Time course of APC capacity and cell-surface expression of costimulatory molecules B7-1 and B7-2 on follicular lymphoma cells stimulated in vitro with CD40L and IL-4. Induction/upregulation of B7-1 and B7-2 was analyzed by FACS using directly conjugated MoAbs or by IC on cytospin preparations of the same cells. Cells were obtained from culture and extensively washed before analysis at time points indicated. (A) FACS analysis. Unshaded area indicates fluorescence of isotype-matched antibody. (B) IC of cytospin preparations. To detect differences in intensity by IC, primary antibodies were used at lower concentrations than described for Fig 2. B7-1 MoAb (EW3.4B2.C4) was used at a 1:2,000 dilution, B7-2 MoAb (HA5.2B7) at a 1:12,000 dilution. (C) Allogeneic T-cell proliferation induced by unstimulated FL cells and CD40-FL cells. FL cells were activated as described in Material and Methods, washed, irradiated, and used at 5 × 104 stimulator cells/well. Highly purified allogeneic T cells (1 × 105 cells/well) were used as responders. T-cell proliferation was measured at day 3 (data not shown) and day 6 by thymidine incorporation for the last 16 hours of the culture period. Approriate controls (T cells, FL cells, CD40-FL cells) were less than 1,800 cpm. One representative experiment (FL4, FL12, FL16) of three experiments is shown.
