In vitro assessment of the effects of MS4A1 variants on CD20 expression and CD20 × CD3 activity. (A) CD20 protein expression by western blot in the SU-DHL-16–engineered cell lines. (B) Detection of CD20 by flow cytometry in SU-DHL-16–engineered cell lines. Intracellular expression was detected after permeabilization using anti-CD20 antibody targeting the C-terminus (H-1, BD-561174), and extracellular expression was detected using anti-CD20 antibody targeting ECL2 (2H7, BD-555623). (C) Immunofluorescence detection of CD20 using an intracellular antibody (ABCAM-78237): red fluorescence = CD20; blue fluorescence = 4′,6-diamidino-2-phenylindole (DAPI). (D) (left) CD20 × CD3–directed CD8-dependent cell killing of SU-DHL-16–engineered cell lines after 48 hours of treatment assessed by spectral flow cytometry. (Right) CD20 × CD3–directed CD8⁺ T-cell activation after 48 hours of treatment. T-cell activation monitored by flow cytometry by detecting activation markers CD69 and CD25. Flow cytometry data are represented as mean ± SD of 3 replicates. EV, empty vector; CD20-CD3, proof-of-concept CD20 × CD3 bispecific molecule; MFI, median fluorescence intensity; N, non-targeting control/CD3 antibody; parent, parental cell line; SD, standard deviation.