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. Author manuscript; available in PMC: 2022 Jul 18.
Published in final edited form as: Leukemia. 2022 Jan 18;36(4):1015–1024. doi: 10.1038/s41375-021-01477-x

Figure 1. Development of prototype BAFF-R/CD19 dual-targeting CAR T cells.

Figure 1.

A. Schematics depict the arrangement of BAFF-R and CD19 targeting scFvs designed for each dual-targeting CAR construct. B. Human CD8 naïve cells from healthy human donors were enriched, activated and transduced with lentiviral vector encoding 4–1BB costimulatory molecules and GFP for single CAR or EGFR for dual CAR. After 7 days in vitro expansion, GFP+ or EGFR+ CAR T cells were enriched and expanded for another 7 days before functional assay. FACS analysis plots show CAR positive T cells following enrichment. CAR reporter genes were gated on T cells: GFP-positive single-targeting CAR T cells (top panel); EGFR positive dual CARs (lower panel). C. FACS histogram show BAFF-R or CD19 expression in Nalm-6 ALL tumor lines, including wild-type, CD19−/−, and BAFF-R−/− variants. Cells were stained by BAFF-R-APC, CD19-APC or isotype control antibodies. D. Calculated specific lysis are plotted from a cytotoxicity assay against Nalm-6 ALL tumor lines, including wild-type (WT), CD19−/−, and BAFF-R−/− at 10:1 of E:T ratios. Effector CAR T cells were CD19-BAFF-R(t) and CD19-BAFF-R(l) dual-targeting CARs and BAFF-R and CD19 single-targeting CARs. Mock T cells were used as negative control. Experiments were conducted in triplicate and analyzed by a Student’s t-test. ***P<0.001, ****P<0.0001, NS: not statistically significant.