Characterization of BM-MDSC–mediated immune suppression. (A) CFSE-labeled PBMCs were stimulated with allogeneic γ-irradiated PBMCs without (left) or with (right) γ-irradiated BM-MDSCs added at a ratio of 1:1. After 7 days, cell cultures were harvested, labeled with anti-CD3ϵ, and analyzed in the CD3ϵ⁺/CFSE⁺ cell gate. The figure shows a representative experiment of cell division analysis of 3 performed. The percentages of the undivided cells are indicated. (B) After 7 days of culture, cultures set up as in panel A were labeled with anti-CD3ϵ, anti-CD8, fixed, and then labeled with anti-CD3ζ. Mean fluorescence intensity (MFI) of CD3ζ was calculated in the CFSE⁺/CD3ϵ⁺/CD8⁺ cell gate. On the left panel, black histogram represents the MFI of stimulated PBMCs without BM-MDSCs, whereas the white histogram refers to MFI of stimulated PBMCs in presence of γ-irradiated BM-MDSCs. On the right panel, MFI values of CD3ζ are presented as mean ± SE of 3 independent experiments; P = .034, Student t test. (C) PBMCs were labeled with CFSE and stimulated with coated anti-CD3 and soluble anti-CD28 (left) and cocultured with BM-MDSCs in the presence (right) or in the absence (center) of a transwell. After 4 days, cells were harvested, labeled with anti-CD3ϵ, and analyzed in the CD3ϵ⁺/CFSE⁺ gate. The figure shows a representative experiment of 3. The percentages of the undivided cells are indicated. (D) Proliferation of alloactivated PBMCs cocultured either with or without γ-irradiated BM-MDSCs was assessed by ³H-thymidine incorporation. White dots represent the proliferation of stimulated PBMCs without BM-MDSCs, and gray dots correspond to the proliferation of alloactivated PBMCs in presence of BM-MDSCs. Twenty independent experiments are shown with proliferation of alloactivated PBMCs < 30 × 10³ cpm (columns 1 and 2) and 15 experiments with proliferation > 30 × 10³ cpm (columns 3 and 4). P = .01 and P < .001, Mann-Whitney U test.