Fig 1.
Cell expansion and viability of PBMC following transduction with LV52.
PBMCs from 3 donors were transduced with the LVV2 (left panels) or LV52 (right panels) using the indicated MOIs. The upper panels showed the total cells recovered, and the lower panels showed the viability of the cultures at the indicated time points. The data shown are the mean ± SEM of 3 independent experiments. *p = 0.0122, **p = 0.0016, ***p = 0.0001, #p = 0.0177, ##p = 0.0052, ###p = 0.0003, ●p = 0.0477, ●●p = 0.0015, ◆◆p = 0.0072; one-way ANOVA.
Fig 2.
The phenotype of LV52 transduced cells.
PBMCs from 3 donors were transduced as indicated and immuno-phenotyped by multicolor flow cytometry. (A) The proportion of T cells (CD3), helper T cells (CD4), and effector T cells (CD8). (B) The proportion of T cell subsets with effector/memory function within the CD4+ and CD8+ populations. The data shown are the mean ± SEM of 3 independent experiments.
Fig 3.
Efficacy of transduction of T cells with anti-CD19 CAR.
PBMCs were mock transduced or transduced with LVV2 or LV52 lentiviral vectors using a range of MOI as indicated. Cells were assessed for anti-CD19 CAR expression using qPCR, and the number of transgene copies was expressed as the average number detected in each cell. Bars indicate the mean ± SEM of 3 independent experiments.
Fig 4.
CAR-CD19 T cells specifically kill CD19-expressing cells.
CAR-CD19 T cells were incubated with the indicated target cells at an E: T ratio of 20:1 for 24 hours, and the viability of target cells was assessed by flow cytometry using a CFSE-7AAD-based cytotoxic assay. (A) Representative flow cytometry panels with CFSE-7-AAD-based cytotoxic assay from one of three independent experiments. (B) The mean ± SEM of normalized cytotoxicity from 3 independent experiments is shown. *p = 0.0206, **p = 0.0018, #p = 0.0136; paired t-test. ****p<0.0001; one-way ANOVA.
Fig 5.
IFN-γ production by CAR-CD19 T cells.
The concentration of IFN-γ in culture supernatants following 24 hours of co-culture between the indicated cell types and mock transduced or T cells transduced with LVV2 or LV52 at the indicated MOI using LEGENDplex™. The data shown are the mean ± SEM of 3 independent experiments.
Fig 6.
Tumor suppression and percent survival of mice engrafted with Raji cells and treated with CAR-CD19 T cells.
Tumor burden in immunocompromised mice engrafted with Raji expressing luciferase as assessed by bioluminescence following treatment with the indicated doses of CAR-CD19 T cells. (A) Normalized bioluminescence intensity was shown are the mean ± SEM. (B) Percent survival of C.B.17/Icr-scid/scidJcl mice engrafted with 5 x 105 Raji cells two weeks before administration of the indicated dose of CAR-CD19 T cells. Note: mice in control groups had been culled due to disease progression prior to week 4. N ≥ 4 mice/group.