Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Synthetically glycosylated antigens induce antigen-specific tolerance and prevent the onset of diabetes

Nature Biomedical Engineering volume 3, pages 817–829 (2019)Cite this article

Subjects

Abstract

Homeostatic antigen presentation by hepatic antigen-presenting cells, which results in tolerogenic T-cell education, could be exploited to induce antigen-specific immunological tolerance. Here we show that antigens modified with polymeric forms of either N-acetylgalactosamine or N-acetylglucosamine target hepatic antigen-presenting cells, increase their antigen presentation and induce antigen-specific tolerance, as indicated by CD4+ and CD8+ T-cell deletion and anergy. These synthetically glycosylated antigens also expanded functional regulatory T cells, which are necessary for the durable suppression of antigen-specific immune responses. In an adoptive-transfer mouse model of type-1 diabetes, treatment with the glycosylated autoantigens prevented T-cell-mediated diabetes, expanded antigen-specific regulatory T cells and resulted in lasting tolerance to a subsequent challenge with activated diabetogenic T cells. Glycosylated autoantigens targeted to hepatic antigen-presenting cells might enable therapies that promote immune tolerance in patients with autoimmune diseases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Antigen–p(GalNAc) and antigen–p(GluNAc) conjugates target hepatic tolerance-inducing antigen-presenting cells.
Fig. 2: Intravenously administered OVA–p(GalNAc) and OVA–p(GluNAc) conjugates enhance antigen presentation and OTI and OTII cell deletion.
Fig. 3: Antigen–p(GalNAc) and antigen–p(GluNAc) conjugates induce CD8+ and CD4+ T-cell tolerance to antigen challenge and augment antigen-specific Treg cells via spleen-independent mechanisms.
Fig. 4: OVA–p(GluNAc) and OVA–p(GalNAc) conjugates suppress adoptively transferred and endogenous T-cell responses via a TGF-β-mediated mechanism.
Fig. 5: OVA–p(GluNAc) conjugates induce tolerogenic memory via CD25+ regulatory T cells.
Fig. 6: p31–p(GluNAc) protects mice from BDC2.5 T-cell-induced diabetes, increases Treg cells and establishes lasting protection against subsequent challenge.

Similar content being viewed by others

Data availability

The authors declare that all data supporting the results in this study are available within the paper and its Supplementary Information. The datasets generated and analysed during the study are available from the corresponding author upon reasonable request.

References

  1. Rosenblum, M. D., Gratz, I. K., Paw, J. S. & Abbas, A. K. Treating human autoimmunity: current practice and future prospects. Sci. Transl. Med. 4, 125sr1–125sr1 (2012).

    Article  Google Scholar 

  2. Sakaguchi, S., Yamaguchi, T., Nomura, T. & Ono, M. Regulatory T cells and immune tolerance. Cell 133, 775–787 (2008).

    Article  CAS  Google Scholar 

  3. Wing, K. & Sakaguchi, S. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat. Immunol. 11, 7–13 (2010).

    Article  CAS  Google Scholar 

  4. Wang, X., Lu, L. & Jiang, S. Regulatory T cells: customizing for the clinic. Sci. Transl. Med. 3, 83ps19–83ps19 (2011).

    Google Scholar 

  5. Berg, M. et al. Cross-presentation of antigens from apoptotic tumor cells by liver sinusoidal endothelial cells leads to tumor-specific CD8+ T cell tolerance. Eur. J. Immunol. 36, 2960–2970 (2006).

    Article  CAS  Google Scholar 

  6. Li, F. & Tian, Z. The liver works as a school to educate regulatory immune cells. Cell. Mol. Immunol. 10, 292–302 (2013).

    Article  CAS  Google Scholar 

  7. Horst, A. K., Neumann, K., Diehl, L. & Tiegs, G. Modulation of liver tolerance by conventional and nonconventional antigen-presenting cells and regulatory immune cells. Cell. Mol. Immunol. 13, 277–292 (2016).

    Article  CAS  Google Scholar 

  8. Knolle et al. IL‐10 down‐regulates T cell activation by antigen‐presenting liver sinusoidal endothelial cells through decreased antigen uptake via the mannose receptor and lowered surface expression of accessory molecules. Clin. Exp. Immunol. 114, 427–433 (1998).

    Article  CAS  Google Scholar 

  9. Knolle, P. A. et al. Endotoxin down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells. J. Immunol. 162, 1401–1407 (1999).

    CAS  PubMed  Google Scholar 

  10. Bissell, D. M., Wang, S. S., Jarnagin, W. R. & Roll, F. J. Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation. J. Clin. Invest. 96, 447–455 (1995).

    Article  CAS  Google Scholar 

  11. Breous, E., Somanathan, S., Vandenberghe, L. H. & Wilson, J. M. Hepatic regulatory T cells and Kupffer cells are crucial mediators of systemic T cell tolerance to antigens targeting murine liver. Hepatology 50, 612–621 (2009).

