Immune responses after vaccination. (A-C) Unprimed ex vivo proliferation assays of fresh CD4⁺ T cells from vaccinated patients. The assays were conducted as described in “Patients, materials, and methods.” The number of weeks are shown on the x-axis. Before indicates before vaccination; +5, after 3 vaccinations; +10, after 5 vaccinations; +24, after 8 vaccinations; and +50, after 11 vaccinations. The stimulation index is shown on the y-axis. Panel A shows stimulation at a 2:1 ratio with b3a2 long peptide at 20 μg/mL; panel B, stimulation at 4:1 ratio with b3a2 long peptide at 20 μg/mL; and panel C, stimulation using total CD4⁺ cells and b3a2 at 20 μg/mL. Medians are shown as horizontal bars. (D) Interferon-gamma ELISPOT on CD4⁺ T cells from vaccinated patients at different time points (see panels A-C for the time points in weeks). (E-F) CD8⁺ interferon-gamma ELISPOT from vaccinated patients. Panel E shows patient 5 before immunization. Black bars show A3 peptide stimulation; white bars, A3/A11 peptide stimulation. The peptide with the APCs in the assay is shown on the x-axis. Panel F shows cells from patient 6 stimulated with CML-A3/A11 peptide before and after 5 immunizations. The peptide used with the APCs in the assay are as follows: black bars, no peptide; hatched bars, CML-A3 peptide; gray bars, CML-A3/A11; and white bars, A24 negative control. Error bars indicate SEM.