MDSCs from bitransgenic mice suppress T-cell proliferation and function. (A) CFSE-labeled splenic CD4⁺ T cells from 3-month doxycycline-treated (+DOX) or untreated (−DOX) c-fms-rtTA/(tetO)₇-CMV-dnPPARγ–bitransgenic mice were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 4 days. Proliferation of labeled CD4⁺ T cells was analyzed by flow cytometry. Peaks represent cell division cycles. PBS stimulation control is shown as the shaded areas. (B) CD4⁺ T cells from the spleens of 3-month doxycycline-treated or untreated bitransgenic mice were cultured and stimulated with anti-CD3 mAb plus anti-CD28 mAb. After 48 hours, CD4⁺ T cells were stained with anti-CD69 and CD4 Abs for the flow cytometry analysis. Percentages of CD69⁺ CD4⁺ T cells were statistically analyzed. (C) Lymphokine production of splenic CD4⁺ T cells in the medium measured by ELISA analysis; (D) CFSE-labeled wild-type 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 spleens of wild-type, doxycycline-untreated, or 3-month doxycycline-treated bitransgenic mice. The ratio of MDSCs to CD4⁺ T cells was 1:5. The proliferation of labeled CD4⁺ T cells was analyzed by flow cytometry. Peaks represent cell division cycles. Top panel, MDSCs and T cells in direct contact coculture study. Bottom panel, MDSCs and T cells in Transwell coculture study. (E) Similar to panel B, CD69⁺ expression on CD4⁺ T cells was analyzed by flow cytometry 48 hours after anti-CD3 mAb plus anti-CD28 mAb stimulation. (F) Lymphokine production of CD4⁺ T cells in the culture medium was measured by ELISA analysis. In all analyses, values were derived from 4 mice in each group (n = 4). *P < .05; **P < .01.