HAdV-5-specific CD4+T cell responses. PBMC were cultured with autologous DC transduced with HAdV-5 TMM2 (baseline) for 1 week, followed by 2 restimulations with HAdV-5 TMM2/DC (weeks 1 and 2) and harvest (week 3). (A and B): CD4+ T cells were purified and tested by direct ELISPOT for recognition of hexon901-930 peptide (A) or full length HAdV-5 (HAdV-5 LacZ, B). The mean frequency of cytokine producing cells from seven donors is shown. These cells were also tested for IFNγ and IL-2 production by flow cytometry, which was found to be low, averaging at 0.05% at week 3 (data not shown). (C). Data from 7 independent HD is shown. Identically cultured melanoma patient cells responding to HAdV-5 LacZ or HAdV-5 TMM2 (HAdV-5 plus melanoma antigens) is shown (n = 2, averaged), for total IFNγ-producing cells and polyfunctional, simultaneous IFNγ + IL-2-producing cells (D). Error bars represent standard error of the mean. *Significant response vs. baseline by single tailed student’s t-test (p < 0.05).