Fig. 2.

Modulation of antitumor immune responses of PBL from HLA-A2 + healthy donors, stimulated by IFN-DC loaded with apoptotic Karpas-422 cells (HLA-A2 + FL cells) in the presence of lenalidomide. a Frequencies of Karpas-422-responsive CD3 +/CD8 + lymphocytes and NK cells (CD3 −/CD56 +), as determined by FACS analysis for IFN-γ intracellular staining and degranulation (CD107a membrane mobilization) in PBL cultured for 14 days with autologous IFN-DC loaded with apoptotic Karpas-422 cells in the presence or not of 2 µM lenalidomide. Representative dot plot graphs from one representative experiment are shown. b Modulation of IFN-γ, TNF-α and IL-10 release as evaluated in culture supernatants by ELISA test at day 7 and 14. The data represent the mean value ± SD of 4 independent experiments. Statistically significant differences were calculated using Wilcoxon test (*p ≤ 0.05). c Frequency of CD25 +/FOXP3 + Tregs in CD4 + lymphocyte, as assessed by flow cytometric analysis. The dot-plot analysis of CD25 +/FOXP3 + expression in CD4 + cells from one representative experiment is shown d Spearman’s rho correlation calculation between Treg frequency and the percentage of degranulating CD8 + cells in response to lymphoma cells, measured as CD107a mobilization to the cell membrane by FACS analysis. An inverse correlation (ρ = − 0.857) is shown in lenalidomide-exposed cultures (ρ = − 0.857). e Cytotoxic activity against Karpas-422 and NK-sensitive K562 target cells as assessed by Calcein-AM assay in PBL cultures, showing Treg frequency reduction (group 1) or irrelevant changes (group 2) in response to lenalidomide treatment (see “Results”)