Immunologic Research

, Volume 55, Issue 1–3, pp 91–99 | Cite as

Mapping I-Ag7 restricted epitopes in murine G6PC2

  • Tao Yang
  • Anita C. Hohenstein
  • Catherine E. Lee
  • John C. Hutton
  • Howard W. Davidson
Immunology in Colorado


G6PC2, also known as islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP), is a major target of autoreactive CD8+ T cells in both diabetic human subjects and the non-obese diabetic (NOD) mouse. However, in contrast to the abundant literature regarding the CD8+ response to this antigen, much less is known about the potential involvement of IGRP-reactive CD4+ T cells in diabetogenesis. The single previous study that examined this question in NOD mice was based upon a candidate epitope approach and identified three I-Ag7-restricted epitopes that each elicited spontaneous responses in these animals. However, given the known inaccuracies of MHC class II epitope prediction algorithms, we hypothesized that additional specificities might also be targeted. To address this issue, we immunized NOD mice with membranes from insect cells overexpressing full-length recombinant mouse IGRP and measured recall responses of purified CD4+ T cells using a library of overlapping peptides encompassing the entire 355-aa primary sequence. Nine peptides representing 8 epitopes gave recall responses, only 1 of which corresponded to any of the previously reported sequences. In each case proliferation was blocked by a monoclonal antibody to I-Ag7, but not the appropriate isotype control. Consistent with a role in diabetogenesis, proliferative responses to 4 of the 9 peptides (3 epitopes) were also detected in CD4+ T cells purified from the pancreatic draining lymph nodes of pre-diabetic female animals, but not from peripheral lymph nodes or spleens of the same animals. Intriguingly, one of the newly identified spontaneously reactive epitopes (P8 [IGRP55–72]) is highly conserved between mice and man, suggesting that it might also be a target of HLA-DQ8-restricted T cells in diabetic human subjects, an hypothesis that we are currently testing.


Autoimmune diabetes NOD IGRP Epitope 



This work was supported by NIH R01 DK052068 (to JCH and HWD), the University of Colorado Health Sciences Center Diabetes and Endocrinology Research Center (P30 DK57516), and American Diabetes Association research grant 1-04-RA-44 (to JCH). Tao Yang gratefully acknowledges support from an American Diabetes Association mentored post-doctoral fellowship (7-04-MN-19).

Supplementary material

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Supplementary material 1 (DOCX 18 kb)


