Tregs typically express high levels of the interleukin

(IL)-2 receptor α-chain CD25, the transcription factor FoxP3 and low levels of the IL-7 receptor CD127 [18-22]. However, both FoxP3 and CD25 can also be expressed by activated non-regulatory AZD9668 T cells. CD39 has also been suggested to be involved in Treg function through the removal of adenosine triphosphate (ATP) and has thus been used to identify subsets of Tregs [23]. Tregs can suppress proliferation and cytokine secretion in a broad range of cell types, including CD4+ and CD8+ T cells, and their dysfunction leads to immunopathology [24]. It has been reported recently that rather than there being a deficiency in Treg numbers, effector T cells (Teff) from patients with T1D are resistant to Treg-mediated suppression [25, 26]. The aim of this work was to investigate whether an increase in cells with a Treg phenotype persisted at 4 years after GAD-alum treatment. In addition, we tested whether GAD-alum treatment affected the suppressive

capacity of Tregs. This study was approved by the Research Ethics Committee at the Faculty of Health Sciences, Linköping University, Sweden. Written informed consent was obtained from participating individuals, and for those aged <18 years also their parents, in accordance with the Declaration of Helsinki. The design and characteristics of the Phase II trial have been described elsewhere [3]. Briefly, 70 T1D children between 10 and 18 years of age with fewer than 18 months of disease duration were recruited at eight Swedish paediatric Regorafenib ic50 3-mercaptopyruvate sulfurtransferase centres. Participants had a fasting serum C-peptide level above 0·1 nmol/l and detectable GADA at inclusion. They were randomized to subcutaneous injections of 20 μg GAD-alum (n = 35) or placebo (n = 35) at day 0 and a booster injection 4 weeks later in a double-blind setting. After 4 years, patients and their parents were asked whether they were willing to participate in a follow-up

study. Fifty-nine patients, of whom 29 had been treated with GAD-alum and 30 received placebo, agreed to participate. Fluorescein isothiocyanate (FITC)-conjugated anti-CD39 (clone A1; Biolegend, San Diego, CA, USA), phycoerythrin (PE)-conjugated anti-FoxP3 (clone PCH101), allophycocyanin (APC)-conjugated anti-CD25 (clone BC96) and FITC- and PE-cyanine 7 (PE-Cy7)-conjugated anti-CD127 (clone eBioRDR5; eBioscience, San Diego, CA, USA), Alexa 700- and Pacific Blue-conjugated anti-CD4 (clone RPA-T4), APC-Cy7-conjugated anti-CD25 (clone M-A251; BD Pharmingen, Franklin Lakes, NJ, USA), and relevant isotype- and fluorochrome-matched control antibodies were used in this study. In addition, 7-amino-actinomycin D (7-AAD; BD Pharmingen) was used to measure cell viability. Peripheral blood mononuclear cells (PBMC) from GAD-alum-treated (n = 24) and placebo-treated (n = 25) patients were isolated from whole blood by Ficoll-Paque (Pharmacia Biotech, Piscataway, NJ, USA) density gradient centrifugation within 24 h after drawing.