Figure 1. Induction of HCMV-specific immune responses following autologous tumor-lysate-pulsed DC vaccination.

(A) Histograms showing HCMV-specific CD8⁺ T cells (CD4/13/19⁻CD3⁺CD8⁺CMVpp65 tetramer⁺) prior to treatment, after one and two DC vaccinations, and at 6 months after DC vaccination. Annotated numbers reflect the percentage of all CD3⁺CD8⁺ T cells. (B) Combined tetramer and intracellular cytokine staining of patient #4-908 PBMC from pre-treatment (Top plots) and after 2 DC vaccinations (bottom plots). PBMC were restimulated for six hours with (right plots) or without (left plots) the immunodominant HCMV peptide in the presence of Brefeldin A, followed by surface antibody labeling, fixation/permeabilization and labeling of intracellular IFN-γ. Annotated boxed numbers reflect the frequency of CMV⁺IFN-γ⁻, CMV⁺IFN-γ⁺ and CMV⁻IFN-γ⁺ T cells gated from the CD3⁺CD8⁺ population.^~ (C) Immunohistochemical localization of HCMV pp65 in glioblastoma. 6 μm sections were deparaffinized in xylene, subjected to an antigen retrieval solution (Dako) and incubated with the pp65-specific mAb (Novocastra) overnight in a humidified chamber at 4°C. Expression was revealed after the incubation with a biotinylated secondary mAb (Vector Labs) and DAB color development (Vector Labs). (D) Serum cytokine levels during the initial bi-weekly DC vaccination regimen. 50 microliters of serum was tested for the presence of TH1 (IL-2, TNF–α, IFN-γ) and T_H2 (IL-4, IL-6, IL-10)-type cytokines by cytometric bead array at the indicated timepoints.