T-lymphocyte activation is driving BM-MDSC proliferation and immune suppression. (A) CellTrace-labeled PBMCs were stimulated with anti-CD3/CD28 in the presence of BM-MDSC CD11b⁺ and CD11b^low/− cell subsets, added at a ratio of 1:1. After 3 days, cell cultures were harvested, labeled with anti-CD3ϵ, and analyzed in the CD3ϵ⁻/CellTrace⁻ gate (M) and in the CD3ϵ⁺/CellTrace⁺ (T) cell gate. The numbers indicated in the top graphs refer to the percentage of cells gated on either T cells (T) or on myeloid cells (M). The central histograms show the profile of CellTrace dilution of either resting or stimulated T cells (gate T) cocultured with BM-MDSCs CD11b⁺ and CD11b^low/− subsets. Black and gray curves refer to undivided and proliferating cells, respectively. The bottom histograms show Ki-67 expression in BM-MDSCs CD11b⁺ and CD11b^low/− subsets (gate M) cocultured with either resting or stimulated T cells. Black histograms indicate isotype control. The figure shows a representative experiment of 3 performed. (B) Flow cytometric evaluation of CD11b, CD16, HLA-DR, CD34, and CD66b markers in CD11b^low/− cell subset sorted from BM-MDSCs either before or after the coculture with resting or anti-CD3/anti-CD28–activated T cells. The expression of these markers was compared with the autofluorescence signal (black histograms). In the figure, 1 representative of 3 independent experiments is presented.