Subcutaneous insulin B:9-23/IFA immunisation induces Tregs that control late-stage prediabetes in NOD mice through IL-10 and IFN[gamma]

Diabetologia (2010) 53:19581970 DOI 10.1007/s00125-010-1777-x

ARTICLE

Subcutaneous insulin B:9-23/IFA immunisation induces Tregs that control late-stage prediabetes in NOD mice through IL-10 and IFN

G. Fousteri & A. Dave & A. Bot & T. Juntti & S. Omid &

M. von Herrath

Received: 6 November 2009 /Accepted: 9 April 2010 /Published online: 20 May 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com

AbstractAims/hypothesis Subcutaneous immunisation with the 923 amino acid region of the insulin B chain (B:9-23) in incomplete Freunds adjuvant (IFA) can protect the majority of 4- to 6-week-old prediabetic NOD mice and is currently in clinical trials. Here we analysed the effect of B:9-23/IFA immunisation at later stages of the disease and the underlying mechanisms.

Methods NOD mice were immunised once s.c. with B:9-23/IFA at 5 or 9 weeks of age, or when blood glucose reached 10 mmol/l or higher. Diabetes incidence was followed in addition to variables such as regulatory T cell (Treg) induction, cytokine production (analysed by Elispot) and emergence of pathogenic CD8+/NRP-V7+ cells. Results A single B:9-23/IFA immunisation protected the majority of NOD mice at advanced stages of insulitis, but not after blood glucose reached 13.9 mmol/l. It increased Treg numbers and lost its protective effect after IFN or IL-10 neutralisation, but not in the absence of IL-4. CD4+CD25+ and to a lesser extent IFN-producing cells from mice protected by B:9-23/IFA induced tolerance upon transfer into new NOD animals, indicating that a dominant Treg-mediated effect was operational. Reduced numbers of CD8+/NRP-V7+ memory T cells coincided with protection from the disease.

Conclusions/interpretation Protection from diabetes after B:9-23/IFA immunisation cannot be achieved once diabetes is fully established, but can be achieved at most prediabetic stages of the disease. Protection is mediated by Tregs that require IFN and IL-10. These findings should provide important guidance for ongoing human trials, especially for the development of suitable T cell biomarkers.

Keywords B:9-23 . IFN . IL-4 . IL-10 . Incomplete Freunds adjuvant . Tregs . Type 1 diabetes

AbbreviationsAPC Antigen-presenting cellB:9-23 9-23 Amino acid region of the insulin B chainCTLA-4 Cytotoxic T lymphocyte antigen 4 FOXP3 Forkhead box p3ICCS Intracellular cytokine stainingIFA Incomplete Freunds adjuvantIGRP Islet-specific glucose-6-phosphatase catalytic subunit-related proteinPDLN Pancreatic draining lymph nodes

Introduction

Type 1 diabetes is one...