Figure 2

CD19BiTE binds to CD19-positive tumor cells and human γ9δ2 T cells. (a) HEK293 cells were transfected without (mock) or with the pcDNA3-CD19BiTE plasmid for 48 h, and then the culture media were subjected to protein purification by Ni-sepharose. The purified products were separated by SDS-PAGE and probed by an anti-6 × -histidine monoclonal antibody (mAb) in the Western blot analysis. A detected major band of the molecular weight of about 55 kDa similar to the predicted size of CD19BiTE was indicated (arrow). (b–l) Immunofluorescence histograms. Four tumor cell lines, Daudi (b), Raji (d), Raji/eGFP-fLu (f), and RPMI-8226 (i), were stained without (dotted black line) or with a PE-labeled anti-CD19 mAb (solid red line). RPMI-8226 (h) and expanded human γ9δ2 T cells (k) were stained without (dotted black line) or with an FITC-labeled anti-CD3 mAb (solid red line). Daudi (c), Raji (e), Raji/eGFP-fLu (g), and RPMI-8226 (j), and expanded human γ9δ2 T cells (l) were pre-incubated with (solid red line) or without (dotted black line) CD19BiTE (0.5 ng/µl) and then stained with a PE-labeled anti-6 × -histidine mAb. The stained cells were analyzed on a flow cytometer, and data were presented as histograms with counts versus fluorescence. The percentage of cell populations with fluorescence intensity higher than the background value was indicated. Images were created using FlowJo software 10.0.0r1.