DC-vaccinated patients with melanoma pretreated with DD have a reduced immune response against the tumor vaccine and a lowered IFNγ response to KLH. (A) DD pretreatment and DC vaccination scheme. Ontak (DD) was given to patients intravenously on 3 consecutive days (days 1-3). On day 4, the first of 4 DC vaccine aliquots (v1-v4) was injected intracutaneously. The remaining aliquots were administered as indicated (every 2-4 weeks). Immunomonitoring was performed with TAA-prestimulated PBMC at Leu1 (L1) and Leu2 (L2) (see panels B-C). Additional monitoring was performed ex vivo with KLH at Leu1 (L1), times of vaccination (v1-v4), and Leu2 (L2) (see panel D). Vacc (vaccination), Leu (leucapheresis). (B) IFNγ EliSpots of peptide-prestimulated PBMC (see “Materials and methods”) against vaccinated TAA after 4 vaccinations (Leu2) compared with Leu1 (prevaccination) assessed separately for MHC class I and II peptides; Note that only those patients reaching Leu2 and able to provide sufficient PBMC could be included. The statistical analysis is based on the paired Student t test, 1-tailed, followed by the Wilcoxon signed rank-sum test. Lines in the scatterplots represent the median of all values. (C) Increase of CD8⁺ T-cell precursors in DD patients and control patients as measured by MLPC limiting dilution after 4 vaccinations. Statistical analysis is as in (B); lines in the scatterplot represent the median of respective values. The increase of spots is expressed as Δ median values. Note that only those patients able to provide enough PBMC at Leu2 could be included. (D) Number of IFNγ ex vivo KLH-specific EliSpots (broken up into 3 groups based on the number of EliSpots) assessed during the course of 4 vaccinations for 16 DD-pretreated patients and 12 control patients at indicated time points if PBMC were available.