  1. 1.
    Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature. 2010;464:1293–300.PubMedCrossRefGoogle Scholar
  2. 2.
    Libman I, Songer T, LaPorte R. How many people in the U.S. have IDDM? Diabetes Care. 1993;16:841–2.PubMedGoogle Scholar
  3. 3.
    Patterson CC, Dahlquist GG, Gyurus E, Green A, Soltesz G. Incidence trends for childhood type 1 diabetes in Europe during 1989–2003 and predicted new cases 2005–20: a multicentre prospective registration study. Lancet. 2009;373:2027–33.PubMedCrossRefGoogle Scholar
  4. 4.
    Soltesz G, Patterson CC, Dahlquist G. Worldwide childhood type 1 diabetes incidence: what can we learn from epidemiology? Pediatr Diabetes. 2007;8(Suppl 6):6–14.PubMedCrossRefGoogle Scholar
  5. 5.
    Todd JA. Etiology of type 1 diabetes. Immunity. 2010;32:457–67.PubMedCrossRefGoogle Scholar
  6. 6.
    Atkinson MA, Bluestone JA, Eisenbarth GS, Hebrok M, Herold KC, Accili D, et al. How does type 1 diabetes develop?: the notion of homicide or beta-cell suicide revisited. Diabetes. 2011;60:1370–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Roep BO. The role of T-cells in the pathogenesis of Type 1 diabetes: from cause to cure. Diabetologia. 2003;46:305–21.PubMedGoogle Scholar
  8. 8.
    van Belle TL, Coppieters KT, von Herrath MG. Type 1 diabetes: etiology, immunology, and therapeutic strategies. Physiol Rev. 2011;91:79–118.PubMedCrossRefGoogle Scholar
  9. 9.
    Kaufman A, Herold KC. Anti-CD3 mAbs for treatment of type 1 diabetes. Diabetes Metab Res Rev. 2009;25:302–6.PubMedCrossRefGoogle Scholar
  10. 10.
    Chatenoud L. Immune therapy for type 1 diabetes mellitus: what is unique about anti-CD3 antibodies? Nat Rev Endocrinol. 2010;6:149–57.PubMedCrossRefGoogle Scholar
  11. 11.
    Peakman M, von Herrath M. Antigen-specific immunotherapy for type 1 diabetes: maximizing the potential. Diabetes. 2010;59:2087–93.PubMedCrossRefGoogle Scholar
  12. 12.
    Shoda LK, Young DL, Ramanujan S, Whiting CC, Atkinson MA, Bluestone JA, et al. A comprehensive review of interventions in the NOD mouse and implications for translation. Immunity. 2005;23:115–26.PubMedCrossRefGoogle Scholar
  13. 13.
    Nakayama M, Abiru N, Moriyama H, Babaya N, Liu E, Miao D, et al. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice. Nature. 2005;435:220–3.PubMedCrossRefGoogle Scholar
  14. 14.
    von Herrath M, Sanda S, Herold K. Type 1 diabetes as a relapsing-remitting disease? Nat Rev Immunol. 2007;7:988–94.CrossRefGoogle Scholar
  15. 15.
    Cobbold SP, Adams E, Nolan KF, Regateiro FS, Waldmann H. Connecting the mechanisms of T-cell regulation: dendritic cells as the missing link. Immunol Rev. 2010;236:203–18.PubMedCrossRefGoogle Scholar
  16. 16.
    DiLorenzo TP. Multiple antigens versus single major antigen in type 1 diabetes: arguing for multiple antigens. Diabetes Metab Res Rev. 2011;27:778–83.PubMedCrossRefGoogle Scholar
  17. 17.
    Shieh JJ, Pan CJ, Mansfield BC, Chou JY. The islet-specific glucose-6-phosphatase-related protein, implicated in diabetes, is a glycoprotein embedded in the endoplasmic reticulum membrane. FEBS Lett. 2004;562:160–4.PubMedCrossRefGoogle Scholar
  18. 18.
    Arden SD, Zahn T, Steegers S, Webb S, Bergman B, O’Brien RM, et al. Molecular cloning of a pancreatic islet-specific glucose-6-phosphatase catalytic subunit-related protein. Diabetes. 1999;48:531–42.PubMedCrossRefGoogle Scholar
  19. 19.
    Efrat S, Linde S, Kofod H, Spector D, Delannoy M, Grant S, et al. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc Natl Acad Sci U S A. 1988;85:9037–41.PubMedCrossRefGoogle Scholar
  20. 20.
    Neophytou PI, Muir EM, Hutton JC. A subtractive cloning approach to the identification of mRNAs specifically expressed in pancreatic beta-cells. Diabetes. 1996;45:127–33.PubMedCrossRefGoogle Scholar
  21. 21.
    Bouatia-Naji N, Rocheleau G, Van Lommel L, Lemaire K, Schuit F, Cavalcanti-Proenca C, et al. A polymorphism within the G6PC2 gene is associated with fasting plasma glucose levels. Science. 2008;320:1085–8.PubMedCrossRefGoogle Scholar
  22. 22.
    Dos Santos C, Bougneres P, Fradin D. A single-nucleotide polymorphism in a methylatable Foxa2 binding site of the G6PC2 promoter is associated with insulin secretion in vivo and increased promoter activity in vitro. Diabetes. 2009;58:489–92.PubMedCrossRefGoogle Scholar
  23. 23.
