Skip to main content
. 2022 May 18;25:335–349. doi: 10.1016/j.omto.2022.05.003

Figure 1.

Figure 1

T cells transduced with WAS promoter-driven LVs mimic the TCR expression pattern

(A) (Left) Timeline of the experiments to study EGFP expression pattern after TCR and CAR stimulation. (Right) Diagrams of the different EGFP-expressing LVs are shown. CMV, SFFV, EF1α, W0.5, and AW promoters were used in CEWP, SE, EFEWP, WE, and AWE LVs, respectively (see Materials and methods for details). (B) CD3 MeFI of T cells stimulated with αCD3/CD28 and measured at the indicated time points is shown. ∗p < 0.05; one-way-ANOVA; n ≥ 5. (C) Representative histograms of CD3 expression on T cells at different times post-stimulation are shown. (D) Representative histograms show EGFP expression levels on T cells transduced with strong-promoter-driven (CEWP, SE, and EFEWP) and WAS-promoter-driven (WE and AWE) LVs at different times post-stimulation with αCD3/CD28. (E) Graphs show fold change in EGFP expression (blue line) and CD3 expression on non-transduced (black lines) and transduced (red lines) T cells along time after αCD3/CD28 stimulation. Two-way ANOVA, multiple comparison post-test, compared with CD3 expression pattern of non-transduced T cells (NT_CD3): ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p< 0.0001 . (F) Diagrams of the EF1α-promoter-driven (ARI) and WAS-promoter-driven (AWARI) LVs expressing the ARI-0001 CAR (αCD19 A3B1-41BB-CD3ζ) are shown. (G) Representative dot plots of CAR expression levels on CAR-T cells generated with the ARI (left) or AWARI (right) LVs measured at different time points after activation with CD19+ cells are shown. MeFIs of total population and CAR+ (gate) populations are indicated in blue at top left or top right of each plot, respectively. (H) Fold change of CAR (solid lines) and CD3 (dashed lines) expression of ARI CAR-T (blue lines) and AWARI CAR-T (red lines) cells at different time points after activation with CD19+ cells (left) or αCD3/CD28 nanomatrix (right) is shown. Data show the MeFI of bulk population at different time points relative to those at 0 h. Populations with similar percentage of CAR+ cells (mean ± SEM; ARI versus AWARI, 33.5% ± 0.9% versus 30.1% ± 1.2%; p = 0.12) and vcn (ARI: 2.7 ± 1 c/c and AWARI: 5.9 ± 2.7 c/c; p = 0.34) were compared. Two-way ANOVA; ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 for ARI_CAR compared with AWARI_CAR and +p < 0.05, +++p < 0.001; ++++p < 0.0001 for comparison of ARI_CAR to ARI_CD3, AWARI_CD3, or NT_CD3 (n ≥ 5).