Proliferative T cell responses to certain recall antigens against which the cell donor was sensitized (e

Proliferative T cell responses to certain recall antigens against which the cell donor was sensitized (e.g. or immediately following the PBL transfer. Without an early rechallenge with antigen immunization Human PBL, derived from buffy coats from healthy donors or heparinized venous blood from selected hepatitis B surface antigen (HBsAg)-vaccinated donors, were isolated by FicollCHypaque (density = 1077 g/ml; Nycomed Pharma, Oslo, Norway) centrifugation and injected intraperitoneally (1C2 107 hu-PBL per mouse in 05 ml PBS) into the recipient mice. For immunization with TT (obtained from Statens Seruminstitut, WHO, Copenhagen, Denmark), TT was injected intraperitoneally (10 g/mouse) together with human PBL. For immunization with HBsAg, 2 g of a commercial HBsAg vaccine (Engerix-B; SmithKline Beecham Biologicals, Brussels, Belgium) were injected subcutaneously into a hind leg 1 day after cell transfer. Cell collection from the humanCmouse chimeras and flow cytometric analysis At days 7 and 14 following cell transfer, mice were bled and subsequently killed (three mice per group) by cervical dislocation. Peritoneal exudate cells (PEC) were obtained by two rounds of peritoneal lavage with 5 ml ice-cold PBS. PEC from three animals of the same experimental group were pooled. Viable mononuclear cells (propidium iodide-negative) in those pooled PEC suspensions were analysed by flow cytometry. To avoid non-specific staining, the murine cells were gated out with cytochrome-conjugated anti-mouse common leucocyte antigen CD45 (30-F11; PharMingen, Hamburg, Germany). Human cells in the PEC were characterized with the following MoAbs: CD45, CD3, CD4, CD8, CD14, CD19, CD45RO, CD25, CD69, CD71, HLA-DR, CD86. Antibodies were conjugated with FITC or PE. All MoAbs were purchased from Becton Dickinson (San Jose, CA) except for CD71 and HLA-DR, which were from Caltag Labs (San Francisco, CA). Cells (1 105) were incubated with antibodies on ice for 30 min, washed and resuspended in PBS containing 1% bovine serum albumin (BSA) and sodium azide (009%). At least 5000 cells were Monomethyl auristatin E analysed on a FACScan (Becton Dickinson). Isotypically matched negative control antibodies were always used. Lymphoproliferation and cytokine assays For functional analysis PEC derived from animals of the same experimental group were pooled and fractionated by FicollCHypaque centrifugation. The cells harvested from the interphase were further examined. For proliferation assays the cells (1 105 cells/well) were suspended in 200 l complete RPMI 1640 medium. This consisted of RPMI 1640 supplemented with 25 mm HEPES, 50 U/ml penicillin, 50 g/ml streptomycin, 2 mm l-glutamine (all from Gibco, Grand Island, NY), 5 10?5 m-mercaptoethanol (Sigma Chemical Co., St Louis, MO) and 10% heat-inactivated human AB+ serum. The cells were cultured for 4 days in the presence of 5 104 irradiated (30 Gy, 60Co source), T-depleted (by CD2-coated Dynabeads; Dynal AS, Oslo, Norway) autologous PBL. These cultures were stimulated with TT (4 g/ml; Statens Seruminstut), rHBsAg (3 g/ml, recombinant, yeast-derived HBV envelope major protein, a gift from SmithKline Beecham Biologicals), PHA (3 g/ml; Sigma), or were left without stimulating antigen (control culture). The quality of the T-depleted PBL was examined by flow cytometry and T cell content was always 2%. All cultures were performed in triplicate. 3H-thymidine (05 Ci/well) was added 18 h before the cultures were harvested using an automatic cell harvester. The incorporation of 3H-thymidine into dividing cells was measured in a liquid scintillation counter (LKB-Wallac 8100 counter; LKB, Bromma, Sweden). The Mela data were expressed as the mean counts of triplicate determinations and stimulation index (SI) calculated as: SI = mean ct/min of antigen-stimulated cultures/mean ct/min of control cultures. Supernatants from the proliferation assays were collected at 72 h and kept Monomethyl auristatin E Monomethyl auristatin E frozen at ?20C until tested. Commercial kits were used to determine human interferon-gamma (IFN-) (MEDGENIX IFN- EASIATM kit; BioSource Europe S.A., Nivelles, Belgium) and IL-5 (Genzyme Human Interleukin-5 Kit). The assays were performed according to the manufacturer’s guidelines. Determination of total human IgG, IgM and antigen-specific antibody Total human IgG and IgM concentrations in chimeric mouse plasma were determined by an in-house ELISA as described previously [26]. The concentration of human TT-specific IgG in human and mouse plasma was Monomethyl auristatin E measured using the Tetanus Toxoid.