Fig. 1. IRU CAR-T cells retained effector functions in the presence of CsA in vitro.
a schematic of CRISPR/Cas9-targeted WT/IRU CAR gene incorporation into the 1st exon of TRAC locus. Top, TRAC locus; bottom, rAAV6 containing the WT/IRU CAR repertoire flanked by homology arms of inserted region. The CNA of IRU CAR was mutated at two points (V314R, Y341F) relative to the WT CAR. b, c Representative flow plots (b), the histogram analysis (c, left), and mean fluorescence intensity (MFI) (c, right, n = 8 biologically independent samples) of WT/IRU CAR expression on human primary T cells after 4 days of gene targeting. d Cytotoxic activity using Nalm6-FFLuc-GFP as target cells (n = 3 biologically independent samples) in 24 hrs cytotoxicity assay at different E:T ratios. Target cell lysis rate was calculated by (1- (RLUsample)/(RLUmax)) ×100 (RLU: relative luminescence units). CD19KO Nalm6-FFLuc-GFP was used as a negative control to validate the specificity of CAR (n = 3 biologically independent samples). e Proliferation of CAR-T cells upon the stimulation with 10-fold irradiated (50 Gy) Nalm6-FFluc-GFP (n = 3 biologically independent samples). f Cytokines (IL-2, IFNγ, and TNFα) expression of CAR-T cells upon stimulation with 10-fold irradiated (50 Gy) Nalm6-FFluc-GFP (n = 3 biologically independent samples). All data are mean ± s.d. P values were determined by two-tailed Unpaired t test (c, f) or Multiple t tests adjusted by the Holm-Sidak method (e). WT CAR-T (-CsA) as blue circle, WT CAR-T (+CsA) as blue square, IRU CAR-T (-CsA) as red circle and IRU CAR-T (+CsA) as red square. TRAV T cell receptor alpha variable, TRAJ T cell receptor joining, TRAC T cell receptor alpha constant, (m)CNA (mutated) calcineurin subunit A, gRNA guide RNA, 2A-self-cleaving peptide-based multi-gene expression, P2A porcine teschovirus1 2A, T2A Thosea asigna virus 2A, V314R valine 314 arginine, Y341F tyrosine 341 phenylalanine.