FIGURE 5.

ATF3 regulates the development of G-MDSCs. A, proportions of the indicated myeloid cell populations in WT and ATF3-KO mice were analyzed by flow cytometry. iMC (CD11b⁺Gr1⁺), Granulo (granulocytes, CD11b⁺Ly6G⁺Ly6C⁻), and Mono (monocytes, CD11b⁺Ly6G⁻Ly6C⁺) are shown. B, proportions of indicated immune cells in WT and ATF3-KO mice were determined by flow cytometric analysis: DC (CD11c⁺MHCII⁺), macrophage (CD11b⁺Gr1⁻F4/80⁺), Treg (CD4⁺CD25⁺), B cell (B220⁺), CD4 T (CD3⁺CD4⁺), and CD8 T (CD3⁺CD8⁺). Con, control. C, allogeneic mixed lymphocytes reaction (MLR). CD3⁺T cells were stimulated with ConA and cocultured with allogeneic splenic CD11b⁺Ly6G⁺ at different ratios for 3 days. T cell proliferation was evaluated by carboxyfluorescein diacetate, succinimidyl ester (CFSE) dilution, and unstimulated T cells were used as negative control. D, BM cells were infected with lentivirus expressing ATF3 or vector (Vec) and cultured in medium containing GM-CSF and IL6. The frequencies of MDSCs (Gr1⁺CD11b⁺), DCs (CD11c⁺MHCII⁺), and macrophages (CD11b⁺F4/80⁺) among GFP ⁺ cells were analyzed by flow cytometry at day 6. Medium alone was used as control. E, splenic MDSCs were cultured with GM-CSF and IL-4 for 3 and 5 days, and the proportions of DCs (CD11c⁺CD11b⁺) and macrophages (CD11b⁺F4/80⁺) were determined. A–D, both representative data and mean ± S.E. from six mice (A) or three independent experiments (B–D) are shown. *, p < 0.05; **, p < 0.01, unpaired t tests. SP, spleen.