MDSC suppression on CD4⁺ T cells in c-fms-rtTA/(TetO)₇-CMV-MMP12 bitransgenic mice. (A) CFSE-labeled CD4⁺ T cells from the spleen of wild-type (WT), doxycycline-treated (+DOX), and untreated (−DOX) mice were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 3 days. Proliferation of labeled CD4⁺ T cells was analyzed by flow cytometry. Peaks represent cell division cycles. (B) Above cultured T cells were stained with anti-CD69 and CD4 Abs and analyzed by flow cytometry. Results are the mean ± SD, n = 5; *P < .05. (C) CFSE-labeled wild-type splenic CD4⁺ T cells were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 4 days in the presence or absence of CD11b⁺/Gr-1⁺ cells from the spleen of wild-type (WT), doxycycline-treated (+DOX), or untreated (−DOX) bitransgenic mice. The ratio between CD11b⁺/Gr-1⁺ cells:CD4⁺ T cells was 1:5. Proliferation of labeled CD4⁺ T cells was analyzed by flow cytometry. Peaks represent cell division cycles. PBS was negative stimulation control. (D) The concentrations of secreted IL-2 and IL-4 in the above cultured medium were measured by ELISA. Results are the mean ± SD, n = 5. (E) CFSE-labeled CD4⁺ T cells were cocultured with CD11b⁺/Gr-1⁺ cells as described in panel C. After 72 hours, cocultured cells were stained with anti-CD69 and CD4 Abs for flow cytometric analysis. PBS was negative stimulation control. (F) Wild-type CD4⁺ T cells were cocultured with CD11b⁺/Gr-1⁺ cells as described in panel C and labeled with annexin V and CD4 Abs for the analysis by flow cytometry. Results are the mean ± SD, n = 5.