    Article  CAS  Google Scholar 

  12. Schon, H.-T. & Weiskirchen, R. Immunomodulatory effects of transforming growth factor-β in the liver. Hepatobiliary Surg. Nutr. 3, 386–406 (2014).

    PubMed  PubMed Central  Google Scholar 

  13. Chen, W. et al. Conversion of peripheral CD4+CD25− naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J. Exp. Med. 198, 1875–1886 (2003).

    Article  CAS  Google Scholar 

  14. Wu, K., Kryczek, I., Chen, L., Zou, W. & Welling, T. H. Kupffer cell suppression of CD8+ T cells in human hepatocellular carcinoma is mediated by B7-H1/programmed death-1 interactions. Cancer Res 69, 8067–8075 (2009).

    Article  CAS  Google Scholar 

  15. Dolina, J. S., Sung, S.-S. J., Novobrantseva, T. I., Nguyen, T. M. & Hahn, Y. S. Lipidoid nanoparticles containing PD-L1 siRNA delivered in vivo enter Kupffer cells and enhance NK and CD8+ T cell-mediated hepatic antiviral immunity. Mol. Ther. Nucleic Acids 2, e72 (2013).

    Article  Google Scholar 

  16. Xia, C.-Q., Campbell, K., Keselowsky, B. & Clare-Salzler, M. in Type 1 Diabetes—Pathogenesis, Genetics and Immunotherapy (ed. Wagner, D.) https://doi.org/10.5772/22113 (InTech, 2011).

  17. Bilyy, R. & Stoika, R. Search for novel cell surface markers of apoptotic cells. Autoimmunity 40, 249–253 (2009).

    Article  Google Scholar 

  18. Duvall, E., Wyllie, A. H. & Morris, R. G. Macrophage recognition of cells undergoing programmed cell death (apoptosis). Immunology 56, 351–358 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Liu, W. et al. Characterization of a novel C-type lectin-like gene, LSECtin: demonstration of carbohydrate binding and expression in sinusoidal endothelial cells of liver and lymph node. J. Biol. Chem. 279, 18748–18758 (2004).

    Article  CAS  Google Scholar 

  20. Apostolopoulos, V., Thalhammer, T., Tzakos, A. G. & Stojanovska, L. Targeting antigens to dendritic cell receptors for vaccine development. J. Drug Deliv. 2013, 869718 (2013).

    Article  Google Scholar 

  21. van Kooyk, Y. C-type lectins on dendritic cells: key modulators for the induction of immune responses. Biochem. Soc. Trans. 36, 1478–1481 (2008).

    Article  Google Scholar 

  22. Ohnishi, H., Oka, K., Mizuno, S. & Nakamura, T. Identification of mannose receptor as receptor for hepatocyte growth factor β-chain: novel ligand-receptor pathway for enhancing macrophage phagocytosis. J. Biol. Chem. 287, 13371–13381 (2012).

    Article  CAS  Google Scholar 

  23. Yang, C.-Y. et al. CLEC4F is an inducible C-type lectin in F4/80-positive cells and is involved in alpha-galactosylceramide presentation in liver. PLoS ONE 8, e65070 (2013).

    Article  CAS  Google Scholar 

  24. Elvevold, K. et al. Liver sinusoidal endothelial cells depend on mannose receptor-mediated recruitment of lysosomal enzymes for normal degradation capacity. Hepatology 48, 2007–2015 (2008).

    Article  CAS  Google Scholar 

  25. Greco, S. H. et al. Mincle signaling promotes Con A hepatitis. J. Immunol. 197, 2816–2827 (2016).

    Article  CAS  Google Scholar 

  26. Maynard, Y. & Baenziger, J. U. Characterization of a mannose and N-acetylglucosamine-specific lectin present in rat hepatocytes. J. Biol. Chem. 257, 3788–3794 (1982).

    CAS  PubMed  Google Scholar 

  27. Kim, S. H., Goto, M. & Akaike, T. Specific binding of glucose-derivatized polymers to the asialoglycoprotein receptor of mouse primary hepatocytes. J. Biol. Chem. 276, 35312–35319 (2001).

    Article  CAS  Google Scholar 

  28. Jackson, D. C., Drummer, H. E., Urge, L., Otvos, L. Jr & Brown, L. E. Glycosylation of a synthetic peptide representing a T-cell determinant of influenza virus hemagglutinin results in loss of recognition by CD4+ T-cell clones. Virology 199, 422–430 (1994).

    Article  CAS  Google Scholar 

  29. Hastings, K. T. & Cresswell, P. Disulfide reduction in the endocytic pathway: immunological functions of gamma-interferon-inducible lysosomal thiol reductase. Antioxid. Redox Signal. 15, 657–668 (2011).

