CMV-specific CD8⁺T cells from CLL patients and HCs are equally effective in forming adequate synapses with APCs. (A-E) Enriched CD8⁺ T cells (1 × 10⁴) from CLL patients and HCs were incubated for 30 minutes with CFSE-labeled CMV-peptide–loaded HLA-I–matched EBV-LCLs (1 × 10⁵) and fixated with 2% paraformaldehyde. Cells were stained for CD8 (CD8-APC), F-actin (PhTR), and a nuclear dye (Hoechst) and acquired using ImageStream X100 (0.5-1.0 × 10⁵ events). As a negative control, enriched CD8⁺ T cells and nonloaded EBV-LCLs were analyzed. (A) T-cell/B-cell aggregates were identified using CFSE vs CD8-APC bivariate plots. (B) Doublets were gated on their content in the DNA-binding dye Hoechst and cell aggregates were discarded. (C) To identify adequate ISs, a synapse score was computed as a ratio of the maximum pixel intensity of the PhTR (Max Px) signal at the T-cell/B-cell interface vs the mean pixel intensity of the PhTR (Mean Px) signal on the CD8⁺ T cell. (D) This score identifies doublets in which the PhTR signal has polarized to the IS. (E) Percentage of doublets displaying an adequately formed ISs (relative to all doublets) corrected for background synapse formation. Dots represent individuals; n = 5 CLL, n = 3 HCs with mean ± SEM.