Wild-type NOD and CD28−/− NOD mice  were bred in our animal facility under specific pathogen-free conditions and with free access to food and water. CD28−/− NOD mice were provided by J. Bluestone (University of California, San Francisco, CA, USA). Blood glucose levels were measured using an Accu-Chek Performa glucometer (Roche Diabetes Care, Meylan, France). A value >13.9 mmol/l signalled diabetes onset. Four to 10 week-old female mice were used in this study. The experiments were approved by the French Ministry of Education and Research (number 04463.02).
In vivo treatments
PV1-PEG was produced by OSE Immunotherapeutics (Nantes, France). Fab′ fragments were obtained from hamster anti-mouse CD28, clone PV1, and subsequently conjugated to a 40 kDa PEG moiety to prolong its half-life in vivo. PV1-PEG was administered to NOD mice at 10 mg/kg i.p. (in PBS), twice weekly for 4 or 10 consecutive weeks. Rapamycin (Sigma-Aldrich, Lyon, France) was reconstituted according to the manufacturer’s instruction and injected i.p. at 1 mg/kg twice weekly for 4 weeks. These treatments were randomly allocated to each group of mice.
Antibodies to CD4 (GK1.5, 1/300), CD8 (53-6.7, 1/300), T cell receptor (TCR) Vβ (H57-597, 1/400), CD44 (IM7, 1/400), CD62L (MEL-14, 1/300) and CD19 (1D3, 1/400) were obtained from BD Biosciences (Le Pont de Claix, France). Antibody to FOXP3 (FJK-16S, 1/200) was obtained from eBioscience (Life Technologies, Saint-Aubin, France). All antibodies were titrated before use. Detection of autoantigen-specific CD8+ T cells was performed using allophycocyanin-labelled MHC class I (H-2Kd) tetramers carrying the proinsulin (PI)15–23 or islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP)206–214 peptides (NIH Tetramer Core Facility, Atlanta, GA, USA). Cells were analysed on a FACSCanto II cytometer using FlowJo software (FlowJo, Ashland, OR, USA).
IFNγ enzyme-linked immunospot
Polyvinylidene fluoride (PVDF) plates (Merck Millipore, Guyancourt, France) were coated with an IFNγ capture antibody (U-CyTech, Utrecht, the Netherlands). Splenocytes were cultured at 2.5 × 105/well with PI15–23 or IGRP206–14 peptides (7 μmol/l). CD3 antibody (145 2C11, 1 μg/ml) was used as a positive control. After a 20 h culture period, IFNγ was detected using biotinylated IFNγ antibody, streptavidin–horseradish peroxidase and SigmaFAST nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) (Sigma-Aldrich). IFNγ spot readouts were expressed as spot-forming units (SFU)/106 cells.
In vitro suppression assay
CD4+CD25− Teffs and CD4+CD25+ Tregs were isolated by magnetic sorting (Miltenyi Biotec, Paris, France). Violet Proliferation Dye (VPD)450-labelled Teffs from untreated NOD mice were incubated with Tregs (5 × 104 cells/well each) isolated from treated NOD mice. Cells were stimulated with CD3 antibody and antigen-presenting cells (APCs: irradiated syngeneic splenocytes, 105 cells/well) for 3 days. Proliferation was evaluated by VPD450 dilution.
Inhibition of proliferation (%) = [1 − (%Teffs that divided more than three times in the presence of Tregs)/(% CD4+CD25− that divided more than three times alone)] ×100.
Analysis of pancreatic T cell infiltrate
Pancreases were perfused via the pancreatic duct with collagenase P (Roche Diagnostics, Mannheim, Germany), recovered and digested for 8 min at 37°C. After washing with Hank’s balanced salt solution (HBSS) + 10% (vol./vol.) FBS and vigorous shaking to allow islet disruption, pellets were resuspended in PBS + 2% FBS before staining.
All statistical analyses were performed using GraphPad Prism 6 software (Graphpad Software, La Jolla, CA, USA). The occurrence of diabetes was plotted using the Kaplan–Meier method. Statistical comparison between the curves was performed using the logrank (Mantel–Cox) test. When appropriate, results were analysed using the Mann–Whitney test.