Figure 6.

EC-induced CD86 trans-costimulation is mediated by CD3⁺CD4⁺ T cells and not by contaminating CD4⁺ dendritic cells. (A) FACS^® analysis of CD4⁺ T cells purified by positive selection and depletion of HLA-DR⁺ and CD14⁺ cells. Double staining of purified CD4⁺ T cells with FITC-conjugated CD3 and PE-conjugated CD4 antibodies. (B) CD3⁺CD4⁺ T cells were cocultured with IFN-γ–treated ECs for 72 h and were repurified by positive selection. FACS^® analysis was then performed by double staining for CD3 and CD86. Histograms show shifts in PE-conjugated CD86 (shaded area) and control PE-isotype antibody staining by CD3⁺ cells gated within regions R1 (scattergram in A). Illustrated are CD86 expression in resting, 72-h EC-activated and PHA (1 μg/ml)-activated CD4⁺ T cells. (C) CD4⁺ T cells were modified by ECs to express CD86 (as above) and were irradiated and cocultured with resting autologous CD4⁺ T cells in the presence of low dose mitogen (PHA 0.3 μg/ml). Anti-CD86 (10 μg/ml) was added to cultures as described in Fig. 4. Shown is one experiment representative of four performed in triplicate cultures ± 1 SD. Irradiated stimulator cells alone failed to proliferate.