Figure 1 from Activation of Cell-Intrinsic Signaling in CAR-T Cells via a Chimeric IL7R Domain

Human primary T cells can be engineered for stable IL7 pathway activation. A, Schematic of CCR and sIL7, as well as transgenes engineered into cells. B, Transduction efficiency of T cells engineered to express GFP only, IL7R + GFP, or to secrete IL7 + GFP was measured by detecting eGFP positivity and CCR expression with flow cytometry. C, Concentration of IL7 measured in the supernatant of sIL7-transduced T cells and control cells (G-o) in cytokine-free culture medium. D, Immunophenotype characterization of T-cell subsets present in healthy donor and transduced T-cell preactivation (CD4 and CD8) and on day 8 of T-cell expansion CCR and sIL7 treated with IL7: +IL7 (G-o); G-o. E, Ratio of STAT5 phosphorylation to total STAT5 expression measured with TR-FRET in indicated cell types following 24 hours of cytokine starvation and following stimulation with IL7 for 30 minutes. Comparison between untreated and IL7-treated cells. F, Percent cells positive for CD127 expression measured by flow cytometry. Statistical comparison is between cell type and control (G-o, no treatment). G, Representative histograms from flow cytometric measurement of �127+ cells of each type. Data representative of n = 3 to 5 independent donors. *, P P P H, MS proteomics analysis of IL7R signaling pathway downstream molecules for total protein and phosphosite abundance. Raw detected protein and phosphoprotein abundance were normalized to the control condition per donor and expressed as mean log2 fold change of three donors. *Note STAT5A pY694 and STAT5B pY699 are indistinguishable due to sequence identity. G-o, GFP-only.