pylori. We have found that H. pylori-stimulated DCs drive Treg proliferation, and impair their suppressive function through the production of IL-1β. This is corroborated by in-vivo data buy PLX4032 showing active division of Tregs in biopsy samples from infected individuals. Dissection of the long-term impact of Treg modulation and dysregulated immunpathology in the context of H. pylori may

provide new insights into the mechanisms underlying the development of H. pylori-associated complications and/or potential targets for the local treatment of inflammation associated with H. pylori in the 15–20% of individuals unresponsive to eradication therapy. Peripheral blood mononuclear cells (PBMCs) were separated from buffy coats provided by the National Blood Transfusion Centre (South Thames, London, UK). CD14+ and CD14− cells were then separated using CD14-Beads (Miltenyi Biotec, Woking, UK), according to the manufacturer’s

instructions. The CD14+ cells were then cultured in RPMI-1640 (Invitrogen, Paisley, UK) PXD101 with 10% fetal calf serum (FCS; SeraQ, East Grinstead, UK), 50 IU/ml penicillin, 50 μg/ml streptomycin and 2 mM L-glutamine (PSG) (PAA Laboratories GmbH, Pasching, Austria). To develop DCs, IL-4 (10 ng/ml) (First Link, Birmingham, UK) and granulocyte–macrophage colony-stimulating factor (GM-CSF) (20 ng/ml) (kindly donated by Dr S. Brett, GlaxoSmithKline, Stevenage, UK) were added every 2 days before the cells were harvested at day 5. T cells were enriched from PBMCs Tideglusib derived from buffy coats by negative selection. CD4+ T cells were purified using a cocktail of antibodies against CD8, CD33, CD14, CD16, CD19, CD56 and γδ-T cell receptor (TCR). The CD4+ T cells were then divided into CD25+ and CD25− cells using anti-CD25 beads (Dynal Biotech, Oslo, Norway). For the CD25hi separation, CD4+ T cells were stained for CD4 and CD25 using anti-CD4-allophycocyanin (APC) (S3·5;

Caltag, Buckingham, UK) and anti-CD25-phycoerythrin (PE) (3G10; Caltag). The CD4+CD25hi (top 2% for expression of CD25) were then separated from the CD4+CD25− T cell population by fluorescence-activated cell sorting (FACS) using a MoFlo high speed multi-laser cell sorter (Cytomation, Fort Collins, CO, USA) running Summit version 3·1 software (Cytomation). Suppression assays were all carried out in complete medium (RPMI with PSG) containing 10% human serum (Biosera, Ringmer, UK) using 2 × 104 T cells with the following conditions: CD25− alone, CD25− : CD25+ at a 1:1 ratio and CD25+ alone. These cells were stimulated with CD3/CD28 expander beads (Dynal Biotech) in the presence of H. pylori. Alternatively, the T cells were stimulated with 2 × 103 allogeneic DCs treated previously with H. pylori, or medium alone for 8 h. Media were supplemented, or not, as described by IL-1 receptor antagonist (IL-1RA) (10 μg/ml, kindly donated by Dr Keith P. Ray, GlaxoSmithKline, Stevenage, UK), anti-IL-6 (10 μg/ml; R&D Systems, Abingdon, UK) or anti-TNFRII (0·2 μg per well; R&D Systems).