Journal: Advanced Science

Article Title: Repurposing a Small Molecule Plant Hormone as a Tunable ON‐Switch for CAR‐T Cell Immunotherapy

doi: 10.1002/advs.77020

Figure Lengend Snippet: Assessment of auxCAR‐T cell performance in Jurkat T cells. (A,B) Representative Flow cytometry (A) and Statistical quantification (B) of early T cell activation as reflected by upregulated CD69 expression on the cell surface. Jurkat T cells transduced with stCAR (left) and auxCAR (right) were co‐cultured with hCD19 + Raji cells or hCD19 − K562 cells, with or without auxin. n = 3 independent biological replicates (mean ± s.d.). The p ‐values were calculated using one‐way analysis of variance (ANOVA). (C,D) Statistical quantification of NFAT‐GFP reporter activity in Jurkat T cells expressing stCAR and auxCAR co‐cultured with tumor cells with hCD19 + Raji cells (C) or A431‐CD19 (D) with the indicated auxin concentration. n = 3 independent technical replicates (mean ± s.d.). The p ‐values were calculated using one‐way analysis of variance (ANOVA). (E) Auxin‐dose‐dependent responses of stCAR T and auxCAR‐T cells examined by IL‐2 production in Jurkat T cells. (F) Architecture of the auxCAR HER2. Upper: cartoon depicting the different components. Bottom: schematic of the R‐ and S‐chains encoded in a single lentiviral vector, each separated by Venus or mScarlet. (G,H) Statistical quantification of NFAT‐GFP reporter activity (G) and IL‐2 production (H) in Jurkat T cells expressing stCAR HER2 or auxCAR HER2 co‐cultured with SK‐OV‐3 tumor cells in the presence of the indicated auxin concentration. n = 3 independent technical replicates (mean ± s.d.). (I) Statistical quantification of NFAT‐GFP reporter activity at the indicated time points following auxin removal in Jurkat T cells expressing auxCAR and co‐cultured with SK‐OV‐3 tumor cells. The auxCAR T cells were induced with 1 µM auxin.

Article Snippet: Recombinant human IL‐2 (Cat# RP01039, ABclonal) was added at a final concentration of 100 Unit/mL −1 to expand T cells.

Techniques: Flow Cytometry, Activation Assay, Expressing, Transduction, Cell Culture, Activity Assay, Concentration Assay, Plasmid Preparation