Acquisition of TGN1412 reactivity by circulating T cells requires interaction with matured monocytes. (A) Before preculture at high density, PBMC subsets were depleted as indicated (−) by magnetic sorting (MACS). The group “All” refers to undepleted PBMCs. Cells were stimulated under standard conditions (Figure 1). In the lower panel, unseparated PBMCs cultured at low density (LDC) were added as a source of accessory cells at a 1:1 ratio. Proliferation was determined by [³H]thymidine incorporation between days 2 and 3. Representation of subsets before/after depletion was (%): B cells (5.79/0.014), monocytes (5.41/0.13), DC (0.68/0.0034), and MHC II⁺ (17.3/0.12). (B) Fresh (CFSE labeled) and high-density precultured PBMCs from the same donor were stimulated for 16 hours in low cell density, either separately or placed in coculture (1:1), with 1 μg/mL TGN1412 (blue) or OKT3 (red). Activation status was assessed by CD69 surface expression, gated on CD4⁺ cells. (C) Fresh CFSE-labeled PBMCs were added to 2-day high-density precultured PBMCs from the same donor and returned to high-density culture conditions for 0, 2, or 4 hours before stimulation under standard conditions with TGN1412 for 2 hours. Activation status was assessed by CD69 surface expression. Results are shown as percentage of CD69-positive cells among the CD4⁺CD45RO⁺ population. (D) Purified CD4⁺ T cells (5 × 10⁵/0.5 mL) were cocultured in 48-well plates at the ratios given with monocytes derived from high-density PBMC cultures, or with monocytes isolated from fresh PBMCs and cultured for 2 days under high-density conditions, and stimulated with TGN1412 or OKT3 (1 μg/mL) for 16 hours. Activation status of CD4⁺ cells was assessed by CD69 surface expression. Gray histograms represent unstimulated cells. HDC indicates high-density cell cultures; LDC, low-density cell cultures; DC, dendritic cells; and MHC II⁺, major histocompatibility complex class II-positive cells. Unpaired t test: *P ≤ .05; **P ≤ .005. Data are mean ± SD of triplicate samples.