Fig. 9

Down-modulation of CD19 full-length protein and CD19 splice variant by CAR19 constructs. Raji cells were co-incubated with CAR T cells at a 1:1 E:T ratio. After overnight incubation, T cells were removed from co-incubated cell populations using magnetic beads. CD19 expression on purified Raji populations was investigated by flow cytometry and Western blot. a Raji cell samples were stained with anti-CD19 antibody and acquired by flow cytometry. Median fluorescence intensity for Raji cells representing each treatment group is shown. Bars depict mean + SD of three independent experiments performed using CAR T cells originating from three different donors. One way ANOVA, Dunnett’s multiple comparisons test *p < 0.05. b Lysates of purified Raji cells from each of the co-incubated groups (CAR-T identity is listed above each line) were resolved on a 4–12% SDS polyacrylamide gel as described in the Methods, and probed with antibodies targeting the C-terminus of CD19 molecule, or β-actin (loading control). c The intensity of specific immunoreactive bands representing full-length CD19 protein (FL CD19), and the exon 2 spliced CD19 variant (Δ2CD19) was quantified using Image Studio software (LI-COR Biosciences). Relative band intensity of CD19 bands was calculated as signal CD19/signal β actin