Qualitative and quantitative upregulation of CD40L on CD4⁺ T cells stimulated with T1(A) and concordant increased activation of DCs to stimulate CTL. (a) CD40L expression on T1-specific CD4⁺ T cells incubated overnight with different concentrations of T1 (dashed line) or T1(A) (solid line) and DCs, or without peptide (shaded area). (b) CD40L expression on gated CD4⁺ T1-specific T cells stimulated 36 hours with indicated concentrations of T1 or T1(A) peptide and naïve A.AL splenic B cells (B220⁺ selected). The bar in the left panel indicates background CD40L without peptide. (c) Percentage CD4⁺/ CD40L⁺ cells plotted versus peptide concentration for Th1 (5 × 10⁴ CD4⁺) cells incubated 24 hours with 5 × 10⁵ BALB.A10 DCs and T1 or T1(A) peptide, or no peptide (open diamond). (d) 5 × 10⁶ DCs of the same preparation as in c were incubated with different concentrations of peptide and 5 × 10⁵ of the same CD4⁺ cells, but in 12 well plates. CD4⁺ cells were depleted by magnetic beads 24 hours later. Remaining DCs were pulsed with 0.5 μM CTL epitope P18IIIB, irradiated, and 5 × 10⁶ added to 5 × 10⁶ purified CD8⁺ cells from spleens of 3 mice immunized 8 weeks earlier with PCLUS3-18IIIB, and rmIL-2 (10 U/ml) was added on day 0. Specific lysis 7 days later on P18IIIB-pulsed P815 targets at a 50:1 E:T ratio is plotted versus Th peptide concentration used in conditioning cultures, or without Th peptide (open diamonds). Similar results were obtained in 2 other experiments. (e) Level of CD40L expression on the Th cells as a function of peptide concentration, using less rested Th cells which were 93% CD40L⁺ even without stimulation. (f) P18-I10/H-2Dd tetramer staining of CD8⁺ cells stimulated with 1 μM P18-pulsed DC conditioned with Th cells from the same experiment as in (e) (less control 3.3% with unconditioned DCs).