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 3

CT increases functional Tregs.

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CT increases functional Tregs. (A) CD25+Foxp3+ cells in PLNs, shown as m...
(A) CD25+Foxp3+ cells in PLNs, shown as mean ± sem within the CD4 gate. (B) Histogram overlays of FR4 (top) and CTLA4 (bottom) by CD4+CD25+Foxp3+ cells isolated from PLNs (end of treatment). Inset values: MFI of FR4 expression and percentage of CTLA4+ cells in CD4+CD25+Foxp3+ gate, respectively. Gray histograms: staining on indicated sample; solid lines: isotype staining on nondiabetic control sample. (C–E) In vitro polyclonal suppressor assay. CD4+CD25– Tresps isolated from normoglycemic NOD mice were dye labeled and stimulated using 0.5 μg/ml soluble anti-CD3 and accessory cells for 72 hours in the presence of CD4+CD25+ Tregs isolated from cured mice at the end of the indicated treatment. Results of assays for in vitro suppressive capacity are shown. (C) Proliferation of Tresps, shown as percentage of Tresps that had undergone 2 or more divisions, normalized to Tresp-only culture. (D) Activation of Tresps, shown as MFI of CD44 expression, normalized to Tresp-only culture. (E) ELISA for IL-10. (F) Assay for in vivo suppressive capacity. CD25-depleted splenocytes from diabetic NOD mice were transferred into NOD/SCID mice without (white circles) or with CD4+CD25+ cells from CT-cured (white squares) or CT-non-cured (black squares) mice. Shown is the diabetes incidence in recipients. Statistical calculation using Mann-Whitney test (#P < 0.05, ##P < 0.01 versus week 6 CT; †P < 0.05, ††P < 0.01, †††P < 0.001 versus week 14 CT), t test (C–E), and Mantel-Cox log-rank (F). Results in C–E are representative of at least 3 experiments (each at least 3 treated mice). *P < 0.05, ***P < 0.001.