Differential expression of costimulatory molecules. Eight-day A.AL DC cultures (5 × 10⁵ cells/well of 96-well round-bottom plate) grown with GM-CSF (10 ng/ml) were conditioned for 20 hours by incubation with 5 × 10⁴ CD4⁺ Th cells and 0.5 μM peptide T1 or T1(A). (a) Gated class II⁺ cells were examined for expression of CD40 and costimulatory molecules B7-1 and B7-2 after stimulation with T1(A) (solid heavy line) compared with T1 (light line). DC cultures incubated with the Th cell without peptide are shown as the shaded area and are similar to cultures incubated without Th cells (not shown). (b) Eight-day BALB.A10 DCs were conditioned similarly for 36 hours. Gated CD11c⁺ shown in the first panel as the shaded area compared with PE-isotype control (dashed line) were examined for costimulatory molecules B7-1, B7-2, ICAM, and CD44H — DCs conditioned with Th cells stimulated with T1(A) (solid heavy line) versus T1 (light line). The shaded area represents DC cultures incubated with resting Th cells not stimulated with peptide. CD40 was maximally expressed under all culture conditions in this experiment. For B7-1 and 2, T1 and T1(A) are shown in separate panels for clarity. Similar results were observed in five additional experiments.