Lin⁻ subset contained within BM-MDSCs shows potent suppressive activity. (A) Flow cytometric analysis of BM cells cultured for 4 days with G-CSF + GM-CSF (BM-MDSCs) or without growth factors (NT BM). At the end of the culture, cells were harvested and labeled, and the percentages of CD11b⁺/CD16⁻ cells were calculated. The figure represents 22 independent experiments; P ≤ .001, Student t test. (B) Flow cytometric profile of CD16 and CD11b expression and May-Grünwald-Giemsa staining on BM-MDSCs before and after immunomagnetic depletion with Lin Ab cocktail. (C) Flow cytometric analysis of the proliferation of allogenic PBMCs, stained with CFSE and activated with anti-CD3 and anti-CD28 for 4 days, in the presence of either BM-MDSCs or the fractions Lin⁺ or Lin⁻ sorted from BM-MDSCs. The figure, in which the percentages of undivided CD3ϵ⁺/CFSE⁺ lymphocytes are shown, represents 1 of 3 independent experiments. (D) Number of CD3ϵ⁺/CFSE⁺ events obtained after activation of PBMCs with anti-CD3/CD28 and cocultured in the presence of BM-MDSCs or the subsets Lin⁺ and Lin⁻ sorted from BM-MDSCs. The figure, in which the black bars refer to undivided cells and the gray bars to divided cells, represent the mean ± SE of 6 independent experiments. The values of P are indicated in the figure, Mann-Whitney U test. (E-F) Evaluation of MFI of CD3ϵ chain expression and percentage of the CD3ϵ⁺/CFSE⁺ cells in PBMCs stimulated with anti-CD3/CD28 in the presence of BM-MDSCs or the Lin⁺ and Lin⁻ fractions. Values are mean ± SE of 6 independent experiments. All comparisons among BM-MDSCs containing cultures versus cultures without BM-MDSCs, P = .041 (E) and P = .009 (F), respectively, Mann-Whitney U test.