Figure 5.
Epo supports the proliferation of EpoRm-CAR-T cells. (A) Survival and expansion of T lymphocytes from 3 donors transduced with CAR, EpoRm-CAR, or GFP only (Control) cocultured with Streck-treated or irradiated OP-1 cells at a 1:1 ratio, in the absence or presence of 10 IU/mL Epo, without exogenous IL-2. Each symbol indicates the percentage of cell recovery compared with the number of input cells. Mean (± SD) of triplicate cultures is shown, except for the day 14 measurement for 1 donor, for whom only the mean of 2 measurements is shown. (B) Proportion of naive (CD45RO‒ CD62L+), effector (TE; CD45RO‒ CD62L‒), central memory (TCM; CD45RO+ CD62L+), and effector memory (TEM; CD45RO+ CD62L‒) T cells in EpoRm-CAR T cells compared with T cells transduced with CAR or GFP only. Shown are mean of 3 experiments with and without Epo (10 IU/mL) after 24-hour culture with RS4;11 cells at a 1:1 ratio. (C) Percentage of T lymphocytes transduced with the various constructs recovered 7 days after coculture with irradiated CD19+ OP-1 at a 1:1 ratio in the presence of 10 IU/mL Epo with 10 IU/mL or 100 IU/mL of IL-2. Each symbol indicates the mean of triplicate cultures. (D) MFI of pSTAT5 in EpoRm-CAR–transduced T cells after stimulation with 10 IU/mL Epo and/or 100 IU/mL IL-2 at the indicated time points. (E) Percentage of GFP+ EpoRm-CAR–transduced T cells expressing pSTAT5 stimulated with the indicated concentrations of Epo and IL-2. *P = .045; **P < .01. (F) Expression of activation and exhaustion markers PD-1, TIM-3, and LAG-3 in T cells expressing EpoRm-CAR cocultured for 6 days with irradiated OP-1 in the presence of 10 IU/mL Epo and/or 10 IU/mL IL-2. Marker expression was analyzed with Diva software, and graphs were plotted with Phyton 3 using Matplotlib package (https://matplotlib.org/). Average measurement of 2 experiments is shown. (G) Percentage of GFP+ cells expressing pSTAT5 in T cells transduced with EpoRm-CAR after exposure to either tofacitinib or ruxolitinib for 1 hour at the indicated doses before stimulation with either 10 IU/mL Epo or 100 IU/mL IL-2. Each dot represents the mean of 2 measurements for tofacitinib and a single measurement for ruxolitinib. (H) Left panel shows the percentage of EpoRm-CAR T cells recovered after 2 days of culture with irradiated RS4;11 cells at a 1:1 ratio in the presence of 10 IU/mL Epo with or without 1 µM ruxolitinib. Cells were then washed to remove ruxolitinib, and cultures were continued for another 2 weeks in the presence of Epo (right); each dot indicates mean (± SD) of triplicate measurements of percentage of cell recovery relative to cell numbers of day 2. ***P < .001. (I) Long-term cytotoxicity of T cells expressing EpoRm-CAR or GFP only (control) against mCherry+ Nalm6 cells at a 1:1 E:T ratio. Epo (10 IU/mL) was added every 2 days, and ruxolitinib (1 µM) or its vehicle (DMSO) were added either at the start of the coculture (left panel) or after 8 hours (right panel). The IncuCyte Zoom System was set to collect the number of viable target cells, expressed as red calibrated units (RCU) × µm3/well, every 4 hours. Shown are mean (± SD) of cultures with T cells from 1 donor. Data from 2 other donors are shown in supplemental Figure 5. ***P < .001.

Epo supports the proliferation of EpoRm-CAR-T cells. (A) Survival and expansion of T lymphocytes from 3 donors transduced with CAR, EpoRm-CAR, or GFP only (Control) cocultured with Streck-treated or irradiated OP-1 cells at a 1:1 ratio, in the absence or presence of 10 IU/mL Epo, without exogenous IL-2. Each symbol indicates the percentage of cell recovery compared with the number of input cells. Mean (± SD) of triplicate cultures is shown, except for the day 14 measurement for 1 donor, for whom only the mean of 2 measurements is shown. (B) Proportion of naive (CD45RO‒ CD62L+), effector (TE; CD45RO‒ CD62L‒), central memory (TCM; CD45RO+ CD62L+), and effector memory (TEM; CD45RO+ CD62L‒) T cells in EpoRm-CAR T cells compared with T cells transduced with CAR or GFP only. Shown are mean of 3 experiments with and without Epo (10 IU/mL) after 24-hour culture with RS4;11 cells at a 1:1 ratio. (C) Percentage of T lymphocytes transduced with the various constructs recovered 7 days after coculture with irradiated CD19+ OP-1 at a 1:1 ratio in the presence of 10 IU/mL Epo with 10 IU/mL or 100 IU/mL of IL-2. Each symbol indicates the mean of triplicate cultures. (D) MFI of pSTAT5 in EpoRm-CAR–transduced T cells after stimulation with 10 IU/mL Epo and/or 100 IU/mL IL-2 at the indicated time points. (E) Percentage of GFP+ EpoRm-CAR–transduced T cells expressing pSTAT5 stimulated with the indicated concentrations of Epo and IL-2. *P = .045; **P < .01. (F) Expression of activation and exhaustion markers PD-1, TIM-3, and LAG-3 in T cells expressing EpoRm-CAR cocultured for 6 days with irradiated OP-1 in the presence of 10 IU/mL Epo and/or 10 IU/mL IL-2. Marker expression was analyzed with Diva software, and graphs were plotted with Phyton 3 using Matplotlib package (https://matplotlib.org/). Average measurement of 2 experiments is shown. (G) Percentage of GFP+ cells expressing pSTAT5 in T cells transduced with EpoRm-CAR after exposure to either tofacitinib or ruxolitinib for 1 hour at the indicated doses before stimulation with either 10 IU/mL Epo or 100 IU/mL IL-2. Each dot represents the mean of 2 measurements for tofacitinib and a single measurement for ruxolitinib. (H) Left panel shows the percentage of EpoRm-CAR T cells recovered after 2 days of culture with irradiated RS4;11 cells at a 1:1 ratio in the presence of 10 IU/mL Epo with or without 1 µM ruxolitinib. Cells were then washed to remove ruxolitinib, and cultures were continued for another 2 weeks in the presence of Epo (right); each dot indicates mean (± SD) of triplicate measurements of percentage of cell recovery relative to cell numbers of day 2. ***P < .001. (I) Long-term cytotoxicity of T cells expressing EpoRm-CAR or GFP only (control) against mCherry+ Nalm6 cells at a 1:1 E:T ratio. Epo (10 IU/mL) was added every 2 days, and ruxolitinib (1 µM) or its vehicle (DMSO) were added either at the start of the coculture (left panel) or after 8 hours (right panel). The IncuCyte Zoom System was set to collect the number of viable target cells, expressed as red calibrated units (RCU) × µm3/well, every 4 hours. Shown are mean (± SD) of cultures with T cells from 1 donor. Data from 2 other donors are shown in supplemental Figure 5. ***P < .001.

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