Fig. 7: MCJ deficiency improves human CD19-BBz CAR-T cell efficacy.
a CD8 cells isolated from the PBMC of a healthy volunteer were transfected by nucleofection with a control siRNA or siMCJ and activated with coated anti-CD3/anti-CD28 for 2 days. MCJ levels were analyzed by Western blot analysis. b CD8 cells from a healthy donor were transfected with siMCJ or control siRNA as in (a), activated for 2 days, expanded with IL-2 for 2 days. MMP of the cells was examined by TMRE staining. The percentage of TMREhigh cells is shown in parenthesis. c CD8 cells from a healthy donor were transfected and activated as in (a), washed, and incubated in medium alone for 48 h. IFNγ levels in the supernatant was determined by ELISA (n = 3 biologically independent samples). d Scheme (created using BioRender.com) showing the human CD19-BBz/shMCJ CAR construct containing (a) the scFv part of human CD19 (hCD19 scFv), (b) the transmembrane domain (TM) of human CD8 (hCD8 TM), (c) the cytoplasmic costimulatory domain of human 4-1BB (h41BB), (d) the cytoplasmic domain of human CD3z (hCD3z) and the shRNA for human MCJ cassette (including the U6 promoter). As a control we used the same construct but with a scramble shRNA. e CD8 cells were isolated from a healthy donor, activated with anti-CD3/anti-CD28 Abs for 2 days, transduced with the lentiviral CD19-BBz/shMCJ-1 CAR or CD19-BBz/shRNA CAR construct, and expanded with IL-2 (100 IU/ml) for 3 expansions. MCJ expression in purified CD19-BBz/shMCJ-1 or CD19-BBz/c-shRNA CAR-T cells after 3 expansions, by Western blot analysis. f After 3 expansions with IL-2, purified CD19-BBz/shMCJ-1 or CD19-BBz/c-shRNA CD8 CAR-T cells were used for Seahorse MitoStress assay (n = 10 replicate well per group). g CD8 cells isolated from two healthy donors (H16 and H19), were activated, transduced and expanded as (e). After the 3rd expansion with IL-2, CD19-BBz/shMCJ-1 or CD19-BBz/c-shRNA CAR-T cells were co-cultured with the human B cell leukemia Nalm6 cells (E:T = 0.125). After 20 h, live Nalm6 cells were counted (n = 3 biologically independent cells). h CD107a expression (MFI) on CD19-BBz/shMCJ or CD19-BBz/c-shRNA CAR-T cells after 4 h of co-culture with Nalm6 cells, as determined by flow cytometry (n = 3 biological independent cells). i Human CD8 cells were activated, transduced with lentiviral construct contained CD19-BBz/shMCJ-2 CAR or CD19-BBz/c-shRNA CAR and expanded with IL-2 for 3 expansions. MCJ expression in purified CAR-T cells, by Western blot analysis. j After 3 expansions with IL-2, purified human CD19-BBz/shMCJ-2 or CD19-BBz/c-shRNA CD8 CAR-T cells were used for Seahorse MitoStress assay (n = 10 replicate wells per group). k CD8 cells isolated from two healthy donors (H16 and H19), were activated, transduced and expanded as (h), and CD19-BBz/shMCJ-2 or CD19-BBz/c-shRNA CAR-T cells were co-cultured with the human B cell leukemia Nalm6 cells (E:T = 0.125). After 20 h, live Nalm6 cells were counted (n = 3 biologically independent cells). l CD107a expression (MFI) on CD19-BBz/shMCJ-2 or CD19-BBz/c-shRNA CAR-T cells after 4 h of co-culture with Nalm6 cells (n = 3 biologically independent cells). m IFNγ production of CD19-BBz/shMCJ-2 or CD19-BBz/c-shRNA CAR-T cells during co-culture with Nalm6 cells at E:T = 0.25, as determined by ELISA (n = 3 biologically independent samples). p was determined by two-sided unpaired t test (c, g, h, k, l) and by two-way ANOVA (f, j, m). Mean ± SD are shown for (c, f–h, j–m).
