T cells from fresh and LD cultured PBMCs respond to TGN1412 in the presence of sufficient FcγRIIb. (A) T and B cells and monocytes were isolated from fresh PBMCs. T cells were then cultured with OKT3 or TGN1412 either alone or in coculture with B cells or monocytes at a B-cell or monocyte:T-cell ratio of 0.3:1. T-cell division was determined on day 4. (B) LD precultured PBMCs were cocultured with B cells or monocytes isolated from HD preculture at B-cell or monocyte:T-cell ratios of 0.02:1 to 1:1 in the presence of TGN1412. Division was determined on day 4. Results show mean and range from 2 donors. (C) FcγRIIb expression on transfected CHO-K1 cells. (D) T cells isolated from fresh PBMCs were cocultured with untransfected or FcγRIIb transfected CHO-K1 cells and TGN1412. Supernatant was taken for cytokine determination at 48 hours, and proliferation was determined on day 4. Results from 5 donors are shown.