, Volume 61, Issue 2, pp 389–398 | Cite as

Oral histone deacetylase inhibitor synergises with T cell targeted immunotherapy to preserve beta cell metabolic function and induce stable remission of new-onset autoimmune diabetes in NOD mice

  • Alix Besançon
  • Tania Goncalves
  • Fabrice Valette
  • Mattias S. Dahllöf
  • Thomas Mandrup-Poulsen
  • Lucienne Chatenoud
  • Sylvaine You



Combination therapy targeting the major actors involved in the immune-mediated destruction of pancreatic beta cells appears to be an indispensable approach to treat type 1 diabetes effectively. We hypothesised that the combination of an orally active pan-histone deacetylase inhibitor (HDACi: givinostat) with subtherapeutic doses of CD3 antibodies may provide ideal synergy to treat ongoing autoimmunity.


NOD mice transgenic for the human CD3ε (also known as CD3E) chain (NOD-huCD3ε) were treated for recent-onset diabetes with oral givinostat, subtherapeutic doses of humanised CD3 antibodies (otelixizumab, 50 μg/day, 5 days, i.v.) or a combination of both drugs. Disease remission, metabolic profiles and autoreactive T cell responses were analysed in treated mice.


We demonstrated that givinostat synergised with otelixizumab to induce durable remission of diabetes in 80% of recently diabetic NOD-huCD3ε mice. Remission was obtained in only 47% of mice treated with otelixizumab alone. Oral givinostat monotherapy did not reverse established diabetes but reduced the in situ production of inflammatory cytokines (IL-1β, IL-6, TNF-α). Importantly, the otelixizumab + givinostat combination strongly improved the metabolic status of NOD-huCD3ε mice; the mice recovered the capacity to appropriately produce insulin, control hyperglycaemia and sustain glucose tolerance. Finally, diabetes remission induced by the combination therapy was associated with a significant reduction of insulitis and autoantigen-specific CD8+ T cell responses.


HDACi and low-dose CD3 antibodies synergised to abrogate in situ inflammation and thereby improved pancreatic beta cell survival and metabolic function leading to long-lasting diabetes remission. These results support the therapeutic potential of protocols combining these two drugs, both in clinical development, to restore self-tolerance and insulin independence in type 1 diabetes.


Beta cells Glucose tolerance HDACi Human CD3 antibodies Humanised NOD mice Insulin secretion Type 1 diabetes 





Histone deacetylase


Forkhead box P3


Histone deacetylase inhibitor


Islet-specific glucose-6-phosphatase catalytic subunit related protein


Lymphocyte-activation gene 3




Pancreatic lymph node


Spot-forming unit


Regulatory type 1 cell


Regulatory T cell



The authors thank M. Bellanger (INSERM U1151, Paris, France) for taking care of the NOD-huCD3ε mouse colony and for providing technical assistance for the experimental mouse work. We are also grateful to E. Panafieu, S. Fonlebeck and Y. Loudin (INSERM U1151, Department of Immunology, Paris, France) for mouse production and maintenance.

Data availability

The data generated during the current study are available from the corresponding author on reasonable request.


This work was supported by grants from the JDRF (#1-2011-654), institutional funding from INSERM and University Paris Descartes and also with the support of Fondation Day Solvay and Fondation Centaure. AB was supported by a doctoral fellowship from INSERM and by a grant from the Société Française d’Endocrinologie et Diabétologie Pédiatrique (grant from Novo Nordisk). The funders had no role in study design, data collection, interpretation or decision to submit the work for publication.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Contribution statement

AB designed experiments, acquired and analysed data, and wrote the manuscript. TG and FV designed and performed experiments, and analysed data. MSD provided research material and contributed to the design of the experiments. LC provided critical advice and help in writing the manuscript. TM-P initiated the study with LC and contributed to planning the protocol and reviewed the manuscript. All authors revised the manuscript and approved the final version to be published. SY designed and directed the study, analysed the data and wrote the manuscript. SY is the guarantor of this work.

