FIGURE 2.

Existence of HLA‐A*0201 restricted YLQPRTFLL‐specific cytotoxic CD8+ T cells in MM and CLL patients. HLA‐A2 positive samples (1‐3 × 10⁶) were incubated with 1 μL of Fixable Viability Stain 700 (BD Biosciences) at room temperature for 15 min. Afterwards, 10 μL of dextramer HLA‐A*0201/YLQPRTFLL‐specific‐PE (Immudex, Copenhagen, Denmark) conjugated complex was added to cells and incubated at room temperature for 10 min. Next, anti‐CD3 BV510, anti‐CD4 APC‐Cy7, anti‐CD8 Percp‐Cy 5.5, anti‐CD45RA BV421, anti‐CCR7 FITC, anti‐CD27 APC and anti‐CD28 Pe‐Cy7 (all BD Biosciences) were added to cells. Cells were analyzed by FACSuite on BD FACS Lyric Flow Cytometer (BD Biosciences, CA, USA). Increase in MdFI levels 2‐5 weeks after second vaccine dose and decrease 12 weeks after second dose in MM (A) and CLL (B) were observed. Statistical comparisons were performed using the Wilcoxon matched‐pairs signed‐rank test and the one‐way analysis of variance (ANOVA). (C) Results were compared to isotypic controls and fluorescence minus one control (FMOC), with the exclusion of dead cells to characterize YLQPRTFLL‐specific CD8+ naïve (CD45RA+CCR7+), central memory—CM (CD45RA‐CCR7+), effector memory—EM (CD45RA‐CCR7−) and terminally differentiated effector memory—TEM (CD45RA+CCR7−) phenotype of specific cytotoxic T‐cells. Distribution of frequencies of YQL‐specific CD8+ T cells in all HLA‐A*0201 positive MM (D) and CLL patients (E) in two‐time points after second vaccine dose [Color figure can be viewed at wileyonlinelibrary.com]