    Article  CAS  Google Scholar 

  30. Arunachalam, B., Phan, U. T., Geuze, H. J. & Cresswell, P. Enzymatic reduction of disulfide bonds in lysosomes: characterization of a gamma-interferon-inducible lysosomal thiol reductase (GILT). Proc. Natl Acad. Sci. USA 97, 745–750 (2000).

    Article  CAS  Google Scholar 

  31. Satyam, A. Design and synthesis of releasable folate-drug conjugates using a novel heterobifunctional disulfide-containing linker. Bioorg. Med. Chem. Lett. 18, 3196–3199 (2008).

    Article  CAS  Google Scholar 

  32. Harvey, D. J., Wing, D. R., Küster, B. & Wilson, I. B. H. Composition of N-linked carbohydrates from ovalbumin and co-purified glycoproteins. J. Am. Soc. Mass Spectrom. 11, 564–571 (2000).

    Article  CAS  Google Scholar 

  33. Kindberg, G. M., Magnusson, S., Berg, T. & Smedsrød, B. Receptor-mediated endocytosis of ovalbumin by two carbohydrate-specific receptors in rat liver cells. The intracellular transport of ovalbumin to lysosomes is faster in liver endothelial cells than in parenchymal cells. Biochem. J. 270, 197–203 (1990).

    Article  CAS  Google Scholar 

  34. Schurich, A. et al. Distinct kinetics and dynamics of cross-presentation in liver sinusoidal endothelial cells compared to dendritic cells. Hepatology 50, 909–919 (2009).

    Article  CAS  Google Scholar 

  35. Rensen, P. C. et al. Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo. J. Biol. Chem. 276, 37577–37584 (2001).

    Article  CAS  Google Scholar 

  36. Eickmeier, I. et al. Influence of CD8 T cell priming in liver and gut on the enterohepatic circulation. J. Hepatol. 60, 1143–1150 (2014).

    Article  CAS  Google Scholar 

  37. Okazaki, T. & Honjo, T. The PD-1–PD-L pathway in immunological tolerance. Trends Immunol. 27, 195–201 (2006).

    Article  CAS  Google Scholar 

  38. Tokita, D. et al. High PD-L1/CD86 ratio on plasmacytoid dendritic cells correlates with elevated T-regulatory cells in liver transplant tolerance. Transplantation 85, 369–377 (2008).

    Article  Google Scholar 

  39. Crespo, J., Sun, H., Welling, T. H., Tian, Z. & Zou, W. T cell anergy, exhaustion, senescence, and stemness in the tumor microenvironment. Curr. Opin. Immunol. 25, 214–221 (2013).

    Article  CAS  Google Scholar 

  40. Carambia, A. et al. TGF-β-dependent induction of CD4+CD25+Foxp3+ Tregs by liver sinusoidal endothelial cells. J. Hepatol. 61, 594–599 (2014).

    Article  CAS  Google Scholar 

  41. Heymann, F. et al. Liver inflammation abrogates immunological tolerance induced by Kupffer cells. Hepatology 62, 279–291 (2015).

    Article  CAS  Google Scholar 

  42. Grinberg-Bleyer, Y. et al. IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells. J. Exp. Med. 207, 20100209 (2010).

    Article  Google Scholar 

  43. Maloy, K. J. & Powrie, F. Fueling regulation: IL-2 keeps CD4+ Treg cells fit. Nat. Immunol. 6, 1071–1072 (2005).

    Article  CAS  Google Scholar 

  44. Schurich, A. et al. Dynamic regulation of CD8 T cell tolerance induction by liver sinusoidal endothelial cells. J. Immunol. 184, 4107–4114 (2010).

    Article  CAS  Google Scholar 

  45. Tye, G. J. et al. The combined molecular adjuvant CASAC enhances the CD8+ T cell response to a tumor-associated self-antigen in aged, immunosenescent mice. Immun. Ageing 12, 659 (2015).

    Article  Google Scholar 

  46. Zelenay, S. & Demengeot, J. Comment on ‘cutting edge: anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4+CD25+ T regulatory cells’. J. Immunol. 177, 2036–2037 (2006).

    Article  CAS  Google Scholar 

  47. Kohm, A. P. et al. Cutting edge: anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4+CD25+ T regulatory cells. J. Immunol. 176, 3301–3305 (2006).

    Article  CAS  Google Scholar 

  48. Baynes, J. W. & Wold, F. Effect of glycosylation on the in vivo circulating half-life of ribonuclease. J. Biol. Chem. 251, 6016–6024 (1976).

    CAS  PubMed  Google Scholar 

  49. Petzold, C. et al. Foxp3+ regulatory T cells in mouse models of type 1 diabetes. J. Diabetes Res. 2013, 940710–940710 (2013).

