Figure 3.

Hypoxia-dependent ON-OFF kinetic characteristic of CAR expression in HiTA-CAR-T cells. (A) Diagram of EF1α-CAR and HiTA-CAR constructs. The Her2-BBξ construct contained an internal Flag tag for the detection of CAR expression. Both EF1α-CAR and HiTA-CAR constructs included an EGFP reporter gene under the control of the constitutively active EF1α promoter for marking the transduced cells. (B) EF1α-CAR, HiTA-CAR and untransduced control T cells were cultured in normoxic or hypoxic (1% O₂) conditions for 24 hours. (C) Example histograms of surface CAR expression on CD3 +T cells were assessed using flow cytometry. (C) Surface CAR expression on EF1α-CAR and HiTA-CAR at the indicated time under hypoxia (1% O₂) or normoxia were assessed using flow cytometry. (n=3 donors, mean±SD of each time point compared with 0 hour in HiTA-CAR is shown, ****p<0.0001, ns: not significant, analyzed using one-way ANOVA). ANOVA, analysis of variance; CAR, chimeric antigen receptor; CMV, cytomegalovirus; EF1α, eukaryotic elongation factor 1-α; EGFP, enhanced green fluorescent protein; HiTA, hypoxia-inducible transcription amplification; ODD, oxygen-dependent degradation domain; UAS, upstream activating sequence; ZFP, zinc finger protein.