Figure 1.
Human CD3 transgenic (huCD3-Tg) T cells demonstrated appropriate functionality when stimulated with T-BsAbs and target cells expressing the corresponding human antigen. (A) Upregulation of activation markers on huCD3-Tg T cells after coculture with target cells in the presence of appropriate T-BsAbs. CD8 T cells from huCD3 transgenic mice were cocultured with huCD20-expressing E2A-PBX1 leukemia cells (B6 syngeneic) along with human CD3 X CD20 T-BsAb or CD3xCD123 T-BsAb (control) for 24 hours. Expression levels of CD69 and CD25 were measured by Flow cytometry. (B) huCD3-Tg T cells proliferate after coculture with target cells in the presence of appropriate T-BsAbs. T cells from huCD3 transgenic mice were labeled with CellTrace Violet and cocultured with huCD20-expressing E2A-PBX1 leukemia cells along with human CD3 X CD20 T-BsAb or CD3xCD123 T-BsAb (control) for 4 days. Cell proliferation was measured by the dilution of CellTrace dye. (C) Production of cytokines by effector huCD3-Tg T cells after being stimulated with appropriate T-BsAb and target cells. huCD3-Tg T cells were differentiated in vitro by stimulation with concanavalin A and then rested for 7 days. The resting effector T cells were then restimulated with CD3xCD20 T-BsAb or control T-BsAb in the presence of huCD20-expressing E2A-PBX1 cells for 5 hours. Intracellular cytokine staining was used to measure the production of cytokines. (D-E) Effector huCD3-Tg T cells demonstrated cytotoxicity against target antigen–expressing cells in the presence of appropriate T-BsAb. Effector T cells, prepared in the same way as in panel C and prelabeled with CellTrace Violet, were cocultured with CFSE-labeled E2A-PBX1-huCD20 cells. The effector-to-target ratio was adjusted to 2:1, and the mixed cells were incubated in triplicate in a 96-well plate with either CD20 T-BsAb or CD123 T-BsAb (as a control). After a 5-hour incubation, cells were stained with annexin V and propidium iodide (PI). The survival of E2A-PBX1-huCD20 target cells was then assessed. (D) Shows representative fluorescence-activated cell sorting (FACS) plots. (E) Displays a scatter plot with mean and standard deviation of calculated cytotoxicity. Data shown are representative of 3 independent experiments.

Human CD3 transgenic (huCD3-Tg) T cells demonstrated appropriate functionality when stimulated with T-BsAbs and target cells expressing the corresponding human antigen. (A) Upregulation of activation markers on huCD3-Tg T cells after coculture with target cells in the presence of appropriate T-BsAbs. CD8 T cells from huCD3 transgenic mice were cocultured with huCD20-expressing E2A-PBX1 leukemia cells (B6 syngeneic) along with human CD3 X CD20 T-BsAb or CD3xCD123 T-BsAb (control) for 24 hours. Expression levels of CD69 and CD25 were measured by Flow cytometry. (B) huCD3-Tg T cells proliferate after coculture with target cells in the presence of appropriate T-BsAbs. T cells from huCD3 transgenic mice were labeled with CellTrace Violet and cocultured with huCD20-expressing E2A-PBX1 leukemia cells along with human CD3 X CD20 T-BsAb or CD3xCD123 T-BsAb (control) for 4 days. Cell proliferation was measured by the dilution of CellTrace dye. (C) Production of cytokines by effector huCD3-Tg T cells after being stimulated with appropriate T-BsAb and target cells. huCD3-Tg T cells were differentiated in vitro by stimulation with concanavalin A and then rested for 7 days. The resting effector T cells were then restimulated with CD3xCD20 T-BsAb or control T-BsAb in the presence of huCD20-expressing E2A-PBX1 cells for 5 hours. Intracellular cytokine staining was used to measure the production of cytokines. (D-E) Effector huCD3-Tg T cells demonstrated cytotoxicity against target antigen–expressing cells in the presence of appropriate T-BsAb. Effector T cells, prepared in the same way as in panel C and prelabeled with CellTrace Violet, were cocultured with CFSE-labeled E2A-PBX1-huCD20 cells. The effector-to-target ratio was adjusted to 2:1, and the mixed cells were incubated in triplicate in a 96-well plate with either CD20 T-BsAb or CD123 T-BsAb (as a control). After a 5-hour incubation, cells were stained with annexin V and propidium iodide (PI). The survival of E2A-PBX1-huCD20 target cells was then assessed. (D) Shows representative fluorescence-activated cell sorting (FACS) plots. (E) Displays a scatter plot with mean and standard deviation of calculated cytotoxicity. Data shown are representative of 3 independent experiments.

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