    Wang Y, Martin CC, Oeser JK, Sarkar S, McGuinness OP, Hutton JC, et al. Deletion of the gene encoding the islet-specific glucose-6-phosphatase catalytic subunit-related protein autoantigen results in a mild metabolic phenotype. Diabetologia. 2007;50:774–8.PubMedCrossRefGoogle Scholar
  24. 24.
    Heni M, Ketterer C, Hart LM, Ranta F, van Haeften TW, Eekhoff EM, et al. The impact of genetic variation in the G6PC2 gene on insulin secretion depends on glycemia. J Clin Endocrinol Metab. 2010;95:E479–84.PubMedCrossRefGoogle Scholar
  25. 25.
    Hu C, Zhang R, Wang C, Yu W, Lu J, Ma X, et al. Effects of GCK, GCKR, G6PC2 and MTNR1B variants on glucose metabolism and insulin secretion. PLoS ONE. 2010;5:e11761.PubMedCrossRefGoogle Scholar
  26. 26.
    Nagata M, Santamaria P, Kawamura T, Utsugi T, Yoon JW. Evidence for the role of CD8+ cytotoxic T cells in the destruction of pancreatic beta-cells in nonobese diabetic mice. J Immunol. 1994;152:2042–50.PubMedGoogle Scholar
  27. 27.
    Santamaria P, Utsugi T, Park BJ, Averill N, Kawazu S, Yoon JW. Beta-cell-cytotoxic CD8+ T cells from nonobese diabetic mice use highly homologous T cell receptor alpha-chain CDR3 sequences. J Immunol. 1995;154:2494–503.PubMedGoogle Scholar
  28. 28.
    Lieberman SM, Evans AM, Han B, Takaki T, Vinnitskaya Y, Caldwell JA, et al. Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes. Proc Natl Acad Sci U S A. 2003;100:8384–8.PubMedCrossRefGoogle Scholar
  29. 29.
    Anderson B, Park BJ, Verdaguer J, Amrani A, Santamaria P. Prevalent CD8(+) T cell response against one peptide/MHC complex in autoimmune diabetes. Proc Natl Acad Sci U S A. 1999;96:9311–6.PubMedCrossRefGoogle Scholar
  30. 30.
    Han B, Serra P, Amrani A, Yamanouchi J, Maree AF, Edelstein-Keshet L, et al. Prevention of diabetes by manipulation of anti-IGRP autoimmunity: high efficiency of a low-affinity peptide. Nat Med. 2005;11:645–52.PubMedCrossRefGoogle Scholar
  31. 31.
    Oeser JK, Parekh VV, Wang Y, Jegadeesh NK, Sarkar SA, Wong R, et al. Deletion of the G6pc2 gene encoding the islet-specific glucose-6-phosphatase catalytic subunit-related protein does not affect the progression or incidence of type 1 diabetes in NOD/ShiLtJ mice. Diabetes. 2011;60:2922–7.PubMedCrossRefGoogle Scholar
  32. 32.
    Ouyang Q, Standifer NE, Qin H, Gottlieb P, Verchere CB, Nepom GT, et al. Recognition of HLA class I-restricted beta-cell epitopes in type 1 diabetes. Diabetes. 2006;55:3068–74.PubMedCrossRefGoogle Scholar
  33. 33.
    Standifer NE, Ouyang Q, Panagiotopoulos C, Verchere CB, Tan R, Greenbaum CJ, et al. Identification of Novel HLA-A*0201-restricted epitopes in recent-onset type 1 diabetic subjects and antibody-positive relatives. Diabetes. 2006;55:3061–7.PubMedCrossRefGoogle Scholar
  34. 34.
    Mallone R, Martinuzzi E, Blancou P, Novelli G, Afonso G, Dolz M, et al. CD8+ T-cell responses identify beta-cell autoimmunity in human type 1 diabetes. Diabetes. 2007;56:613–21.PubMedCrossRefGoogle Scholar
  35. 35.
    Jarchum I, Nichol L, Trucco M, Santamaria P, DiLorenzo TP. Identification of novel IGRP epitopes targeted in type 1 diabetes patients. Clin Immunol. 2008;127:359–65.PubMedCrossRefGoogle Scholar
  36. 36.
    Martinuzzi E, Novelli G, Scotto M, Blancou P, Bach JM, Chaillous L, et al. The frequency and immunodominance of islet-specific CD8+ T-cell responses change after type 1 diabetes diagnosis and treatment. Diabetes. 2008;57:1312–20.PubMedCrossRefGoogle Scholar
  37. 37.
    Mukherjee R, Wagar D, Stephens TA, Lee-Chan E, Singh B. Identification of CD4+ T cell-specific epitopes of islet-specific glucose-6-phosphatase catalytic subunit-related protein: a novel beta cell autoantigen in type 1 diabetes. J Immunol. 2005;174:5306–15.PubMedGoogle Scholar
  38. 38.
    Yang J, Danke NA, Berger D, Reichstetter S, Reijonen H, Greenbaum C, et al. Islet-specific glucose-6-phosphatase catalytic subunit-related protein-reactive CD4+ T cells in human subjects. J Immunol. 2006;176:2781–9.PubMedGoogle Scholar
  39. 39.
    Nielsen M, Lund O, Buus S, Lundegaard C. MHC class II epitope predictive algorithms. Immunology. 2010;130:319–28.PubMedCrossRefGoogle Scholar
  40. 40.
    Schneider I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 1972;27:353–65.PubMedGoogle Scholar
  41. 41.
    Bunch TA, Grinblat Y, Goldstein LS. Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res. 1988;16:1043–61.PubMedCrossRefGoogle Scholar
  42. 42.
