Soluble costimulation enhances the alloantigen-specific CD154 signal in both intracellular staining protocol and live assay and kinetics of CD154-expressing CD4⁺ T-cell subsets. (A) Representative dot plot of the CD154 signal in CD4⁺ T cells using the CD154 live assay. Donor PBMCs were stimulated with CFSE-labeled donor PBMCs in medium only (unstimulated, i) or with costimulation (cos only, ii) provided by the combination of soluble anti-CD28 and anti-CD49d monoclonal antibodies for 24 hours. The MLR (inducing class II mismatches) was performed in the presence of costimulatory antibodies (alloantigen stimulated, iii). Next, the percentage of CD154⁺ cells within the different CD4⁺ T-cell subsets was measured, comparing the intracellular assay using brefeldin A (BFA) with the CD154 live assay using αCD40 (B). The closed and open bars represent the net CD154 signal (corrected for the background obtained for autologous stimulation) with and without costimulation, respectively. The results of 4 to 6 independent experiments for every condition are shown. The expression in time of CD154 on alloreactive (C) and CMV-specific (D) CD4⁺ T cells was monitored using the CD154 live assay with costimulation. Total CD4⁺ T cells (■), naive CD4⁺ T lymphocytes (▼; CD45RO⁻CCR7⁺; Tnaive), and memory CD4⁺ T lymphocytes (●; CD45RO⁺; Tmem) expressing CD154. A representative example of 4 separate experiments is shown.