BsAbs induce clustering of T cells and B cells. (A) Schematic overview of the confocal microscopy experimental set-up. (B-F) Lymph node–derived lymphocytes (n = 27 biologically independent samples) were seeded into 384-well microscopy plates and incubated for 7 days without (w/o) or with CD19-BsAb at 4 different concentrations (C1-C4). After 7 days, cells were stained with Hoechst, calcein, anti-CD20 PE-conjugated, and anti-CD3 APC-conjugated antibodies. Shown are representative images for the untreated condition in panel B and the CD19-BsAb–treated condition in panel C separated by channel as indicated. The filled area of clusters was quantified (panel D) and the intensity of CD20-PE (panel E) or CD3-APC (panel F) within these clusters was determined. All conditions were tested for significance vs untreated control (w/o) using the two-sided Wilcoxon’s test. P values were corrected for multiple testing by using the Benjamini-Hochberg procedure. *** ≙ P ≤ .001; ** ≙ P ≤ .01; * ≙ P ≤ .05; missing asterisks and/or horizontal brackets indicate P > .05. APC: allophycocyanin; PE: phycoerythrin.