Figure 2
BMMC cultures depleted of contaminating APC were fully capable of antigen presentation. 1 × 105 pure BMMC depleted of B cells/macrophages or DC were incubated for 72 hr with 105 3Do-54.8 in the presence of IgE-TNP-OVA, TNP-OVA (Panel A), OVA (Panel B) or OVA peptide (Panel C). The residual contaminating APC eluted from the anti-B220/F480 and CD11c MACS columns from bulk cultures (~2 × 108 cells in order to harvest around 1% contaminating cells for experiments), were prepared. 0.4 × 105 B cells/macrophages (Panel D) or 0.4 × 105 DC (Panel E) were incubated for 72 hr with 105 3Do-54.8 in the presence of IgE-TNP-OVA, TNP-OVA, OVA, or OVA peptide. Next, in the titration experiment, pure mast cells were similarly prepared as above by MACD columns, along with column eluted, residual contaminating APC of these bulk cultures. The different cell types were then incubated with 105 3Do-54.8 in the presence of OVA peptide, at a APC to T-cell ratios ranged from 1:1 to 0.0025:1 at a serial 2- to 2.5-fold dilutions (Panel F). Levels of IL-2 in 72 hr supernatants were determined by stimulating 3H-thymidine incorporation and proliferation of IL-2 dependent NK-3 cells as described in Material and methods.