Longitudinal analysis of T-cell immune synapse function during PCR induction-lenalidomide (Len.) consolidation therapy. (A) Confocal images of T-cell synapse formation for a representative patient: CD3⁺ T cells from each treatment time point indicated were conjugated with baseline untreated CLL B cells (pulsed with superantigen [sAg] and dyed blue with CellTracker). These identical APCs used for all time points allowed evaluation of changes in T-cell F-actin (dyed red with rhodamine phallodin) synapse activity (indicated with white arrows) during treatment. T-cell conjugates formed were analyzed by immunofluorescence and confocal microscopy. Blinded images (n = 10 per patient treatment sample) were analyzed using AxioVision (ASSAYbuilder) image analysis software (Zeiss). This measures the total area (μm²) of F-actin (red fluorescent channel) accumulation at all T-cell contact sites and synapses with CLL cells (minimum n = 50 synapses assessed per patient at each timepoint). (B) Mean change in T-cell synapse activity of 28 patients following PCR induction therapy compared with their respective (matched) baseline activity (C) Changes in T-cell synapse activity for a representative patient. Each data point represents the area (μm²) of F-actin polymerization at a T-cell:APC synapse contact site (n = 50 synapses per patient at each time point) measured using Axiovision software. The mean ± standard error of the mean is shown for each treatment time point in a representative patient (baseline, data points shown as black dots; post-PCR, blue; post-Len. 3 months, orange; post-Len. 6 months, red; matching colored squares denote the bar chart data shown in E and F), and (D) the mean change in T-cell synapse activity of 11 patients after 6 months (cycles) of lenalidomide consolidation compared with their respective (matched) post-PCR activity. Each data point represents the mean area (μm²) of T-cell F-actin synapse formation from ≥50 synapses for each patient. The mean for all patients is indicated by a bar. (E) Quantitative analysis of the percent of T-cell:APC conjugates (n = 50) showing phosphotyrosine synapse signaling by immunofluorescence (shown in yellow) comparing PCR induction therapy to the respective (matched) baseline activity (28 patients) and (F) cytolytic T lymphocyte granzyme B protein accumulation (shown in yellow) at the synapse site comparing 6 months (cycles) of lenalidomide consolidation to the respective (matched) post-PCR activity (11 patients). Data represent the combined mean ± standard deviation (baseline, black bar chart; post-PCR, blue; post-Len. 6 months, red). Original magnification, ×63. **P < .01 values are shown.