    Kelemen K, Gottlieb PA, Putnam AL, Davidson HW, Wegmann DR, Hutton JC. HLA-DQ8-associated T cell responses to the diabetes autoantigen phogrin (IA-2 beta) in human prediabetes. J Immunol. 2004;172:3955–62.PubMedGoogle Scholar
  43. 43.
    Oi VT, Jones PP, Goding JW, Herzenberg LA. Properties of monoclonal antibodies to mouse Ig allotypes, H-2, and Ia antigens. Curr Top Microbiol Immunol. 1978;81:115–20.PubMedGoogle Scholar
  44. 44.
    Shameli A, Yamanouchi J, Thiessen S, Santamaria P. Endoplasmic reticulum stress caused by overexpression of islet-specific glucose-6-phosphatase catalytic subunit-related protein in pancreatic Beta-cells. Rev Diabet Stud. 2007;4:25–32.PubMedCrossRefGoogle Scholar
  45. 45.
    Boitard C, Bendelac A, Richard MF, Carnaud C, Bach JF. Prevention of diabetes in nonobese diabetic mice by anti-I-A monoclonal antibodies: transfer of protection by splenic T cells. Proc Natl Acad Sci U S A. 1988;85:9719–23.PubMedCrossRefGoogle Scholar
  46. 46.
    Burtles SS, Trembleau S, Drexler K, Hurtenbach U. Absence of T cell tolerance to pancreatic islet cells. J Immunol. 1992;149:2185–93.PubMedGoogle Scholar
  47. 47.
    Bendelac A, Carnaud C, Boitard C, Bach JF. Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates. Requirement for both L3T4+ and Lyt-2+ T cells. J Exp Med. 1987;166:823–32.PubMedCrossRefGoogle Scholar
  48. 48.
    Wicker LS, Miller BJ, Mullen Y. Transfer of autoimmune diabetes mellitus with splenocytes from nonobese diabetic (NOD) mice. Diabetes. 1986;35:855–60.PubMedCrossRefGoogle Scholar
  49. 49.
    Li R, Perez N, Karumuthil-Melethil S, Vasu C. Bone marrow is a preferential homing site for autoreactive T-cells in type 1 diabetes. Diabetes. 2007;56:2251–9.PubMedCrossRefGoogle Scholar
  50. 50.
    Stratmann T, Apostolopoulos V, Mallet-Designe V, Corper AL, Scott CA, Wilson IA, et al. The I-Ag7 MHC class II molecule linked to murine diabetes is a promiscuous peptide binder. J Immunol. 2000;165:3214–25.PubMedGoogle Scholar
  51. 51.
    Gowthaman U, Agrewala JN. In silico tools for predicting peptides binding to HLA-class II molecules: more confusion than conclusion. J Proteome Res. 2008;7:154–63.PubMedCrossRefGoogle Scholar
  52. 52.
    Moudgil KD, Sercarz EE, Grewal IS. Modulation of the immunogenicity of antigenic determinants by their flanking residues. Immunol Today. 1998;19:217–20.PubMedCrossRefGoogle Scholar
  53. 53.
    O’Brien C, Flower DR, Feighery C. Peptide length significantly influences in vitro affinity for MHC class II molecules. Immunome Res. 2008;4:6.PubMedCrossRefGoogle Scholar
  54. 54.
    Carrasco-Marin E, Shimizu J, Kanagawa O, Unanue ER. The class II MHC I-Ag7 molecules from non-obese diabetic mice are poor peptide binders. J Immunol. 1996;156:450–8.PubMedGoogle Scholar
  55. 55.
    Standifer NE, Burwell EA, Gersuk VH, Greenbaum CJ, Nepom GT. Changes in autoreactive T cell avidity during type 1 diabetes development. Clin Immunol. 2009;132:312–20.PubMedCrossRefGoogle Scholar
  56. 56.
    Amrani A, Verdaguer J, Serra P, Tafuro S, Tan R, Santamaria P. Progression of autoimmune diabetes driven by avidity maturation of a T-cell population. Nature. 2000;406:739–42.PubMedCrossRefGoogle Scholar
  57. 57.
    Delong T, Baker RL, Reisdorph N, Reisdorph R, Powell RL, Armstrong M, et al. Islet amyloid polypeptide is a target antigen for diabetogenic CD4+ T cells. Diabetes. 2011;60:2325–30.PubMedCrossRefGoogle Scholar
  58. 58.
    Chang KY, Suri A, Unanue ER. Predicting peptides bound to I-Ag7 class II histocompatibility molecules using a novel expectation-maximization alignment algorithm. Proteomics. 2007;7:367–77.PubMedCrossRefGoogle Scholar
  59. 59.
    Suri A, Walters JJ, Gross ML, Unanue ER. Natural peptides selected by diabetogenic DQ8 and murine I-A(g7) molecules show common sequence specificity. J Clin Invest. 2005;115:2268–76.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Tao Yang
    • 1
    • 3
  • Anita C. Hohenstein
    • 1
  • Catherine E. Lee
    • 1
  • John C. Hutton
    • 1
  • Howard W. Davidson
    • 1
    • 2
  1. 1.Barbara Davis Center for Childhood DiabetesUniversity of Colorado Anschutz Medical CampusAuroraUSA
  2. 2.Integrated Department of ImmunologyUniversity of Colorado Anschutz Medical CampusAuroraUSA
  3. 3.Department of EndocrinologyThe First Affiliated Hospital of Nanjing Medical UniversityNanjingChina

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