Figure 4.

EC-modified CD4⁺ T cells provide CD86-mediated trans-costimulation to autologous CD4⁺ T cells. (A) CD4⁺ T cells were cultured on a monolayer of IFN-γ–treated endothelial cells for 72 h and were reisolated from the coculture by positive selection using CD4-coated magnetic beads. These cells were then irradiated (1,750 rads) and 10⁵ cells were cultured with autologous CD4⁺ T cells (5 × 10⁵) in the presence of low dose PHA (0.3 μg/ml) for 4 d. Shown are the proliferative responses of CD4⁺ T cells. Anti-CD86 (10 μg/ml, IT2.2 clone), anti-CD80 (10 μg/ml, BB1-1 clone), or CTLA4 Ig (10 μg/ml) were added as indicated. Illustrated is one experiment representative of three similar experiments in triplicate cultures ± 1 SD. Anti–HLA-DR mAbs (LB3.1) do not inhibit CD4⁺ T cell proliferation (data not shown). (B and C) To determine the EC cell surface molecule(s) that mediates induction of CD4⁺ T cell CD86 expression, anti–HLA-DR (LB3.1), anti–LFA-3 (1E6), anti–ICAM-1 (RR1/1), anti-CD40(220), or anti-OX40 (Ancell) were added to the primary CD4⁺ T cell–EC coculture at optimal blocking concentrations. CD4⁺ T cells were reisolated, irradiated, and added to autologous CD4⁺ T cells in secondary cultures in the presence of low dose PHA as described above. Proliferation was assessed by [³H]thymidine incorporation for the final 18 h of a 6-d culture. Data is a representative of three experiments performed in triplicate cultures ± 1 SD. Irradiated stimulator cells alone failed to proliferate, as illustrated in A.