Figure 6 from Activation of Cell-Intrinsic Signaling in CAR-T Cells via a Chimeric IL7R Domain

Single-chain IL7Rα signaling stimulates STAT5 activation in CAR-T cells. A, Schema of the compared T-cell conditions. A: CAR and sIL7; B: CAR with the distal IL7Rα domain; C: CAR with the proximal IL7Rα domain. B, Levels of STAT5 phosphorylation before and after exposure to plate-bound recombinant antigen (rhCD123) for 30 minutes, measured with a TR-FRET assay. Comparison between conditions with and without rhCD123 stimulation. n = 3 donors, performed in duplicate. C, Relative ECAR and (D) relative OCR of T cells measured after 24-hour rhCD123 stimulation in cytokine-free media. n = 4 individual donors tested in five replicates per assay. For each donor, normalization of all values to the baseline (NT control) was performed. E, Cytotoxic activity measured by luminescence-based assay. CD123-negative K562 cell line and NT T cells included as controls. n = 4 T-cell donors, E:T. Comparison of cytotoxicity of engineered T cells against all CD123-expressing targets with that from NT T cells was very significant (P P P F, Long-term cytotoxicity measured with six 48-hour serial stimulations with indicated NLS eGFP-modified AML cell lines with initial plating at 1:1 E:T (n = 4 individual donors in technical triplicate each normalized to timepoint 0). G, Half-life decay of each target cell line calculated at each stimulation. Shaded bars, 95% confidence intervals for decay calculated at each timepoint. NT, nontransduced.