Supplementary material

125_2017_4459_MOESM1_ESM.pdf (291 kb)
ESM Figures (PDF 290 kb)


  1. 1.
    Ogawa N, List JF, Habener JF, Maki T (2004) Cure of overt diabetes in NOD mice by transient treatment with anti-lymphocyte serum and exendin-4. Diabetes 53:1700–1705CrossRefPubMedGoogle Scholar
  2. 2.
    Sherry NA, Chen W, Kushner JA et al (2007) Exendin-4 improves reversal of diabetes in NOD mice treated with anti-CD3 monoclonal antibody by enhancing recovery of beta-cells. Endocrinology 148:5136–5144CrossRefPubMedGoogle Scholar
  3. 3.
    Xue S, Posgai A, Wasserfall C et al (2015) Combination therapy reverses hyperglycemia in NOD mice with established type 1 diabetes. Diabetes 64:3873–3884CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ding L, Gysemans CA, Stange G et al (2014) Combining MK626, a novel DPP-4 inhibitor, and low-dose monoclonal CD3 antibody for stable remission of new-onset diabetes in mice. PLoS One 9:e107935CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Tian J, Dang H, Nguyen AV, Chen Z, Kaufman DL (2014) Combined therapy with GABA and proinsulin/alum acts synergistically to restore long-term normoglycemia by modulating T cell autoimmunity and promoting beta-cell replication in newly diabetic NOD mice. Diabetes 63:3128–3134CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ablamunits V, Henegariu O, Hansen JB et al (2012) Synergistic reversal of type 1 diabetes in NOD mice with anti-CD3 and interleukin-1 blockade: evidence of improved immune regulation. Diabetes 61:145–154CrossRefPubMedGoogle Scholar
  7. 7.
    Glauben R, Batra A, Fedke I et al (2006) Histone hyperacetylation is associated with amelioration of experimental colitis in mice. J Immunol 176:5015–5022CrossRefPubMedGoogle Scholar
  8. 8.
    Joosten LA, Leoni F, Meghji S, Mascagni P (2011) Inhibition of HDAC activity by ITF2357 ameliorates joint inflammation and prevents cartilage and bone destruction in experimental arthritis. Mol Med 17:391–396CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Leoni F, Fossati G, Lewis EC et al (2005) The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo. Mol Med 11:1–15CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Larsen L, Tonnesen M, Ronn SG et al (2007) Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells. Diabetologia 50:779–789CrossRefPubMedGoogle Scholar
  11. 11.
    Lewis EC, Blaabjerg L, Storling J et al (2011) The oral histone deacetylase inhibitor ITF2357 reduces cytokines and protects islet beta cells in vivo and in vitro. Mol Med 17:369–377CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Christensen DP, Gysemans C, Lundh M et al (2014) Lysine deacetylase inhibition prevents diabetes by chromatin-independent immunoregulation and beta-cell protection. Proc Natl Acad Sci U S A 111:1055–1059CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Xu G, Stoffers DA, Habener JF, Bonner-Weir S (1999) Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats. Diabetes 48:2270–2276CrossRefPubMedGoogle Scholar
  14. 14.
    Stoffers DA, Kieffer TJ, Hussain MA et al (2000) Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes 49:741–748CrossRefPubMedGoogle Scholar
  15. 15.
    Fu W, Farache J, Clardy SM et al (2014) Epigenetic modulation of type-1 diabetes via a dual effect on pancreatic macrophages and beta cells. eLife 3:e04631CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chatenoud L, Bluestone JA (2007) CD3-specific antibodies: a portal to the treatment of autoimmunity. Nat Rev Immunol 7:622–632CrossRefPubMedGoogle Scholar
  17. 17.
    Chatenoud L, Thervet E, Primo J, Bach JF (1994) Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. Proc Natl Acad Sci U S A 91:123–127CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Belghith M, Bluestone JA, Barriot S, Megret J, Bach JF, Chatenoud L (2003) TGF-beta-dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat Med 9:1202–1208CrossRefPubMedGoogle Scholar
