Expression and function of EpoRm-CAR in T cells. (A) Schematic representation of the EpoRm-CAR construct. (B) Flow cytometric dot-plots illustrate surface expression of CAR and EpoR in T cells transduced with EpoRm-CAR; cells transduced with GFP only were used as control. Percentage of cells in each quadrant is shown. Cells were stained with EpoR-PE antibody to detect EpoR expression; CAR expression was detected with goat anti-mouse F(ab′)₂ antibody and streptavidin-APC (Jackson ImmunoResearch Laboratories). (C) Percentage of GFP⁺ cells expressing CAR (left) and EpoR (right) among T lymphocytes transduced with CAR, EpoRm, EpoRm-CAR, or GFP only. Each symbol corresponds to a measurement for one transduction. Horizontal bars indicate median value. (D) Percentage of GFP⁺ T lymphocytes expressing pSTAT5 after stimulation with 10 IU/mL Epo or pretreatment with 10 µM ruxolitinib before Epo stimulation. Each symbol corresponds to a measurement for one transduction with the construct shown. Horizontal bars indicate median value. (E) Cytotoxicity of T lymphocytes transduced with CAR, EpoRm-CAR, or GFP only against the CD19⁺ cell lines OP-1, RS4;11, and Nalm6. Bars represent mean (± SD) of triplicate experiments in a 4-hour cytotoxicity assay at a 1:1 E:T ratio. (F) Long-term cytotoxicity of T lymphocytes transduced with various constructs against mCherry⁺ OP-1 cells at the indicated E:T ratios in the presence of 10 IU/mL Epo. The number of viable target cells was collected every 4 hours with the IncuCyte Zoom System (Essen Bioscience) and expressed as red calibrated units (RCU) × µm³/well. Symbols represent the mean (± SD) percentage cytotoxicity measured at the indicated time points. *P = .03; ***P < .001.