Extended Data Fig. 3: CCT fusion enables enhanced killing with low production of proinflammatory cytokines.
a. Schematics of lentiviral constructs to make CAR-T cells targeting human CD19 (FMC63-CD28-41BB-CD3zeta). CD19-CAR (19CAR) constructs were made by replacing the m971 scFv with the FMC63 scFv from Extended Data Fig. 1a. b. Representative flow cytometry analysis of 19CAR-T killing capability in vitro. c. Quantification of (b) showing CD19-CAR percentage (left panel), and counts (right panel), n = 3. d. Quantification of (b) showing NALM6GL percentage (left panel), and counts (right panel), n = 3. e. Quantification of 13 granule molecules and cytokines in CAR-T and NALM6GL cocultures. CAR-T cells were co-cultured with one round of NALM6GL cells at E/T = ½ for 24 hours. Co-culture supernatant was then collected and measured using LEGENDplex™ Human CD8/NK Panel, n = 3. f. Schematics of lentiviral constructs to make CD22 targeting CAR-T cells. In addition to the four constructs shown in Extended Data Fig. 1a, mCMV-CAR construct was generated by replacing the EFS promoter in the CAR construct with a minimal CMV promoter (mCMV). g. Representative flow cytometry analysis of CAR-T cells, pre-gated on mScarlet+ populations, showing surface expression level of CAR, indicated by Flag staining. h. Quantification of (g) showing surface flag expression in MFI, n = 3. For all bar plot figures, data are shown as mean ± s.e.m. One-way ANOVA with Dunnett’s multiple-comparisons test is used to assess significance. Exact p values are labeled. All numbers defined by ‘n’ indicate the number of biologically independent samples. Data are representative of two independent experiments performed with biological repeats.