  19. 19.
    You S, Candon S, Kuhn C, Bach JF, Chatenoud L (2008) CD3 antibodies as unique tools to restore self-tolerance in established autoimmunity their mode of action and clinical application in type 1 diabetes. Adv Immunol 100:13–37CrossRefPubMedGoogle Scholar
  20. 20.
    Kuhn C, You S, Valette F et al (2011) Human CD3 transgenic mice: preclinical testing of antibodies promoting immune tolerance. Sci Transl Med 3:68ra10CrossRefPubMedGoogle Scholar
  21. 21.
    Keymeulen B, Vandemeulebroucke E, Ziegler AG et al (2005) Insulin needs after CD3-antibody therapy in new-onset type 1 diabetes. N Engl J Med 352:2598–2608CrossRefPubMedGoogle Scholar
  22. 22.
    Keymeulen B, Walter M, Mathieu C et al (2010) Four-year metabolic outcome of a randomised controlled CD3-antibody trial in recent-onset type 1 diabetic patients depends on their age and baseline residual beta cell mass. Diabetologia 53:614–623CrossRefPubMedGoogle Scholar
  23. 23.
    Herold KC, Hagopian W, Auger JA et al (2002) Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. N Engl J Med 346:1692–1698CrossRefPubMedGoogle Scholar
  24. 24.
    Sherry N, Hagopian W, Ludvigsson J et al (2011) Teplizumab for treatment of type 1 diabetes (Protégé study): 1-year results from a randomised, placebo-controlled trial. Lancet 6736:60931–60938Google Scholar
  25. 25.
    Friend PJ, Hale G, Chatenoud L et al (1999) Phase I study of an engineered aglycosylated humanized CD3 antibody in renal transplant rejection. Transplantation 68:1632–1637CrossRefPubMedGoogle Scholar
  26. 26.
    Enee E, Martinuzzi E, Blancou P, Bach JM, Mallone R, van Endert P (2008) Equivalent specificity of peripheral blood and islet-infiltrating CD8+ T lymphocytes in spontaneously diabetic HLA-A2 transgenic NOD mice. J Immunol 180:5430–5438CrossRefPubMedGoogle Scholar
  27. 27.
    Penaranda C, Tang Q, Bluestone JA (2011) Anti-CD3 therapy promotes tolerance by selectively depleting pathogenic cells while preserving regulatory T cells. J Immunol 187:2015–2022CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Besancon A, Baas M, Goncalves T et al (2017) The Induction and maintenance of transplant tolerance engages both regulatory and anergic CD4+ T cells. Front Immunol 8:218CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Gagliani N, Magnani CF, Huber S et al (2013) Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat Med 19:739–746CrossRefPubMedGoogle Scholar
  30. 30.
    Dahllof MS, Christensen DP, Lundh M et al (2012) The lysine deacetylase inhibitor Givinostat inhibits beta-cell IL-1beta induced IL-1beta transcription and processing. Islets 4:417–422CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Dahllof MS, Christensen DP, Harving M, Wagner BK, Mandrup-Poulsen T, Lundh M (2015) HDAC inhibitor-mediated beta-cell protection against cytokine-induced toxicity is STAT1 Tyr701 phosphorylation independent. J Interf Cytokine Res 35:63–70CrossRefGoogle Scholar
  32. 32.
    Leus NG, Zwinderman MR, Dekker FJ (2016) Histone deacetylase 3 (HDAC 3) as emerging drug target in NF-kappaB-mediated inflammation. Curr Opin Chem Biol 33:160–168CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Di Liddo R, Valente S, Taurone S et al (2016) Histone deacetylase inhibitors restore IL-10 expression in lipopolysaccharide-induced cell inflammation and reduce IL-1beta and IL-6 production in breast silicone implant in C57BL/6J wild-type murine model. Autoimmunity.  https://doi.org/10.3109/08916934.2015.1134510
  34. 34.