    Article  Google Scholar 

  50. Delong, T. et al. Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion. Science 351, 711–714 (2016).

    Article  CAS  Google Scholar 

  51. Carambia, A. et al. Nanoparticle-based autoantigen delivery to Treg-inducing liver sinusoidal endothelial cells enables control of autoimmunity in mice. J. Hepatol. 62, 1349–1356 (2015).

    Article  CAS  Google Scholar 

  52. Estey, T., Kang, J., Schwendeman, S. P. & Carpenter, J. F. BSA degradation under acidic conditions: a model for protein instability during release from PLGA delivery systems. J. Pharm. Sci. 95, 1626–1639 (2006).

    Article  CAS  Google Scholar 

  53. Prior, S. et al. In vitro phagocytosis and monocyte-macrophage activation with poly(lactide) and poly(lactide-co-glycolide) microspheres. Eur. J. Pharm. Sci. 15, 197–207 (2002).

    Article  CAS  Google Scholar 

  54. Wisse, E., Jacobs, F., Topal, B., Frederik, P. & De Geest, B. The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer. Gene Ther. 15, 1193–1199 (2008).

    Article  CAS  Google Scholar 

  55. Perdicchio, M. et al. Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells. Proc. Natl Acad. Sci. USA 113, 3329–3334 (2016).

    Article  CAS  Google Scholar 

  56. Chen, P. et al. Dendritic cell targeted vaccines: recent progresses and challenges. Hum. Vaccin. Immunother. 12, 612–622 (2015).

    Article  Google Scholar 

  57. Harding, F. A., Stickler, M. M., Razo, J. & DuBridge, R. B. The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions. MAbs 2, 256–265 (2010).

    Article  Google Scholar 

  58. Knolle, P. A. & Wohlleber, D. Immunological functions of liver sinusoidal endothelial cells. Cell. Mol. Immunol. 13, 347–353 (2016).

    Article  CAS  Google Scholar 

  59. Tang, L. et al. Liver sinusoidal endothelial cell lectin, LSECtin, negatively regulates hepatic T-cell immune response. Gastroenterology 137, 1498–1508.e5 (2009).

    Article  CAS  Google Scholar 

  60. Domínguez-Soto, A. et al. The pathogen receptor liver and lymph node sinusoidal endotelial cell C-type lectin is expressed in human Kupffer cells and regulated by PU.1. Hepatology 49, 287–296 (2009).

    Article  Google Scholar 

  61. Daniels, C. K., Schmucker, D. L. & Jones, A. L. Hepatic asialoglycoprotein receptor-mediated binding of human polymeric immunoglobulin A. Hepatology 9, 229–234 (1989).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Flow Cytometry Core Facility of EPFL for technical assistance and E. Simeoni (EPFL) for helpful discussion on the research and guidance on animal work. D.S.W. was supported by a fellowship from the Whitaker Foundation. This study was supported by the School of Life Sciences, EPFL, the University of Chicago, the Juvenile Diabetes Research Foundation and Anokion.

Author information

Author notes

  1. These authors contributed equally: D. Scott Wilson, Martina Damo.

Authors and Affiliations

  1. Institute for Molecular Engineering, University of Chicago, Chicago, IL, USA

    D. Scott Wilson, Martina Damo, Michal M. Raczy, Kym Brünggel & Jeffrey A. Hubbell

  2. Institute for Bioengineering, Ecole Polytechnique Fédérale de Lausanne, EPFL-SV-IBI, Lausanne, Switzerland

    D. Scott Wilson, Martina Damo, Sachiko Hirosue, Michal M. Raczy, Kym Brünggel, Giacomo Diaceri, Xavier Quaglia-Thermes & Jeffrey A. Hubbell

Authors
  1. D. Scott Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martina Damo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sachiko Hirosue
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michal M. Raczy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kym Brünggel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Giacomo Diaceri
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xavier Quaglia-Thermes
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jeffrey A. Hubbell
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.S.W. and J.A.H. designed the research; D.S.W. and M.M.R. performed synthesis; D.S.W., M.D., S.H., K.B., G.D. and X.Q.-T. performed biological research; D.S.W. analysed data and D.S.W. and J.A.H. wrote the paper.

Corresponding author

Correspondence to Jeffrey A. Hubbell.

Ethics declarations

Competing interests

The EPFL has filed for patent protection on the p(GalNAc) and p(GluNAc) delivery platforms and D.S.W. and J.A.H. are named as inventors on the patents. Anokion and Kanyos Bio have licensed the patents and J.A.H. and D.S.W. participate in equity in these companies.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary methods and figures.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wilson, D.S., Damo, M., Hirosue, S. et al. Synthetically glycosylated antigens induce antigen-specific tolerance and prevent the onset of diabetes. Nat Biomed Eng 3, 817–829 (2019). https://doi.org/10.1038/s41551-019-0424-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-019-0424-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research