Fig. 4
Generation and functional validation of MU-CAR-T cells. A Flowchart illustrating the protocol for generating MU-CAR-T cells. Briefly, α-CD3/α-CD28-activated CD8+ T cells were electroporated with Cas9 mRNA and sgRNAs targeting B2M and TRAC on Day 3 and Day 4. On Day 6, TCR−/HLA-I− T cells were enriched. Then, enriched T cells were transduced with SdΔN17-28BBZ3-tCD19 through lentiviral infection on Day 7. CD19-positive selection was performed on Day 9. After incubation with the specific Gv-scFv, the resulting products were identified as MU-CAR-T cells. B and C The cytotoxicity of MU-CAR-T cells against Jurkatgp160 and Karpas 299 target cells was observed upon being conjugated with VRC01 scFv (B) and CD5-CD30 scFvs (C) respectively. Data represented as mean ± SD. N = 3 independent biological replicates. D Control CD8+ T, SdΔN17-28BBZ3-expressing CD8+ T, conventional CAR-T and MU-CAR-T cells were co-cultured with target cells at a 4:1 ratio respectively. IFN-γ secretion was detected utilizing ELISPOT assay. PHA-stimulated effector cells were treated as the positive control (PC), and effector cells-only group was treated as the negative control (NC). Data represented as mean ± SD. N = 3 independent biological replicates. (E, F and G) The production of cytokines including Granzyme B (E), IFN-γ (F) and TNFα (G) was assessed upon co-culturing of control CD8+ T cells and CAR-T cells with different target cells at the ratio of 4:1. Data represented as mean ± SD. N = 3 independent biological replicates. Data in (B) and (C) were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. Data in (D-G) were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001