CD4⁺ proliferation. (A) CD4⁺ T cells from pre (i), postvaccine 3 (ii), and postvaccine 6 (iii) vaccinations from patient 5 (A0201⁺) were incubated with indicated peptides at 20 μg/mL or 50 μg/mL for 5 days, and 1 μCi [³H]-thymidine was added to the cultures for 20 hours. The cell proliferation was determined by [³H]-thymidine incorporation. Data are mean ± SD from quadruplicate cultures. After 3 vaccinations, cell proliferation increased 54-fold to 331, 37-fold to 427, 4.2-fold to 122A1, and 2.6-fold to 122A (P = .032) at a concentration of 50 μg/mL peptides tested. There was no significant dose dependency of the peptides, and similar responses were also seen after 6 vaccinations. (B) Time course of CD4⁺ response: CD4⁺ T-cell responses of 3 patients who completed 12 vaccinations were calculated by the fold increase of the CD4⁺ T-cell proliferation against 331, 427, and 122A1 peptides over irrelevant peptide B2A2 long at a concentration of 50 μg/mL. Responses at T9 and T12 were not tested for patient 2 (A0201⁺) because of clinical relapse before those time points. CD4⁺ T-cell responses were elicited and maintained throughout vaccination, although the magnitude to each peptide with respect to vaccination times varied among patients.