    Lundh M, Christensen DP, Damgaard Nielsen M et al (2012) Histone deacetylases 1 and 3 but not 2 mediate cytokine-induced beta cell apoptosis in INS-1 cells and dispersed primary islets from rats and are differentially regulated in the islets of type 1 diabetic children. Diabetologia 55:2421–2431CrossRefPubMedGoogle Scholar
  35. 35.
    Baas M, Besancon A, Goncalves T et al (2016) TGFbeta-dependent expression of PD-1 and PD-L1 controls CD8+ T cell anergy in transplant tolerance. eLife 5:e08133CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Long AA, Thorpe J, DeBerg HA et al (2016) Partial exhaustion of CD8 T cells and clinical response to teplizumab in new-onset type 1 diabetes. Sci Immunol 1:eaai7793Google Scholar
  37. 37.
    Wallberg M, Recino A, Phillips J et al (2017) Anti-CD3 treatment up-regulates programmed cell death protein-1 expression on activated effector T cells and severely impairs their inflammatory capacity. Immunology 151:248–260CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Wardell SE, Ilkayeva OR, Wieman HL et al (2009) Glucose metabolism as a target of histone deacetylase inhibitors. Mol Endocrinol 23:388–401CrossRefPubMedGoogle Scholar
  39. 39.
    Groux H, O’garra A, Bigler M et al (1997) A CD4+ T cell subset inhibits antigen-specific T cell responses and prevents colitis. Nature 389:737–742CrossRefPubMedGoogle Scholar
  40. 40.
    Roncarolo MG, Gregori S, Bacchetta R, Battaglia M (2014) Tr1 cells and the counter-regulation of immunity: natural mechanisms and therapeutic applications. Curr Top Microbiol Immunol 380:39–68PubMedGoogle Scholar
  41. 41.
    Vojinovic J, Damjanov N, D’Urzo C et al (2011) Safety and efficacy of an oral histone deacetylase inhibitor in systemic-onset juvenile idiopathic arthritis. Arthritis Rheum 63:1452–1458CrossRefPubMedGoogle Scholar
  42. 42.
    Chou DH, Holson EB, Wagner FF et al (2012) Inhibition of histone deacetylase 3 protects beta cells from cytokine-induced apoptosis. Chem Biol 19:669–673CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Plaisance V, Rolland L, Gmyr V et al (2014) The class I histone deacetylase inhibitor MS-275 prevents pancreatic beta cell death induced by palmitate. J Diabetes Res 2014:195739PubMedPubMedCentralGoogle Scholar
  44. 44.
    Lundh M, Galbo T, Poulsen SS, Mandrup-Poulsen T (2015) Histone deacetylase 3 inhibition improves glycaemia and insulin secretion in obese diabetic rats. Diabetes Obes Metab 17:703–707CrossRefPubMedGoogle Scholar
  45. 45.
    Wagner FF, Lundh M, Kaya T et al (2016) An isochemogenic set of inhibitors to define the therapeutic potential of histone deacetylases in beta-cell protection. ACS Chem Biol 11:363–374CrossRefPubMedGoogle Scholar
  46. 46.
    Remsberg JR, Ediger BN, Ho WY et al (2017) Deletion of histone deacetylase 3 in adult beta cells improves glucose tolerance via increased insulin secretion. Mol Metab 6:30–37CrossRefPubMedGoogle Scholar
  47. 47.
    Daneshpajooh M, Bacos K, Bysani M et al (2017) HDAC7 is overexpressed in human diabetic islets and impairs insulin secretion in rat islets and clonal beta cells. Diabetologia 60:116–125CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Alix Besançon
    • 1
    • 2
    • 3
  • Tania Goncalves
    • 1
    • 2
    • 3
  • Fabrice Valette
    • 1
    • 2
    • 3
  • Mattias S. Dahllöf
    • 4
  • Thomas Mandrup-Poulsen
    • 4
  • Lucienne Chatenoud
    • 1
    • 2
    • 3
  • Sylvaine You
    • 1
    • 2
    • 3
  1. 1.University Paris Descartes, Sorbonne Paris CitéParisFrance
  2. 2.INSERM U1151, Institut Necker-Enfants Malades, Hôpital NeckerParisFrance
  3. 3.CNRS UMR 8253, Institut Necker-Enfants MaladesParisFrance
  4. 4.Laboratory for Immuno-Endocrinology, Department of Biomedical SciencesUniversity of CopenhagenCopenhagenDenmark

Personalised recommendations