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

Fig. 5. BAFF CAR-T cell activation, degranulation, and cytokine release are stimulated by different B cell cancers.

Fig. 5

a T cells were co-cultured with labeled Jeko-1 or rs4;11 cells at 5:1 E:T ratio for 24 h, RPMI-8226 cells at 1:5 E:T ratio for 5 h, or U266 cells at 5:1 E:T ratio for 5 h, then stained for the activation marker CD69. % CD69+ T cells were measured via flow cytometry after gating on live cells and excluding labeled cancer cells; for CAR-T samples, additional gating over GFP+ cells was applied to exclude unmodified cells. ***P < 0.001, ****P < 0.0001. Mean±SD, n = 3 biologically independent co-cultures, unpaired two-tailed t-test for each cell line. Experiment was repeated with 2 different T cell donors. b T cells were co-cultured with Jeko-1, rs4;11, RPMI-8226, or U266 cells at 5:1 E:T ratio for 6 h while staining for the degranulation marker CD107a. % CD107a+ T cells were measured via flow cytometry after gating on CD3+ cells; for CAR-T samples, additional gating over GFP+ cells was applied to exclude unmodified cells. ****P < 0.0001. Mean±SD, n = 3 biologically independent co-cultures, unpaired two-tailed t-test for each cell line. Experiment was repeated with two different T cell donors. c T cell and Jeko-1, rs4;11, or RPMI-8226 co-culture supernatant was collected from cytotoxicity assays to measure T cell release of various pro-inflammatory cytokines and lytic enzymes using a multiplex cytokine release assay. **P < 0.01, ***P < 0.001, ****P < 0.0001. For Jeko-1, Unmodified Control vs CAR-T: TNF-α: P = 1e-6; IFN-γ: P = 7e-6; sFasL: P = 3e-6; Granzyme A: P = 2.751e-3; Granzyme B: P = 2.9e-4; Perforin: P = 3.834e-3; Granulysin: P = 2.9e-4. For rs4;11, Unmodified Control vs CAR-T: TNF-α: P = 9e-6; IFN-γ: P = 5.9e-5; sFasL: P = 2.2e-5; Granzyme A: P = 2.19e-4; Granzyme B: P = 2.9e-5; Perforin: P = 2.19e-4; Granulysin: P = 9e-6. For RPMI-8226: TNF-α: P < 1e-6; IFN-γ: P < 1e-6; sFasL: P = 1.1e-5; Granzyme A: P < 1e-6; Granzyme B: P < 1e-6; Perforin: P = 7e-6; Granulysin: P = 7e-6. Mean±SD, n = 3 biologically independent co-culture samples, multiple unpaired two-tailed t-tests with Holm-Šídák correction for multiple comparisons. Experiment was repeated with two different T cell donors. Source data for all graphs are provided as a Source Data file.