, USA.

    • John W Kappler
  7. Present address: Department of Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

    • Jon D Piganelli


B.D.S., T.D., N.R., R.R., J.W.K. and K.H. designed the experiments; B.D.S., T.D., R.L.P. and M.A. did most of the experiments, assisted by G.B., B.B. and F.C.; J.D.P. initially suggested ChgA as a candidate autoantigen; S.K.M. provided the Chga−/− and Chga+/+ mice; B.D.S., T.D., N.R., R.R., P.M., J.W.K. and K.H. analyzed and interpreted the data; B.D.S., T.D., J.W.K. and K.H. wrote the manuscript and prepared the figures; and N.R., R.R. and P.M. helped edit the manuscript.

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