T-cell decrease in c-fms-rtTA/(TetO)₇-CMV-MMP12 bitransgenic mice. (A) Flow cytometric analysis of CD4⁺ and CD8⁺ cells from the spleen of 3-month doxycycline-treated wild-type (WT) mice, doxycycline-treated (+DOX), doxycycline-untreated (−DOX) bitransgenic mice, and doxycycline-treated (for 2 months) followed by doxycycline-removal (for 1−month) bitransgenic mice (on/off). Results are the mean ± SD, n = 5; *P < .05. (B) A representative flow cytometric analysis showing the FoxP3 and CD25 profiles among total CD4⁺ T cells from the spleen of 3-month doxycycline-treated wild-type (WT) mice, doxycycline-treated (+DOX), untreated (−DOX) bitransgenic mice and doxycycline-treated (for 2 months) followed by doxycycline-removal (for 1 month) bitransgenic mice (on/off). (C) Absolute cell numbers of FoxP3⁺ Treg cells among total CD4⁺ T cells were calculated based on analyses of the above experimental groups. Results are the mean ± SD, n = 5; *P < .05. (D) CFSE-labeled CD4⁺ T cells were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 4 days in the presence or absence of Treg cells isolated from the spleens of wild-type (WT), doxycycline-treated (+DOX) or untreated (−DOX) bitransgenic mice. The ratio between Treg:CD4⁺ T cells was 1:1. Proliferation of labeled CD4⁺ T cells was analyzed by flow cytometry. Peaks represent cell division cycle. (E) Wild-type CD4⁺ T cells from the spleen were cultured and stimulated with anti-CD3 mAb plus anti-CD28 mAb in the absence and presence of inactivated (inact) or activated (act) MMP12. After 72 hours, T cells were stained with anti-CD69 and CD4 Abs. A representative flow cytometric analysis is demonstrated. (F) The concentrations of secreted IL-2, IL-4, and IFNγ in the above cultured medium were measured by ELISA. Results are the mean ± SD, n = 5.