Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice
Tatiana Takiishi, Hannelie Korf, Tom L. Van Belle, Sofie Robert, Fabio A. Grieco, Silvia Caluwaerts, Letizia Galleri, Isabella Spagnuolo, Lothar Steidler, Karolien Van Huynegem, Pieter Demetter, Clive Wasserfall, Mark A. Atkinson, Francesco Dotta, Pieter Rottiers, Conny Gysemans, Chantal Mathieu
Tatiana Takiishi, Hannelie Korf, Tom L. Van Belle, Sofie Robert, Fabio A. Grieco, Silvia Caluwaerts, Letizia Galleri, Isabella Spagnuolo, Lothar Steidler, Karolien Van Huynegem, Pieter Demetter, Clive Wasserfall, Mark A. Atkinson, Francesco Dotta, Pieter Rottiers, Conny Gysemans, Chantal Mathieu
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Research Article Autoimmunity

Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice

Abstract

Current interventions for arresting autoimmune diabetes have yet to strike the balance between sufficient efficacy, minimal side effects, and lack of generalized immunosuppression. Introduction of antigen via the gut represents an appealing method for induction of antigen-specific tolerance. Here, we developed a strategy for tolerance restoration using mucosal delivery in mice of biologically contained Lactococcus lactis genetically modified to secrete the whole proinsulin autoantigen along with the immunomodulatory cytokine IL-10. We show that combination therapy with low-dose systemic anti-CD3 stably reverted diabetes in NOD mice and increased frequencies of local Tregs, which not only accumulated in the pancreatic islets, but also suppressed immune response in an autoantigen-specific way. Cured mice remained responsive to disease-unrelated antigens, which argues against excessive immunosuppression. Application of this therapeutic tool achieved gut mucosal delivery of a diabetes-relevant autoantigen and a biologically active immunomodulatory cytokine, IL-10, and, when combined with a low dose of systemic anti-CD3, was well tolerated and induced autoantigen-specific long-term tolerance, allowing reversal of established autoimmune diabetes. Therefore, we believe this method could be an effective treatment strategy for type 1 diabetes in humans.

Authors

Tatiana Takiishi, Hannelie Korf, Tom L. Van Belle, Sofie Robert, Fabio A. Grieco, Silvia Caluwaerts, Letizia Galleri, Isabella Spagnuolo, Lothar Steidler, Karolien Van Huynegem, Pieter Demetter, Clive Wasserfall, Mark A. Atkinson, Francesco Dotta, Pieter Rottiers, Conny Gysemans, Chantal Mathieu

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Figure 4

Local accumulation and autoAg-specific suppression by Tregs upon CT.

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Local accumulation and autoAg-specific suppression by Tregs upon CT. (A)...
(A) Real-time PCR analysis of indicated mRNA isolated from CD4+CD25+ T cells sorted from spleen and PLN of long-standing normoglycemic, diabetic, and CT-cured NOD mice. (B) Number of Foxp3-expressing cells in or around the pancreatic islets as determined by manual counting of Foxp3+ cells on immunostained cryosections as shown in Supplemental Figure 9. (C) Representative microphotographs of immunostaining for Ki-67 and Foxp3 and nuclear staining using DAPI on paraffin sections of the pancreas of CT-cured NOD mice. White arrows indicate Ki-67+Foxp3+ double-positive cells, indicating proliferating Foxp3+ Tregs. Original magnification, ×63; detail, ×252. Statistical significance was calculated using Mann-Whitney U test (*P < 0.05, **P < 0.01, ***P < 0.001).