Expanded Tregs and Teffs were thawed and incubated

in AIM-V 10% HS at 37°C, 5% CO2 overnight, then resuspended at 0·5 × 105 cells/ml. Teffs were plated into 96-well U-bottomed plates at a density of 5 × 104 cells per well, while Tregs were plated into Teff-containing wells at Treg-to-Teff ratios of 1:1, 1:2, 1:4, 1:8 and 1:16. Treg/Teff cultures were stimulated with 5 μg/ml soluble anti-CD3 and 1 μg/ml soluble anti-CD28 antibodies. Unstimulated wells were included as negative controls, both from patients and interassay control healthy Teffs. IL-2 (1 U/ml) was added to all wells. Supernatants were collected after 3 days of culture and FG4592 cells were incubated with 0·2 μCi [3H]-thymidine (PerkinElmer, Waltham, MA, USA) for 18 h before harvesting. Thymidine incorporation was measured using a 1450 Wallac MicroBeta counter (PerkinElmer). C-peptide levels were measured in serum samples with a time-resolved fluoroimmunoassay (AutoDELFIATM C-peptide kit, Wallac; PerkinElmer), as described [3]. Stimulated C-peptide was measured during a mixed meal tolerance test (MMTT) in GAD-alum- (n = 21) and placebo- (n = 10) treated patients who had a maximal C-peptide response check details of >0·20 nmol/l at the 30-month follow-up. Clinical effect of treatment was defined by changes in stimulated

C-peptide measured as area under the curve (AUC) from baseline to 48 months. Statistically significant differences were determined using the Mann–Whitney two-tailed U-test for unpaired observations, as

data were determined to be significantly different from a Gaussian distribution. Wilcoxon’s signed-rank test was used to compare ever paired samples. Linear regression was used to compare slope and Y-intercept of suppression curves, and correlations were determined with Spearman’s rank correlation coefficient test. A probability level of <0·05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism software, version 5·04 (GraphPad Software, Inc., La Jolla, CA, USA). We have demonstrated previously that in-vitro stimulation with GAD65 induced CD4+CD25hi FoxP3+ cells in PBMC from GAD-alum-treated patients [9]. To determine whether this effect persisted 4 years after treatment, we analysed CD25hiCD127lo cells and used FoxP3 and CD39 as additional markers to discriminate Tregs from activated T cells more accurately. Thus, the expression of CD25, CD127, FoxP3 and CD39 on CD4+ lymphocytes was analysed in PBMC after 7 days of incubation with or without GAD65. Gates used for analysis and representative PBMC samples describing the expression of CD4, CD25 and CD127 are shown in Fig. 1a,b. The frequency of CD25hiCD127lo cells in the CD4+ population was increased significantly upon GAD65 stimulation in GAD-alum-treated patients compared to unstimulated cells (7·4% and 4·5%, respectively), but not in the placebo group (Fig. 1c).