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. 2022 Jan 11;13:217. doi: 10.1038/s41467-021-27853-w

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Cell lines of different B cell malignancies were stained for BAFF-R, BCMA, TACI, and CD19 expression. Red = Jeko-1 (MCL); blue = rs4;11 (ALL); cyan = RPMI-8226 (MM); orange = U266 (MM); purple = MM.1s (MM); black = unstained cells. b CAR-T cells or unmodified T cells were co-cultured with fluorescently-labeled cancer cells at indicated E:T ratios for 16 h (Jeko-1, rs4;11, MM.1s) or 40 h (RPMI-8226, U266). Cytotoxicity was measured via PI staining and gating on labeled target cells. Circle = Unmodified Control, triangle = CAR-T. **P < 0.01, ***P < 0.001, ****P < 0.0001. Jeko-1: P = 3.34e-4 (10:1), P = 1.92e-4 (5:1), P = 6.72e-4 (1:1). rs4;11: P = 9e-6 (10:1), P = 3e-6 (5:1), P = 8.8e-5 (1:1). RPMI-8226: P = 2.1e-5 (1:5), P = 2.85e-4 (1:10), P = 2.919e-3 (1:20). U266: P = 7.2e-5 (1:5), P = 3e-6 (1:10), P = 2.179e-3 (1:20). MM.1s: P = 6.6e-5 (5:1), P = 3e-6 (3:1), P = 1.29e-4 (1:1). Mean ± SD, n = 3 biologically independent co-cultures, multiple unpaired two-tailed t-tests with Holm-Šídák correction for multiple comparisons. c CRISPR was used to knock out expression of either BAFF-R, TACI, or both BAFF-R and TACI in Jeko-1 cells. Fluorescently-labeled cancer cells were co-cultured with BAFF CAR-T cells or unmodified T cells 16 h at 3:1 E:T ratio. Parental Jeko-1 cells served as negative control. Cytotoxicity was measured as above. ns not significant, ****P < 0.0001. Mean ± SD, n = 3 biologically independent co-cultures, two-way ANOVA with Tukey’s multiple comparisons test. d CRISPR was used to knock out expression of either BCMA, TACI, or both BCMA and TACI in RPMI-8226 cells. Fluorescently-labeled cancer cells were co-cultured with BAFF CAR-T cells or unmodified T cells 36 h at 1:10 E:T ratio. Parental RPMI-8226 cells served as negative control. Cytotoxicity was measured as above. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P = 0.0015 (Parental vs TACI KO), P < 0.0001 (Parental vs BCMA/TACI Dual KO), P = 0.0006 (BCMA KO vs TACI KO), P < 0.0001 (BCMA KO vs BCMA/TACI Dual KO), P = 0.0137 (TACI KO vs BCMA/TACI Dual KO). Mean ± SD, n = 3 biologically independent co-cultures, two-way ANOVA with Tukey’s multiple comparisons test. Source data for all graphs are provided as a Source Data file. All cytotoxicity experiments were repeated with two different T cell donors.