CD19/CD3-scFv-Fc promotes synapse formation between CLL cells and autologous T cells. CLL PBMCs from treatment-naïve patients were cultured with either CD19/CD3-scFv-Fc or HER2/CD3-scFv-Fc for 15 minutes. Cells were then stained with labels against CD8, CD4, and CD20, and frequency of CLL cell/T-cell synapses was measured using multispectral fluorescence imaging. (A) Schematic of CD19/CD3-scFv-Fc, illustrating the combination of human anti-human CD19 scFv and humanized mouse anti-human CD3 scFv via a mutated Fc domain of human immunoglobulin G1. The CH2 aglycosylation (N297A) and CH3 knob (S354C and T366W) and hole (Y349C, T366S, L368A, and Y407V) mutations in the Fc region are indicated. (B) Representative images of CLL cell/T-cell doublets, or cells captured in the same imaging frame not considered to be in synapse (doublets), and true CLL cell/T-cell synapses with overlapping CD20 and CD8 or CD4 signals (synapse+), at ×60 magnification. (C) Quantitative results depicting the percent of CD8 or CD4 T cells found in synapse with CLL cells by treatment condition. Mean and 95% CI are shown. Asterisks indicate statistical significance using paired Student t tests. **P < .01; ***P < .001; ****P < .0001. CD19/CD3, CD19/CD3-scFv-Fc; H2/CD3, HER2/CD3-scFv-Fc.
