Thyroglobulin elicited CD4+ T-cell proliferation kinetics characteristic of a memory response, but unlike TT the autoantigen provoked the persistent production of IL-10. Though monocytes were the primary producers of IL-10, their IL-10 release depended

on small numbers of IL-10-secreting CD4+ T cells, predominantly of the CD45RO+ memory phenotype. The implications of these findings are discussed. Blood samples were collected after obtaining informed consent in 10-ml PLX3397 BD Vacutainer™ heparin tubes and 10-ml BD Vacutainer™ non-coagulant containing tubes (BD Bioscience, Brøndby, Denmark) Ipilimumab in vivo from 19 healthy volunteers (seven women and 12 men; median age = 37 years, range 20–65) attending the blood banks of Odense University Hospital and Copenhagen University

Hospital, all of whom fulfilled the criteria recommended by the Council of Europe for blood donation. The study was approved by the local ethical committee. Tetanus toxoid (molecular weight 150 000) was purchased from the State Serum Institute, Copenhagen, Denmark. The crude TT extract was purified by filtration on Sephacryl S-300, sterile filtered and diluted to 1 mg/ml in phosphate-buffered saline (PBS). Lyophilized KLH (molecular weight 800 000–900 000), purchased from Sigma-Aldrich, (Vallensbæk, Denmark), was dissolved in

water at a concentration of 1 mg/ml. The TG (molecular weight 670 000), purified from human thyroid tissue, was purchased from Biogenesis Ltd. (Poole, UK). The preparation was > 99% pure with immunoglobulin G (IgG); < 0·4%), IgA (< 0·3%) and IgM O-methylated flavonoid (< 0·3%) as the primary contaminants. The TG was diluted to 1 mg/ml in either PBS or medium (RPMI-1640). Boiling TG has previously been shown to abrogate both the cell-proliferative and cytokine responses induced by this antigen, thereby excluding contaminant LPS as the causative agent.12,13 Phycoerythrin (PE) -conjugated anti-human CD3, peridinin chlorophyll-a protein (PerCP) -conjugated anti-human CD4 and CD14, allophycocyanin (APC) – conjugated anti-human CD45RO and fluorescein isothiocyanate (FITC) -conjugated anti-human CD45RA, all of mouse origin, were purchased from BD Biosciences (Brøndby, Denmark). Peripheral blood mononuclear cells (PBMC) were harvested by density centrifugation (814 g, 30 min) over Lymphoprep© (Axis-Shield Poc AS. Oslo, Norway).