Transduction with the OKT3::CD14 construct resulted in Bw5147 cells expressing high levels of membrane-bound anti-CD3 antibody fragment on their surface and were thus termed Bw-anti-CD3high stimulator cells. Single cell clones were obtained from both Bw lines and cell clones expressing homogenous amounts of membrane-bound anti-CD3 antibodies were selected for further use. cDNAs encoding human CD80, CD58, CD54, CD150, EPZ015666 manufacturer TL1A, 41BB-L and ICOS-L were PCR amplified from a human dendritic cell library and cloned into the retroviral expression vector pCJK2 generated in our laboratory. Integrity of these expression plasmids was confirmed by
DNA sequencing. Using retroviral transduction these molecules were expressed on the T cell stimulator cells as described (Steinberger et al., 2004). Control stimulator cell lines expressing no human molecule were generated by treating T cell stimulator cells with supernatants derived from retroviral producer cell lines transfected
with empty vector DNA or a vector encoding GFP. All T cell proliferation assays were done in triplicates, means and SD are shown. For T cell proliferation assays human T cells (1 × 105/well) were co-cultured with irradiated (6000 rad) T cell stimulator cells (2 × 104/well) for 72 h. In some experiments Adalimumab Sirolimus manufacturer (Humira, Abbott Laboratories, Chicago, IL) or Beriglobin P as control (CSL Behring GmbH, Marburg, Germany), was added at a final concentration of 10 μg/ml at the onset of culture. To assess T cell proliferation methyl-3[H]-thymidine (final concentration: 0.025 mCi; Perkin Elmer/New England Nuclear Corporation, Wellesley, MA) was added for the last 18 h prior harvesting of the cells. Methyl-3[H]-thymidine uptake was measured as described (Pfistershammer et al., 2004). Purified human T cells (5 × 105/well) were co-cultured in 1 ml medium with 1.2 × 105 irradiated anti-CD3high T cell stimulator cells expressing human
costimulatory molecules as indicated. Following 7 days of culture, T cells were harvested, counted and analyzed for CD8+ expression. 5 × 105 T cells were re-cultured with 1.2 × 105 irradiated stimulator cells as described above. Five rounds of stimulation were performed. For each round of stimulation the T cell expansion factor was calculated by dividing the starting cell Liothyronine Sodium number by the cell number obtained after 7 days of stimulation. Cytotoxic activity of expanded T cells was measured using a europium release assay kit (Delfia, Perkin Elmer) following the manufacturer’s protocol. Briefly, expanded T cells (1 × 105/well) were incubated with the labeled target cells (5 × 103/well; Bw-anti-CD3high cells or Bw cells not expressing anti-CD3 as control) for 2 h at 37 °C. For detection of cell lysis-associated europium release 20 μl of supernatant was transferred to a 96-well flat bottom plate and 200 μl enhancement solution was added.