CD4 HLA-G^pos T cells show reduced proliferation to allogeneic or polyclonal stimuli. (A) Isolation of CD4 HLA-G^pos T cells by sequential magnetic-activated cell sorting (MACS) bead separation and analysis of the different steps of the isolation by flow cytometry. From top to bottom, dot plots show staining for whole PBMCs before separation, purified CD4 T cells, the CD4 HLA-G^neg T-cell fraction, and the CD4 HLA-G^pos T-cell fraction. Counterstaining for CD14 indicates that there are no contaminating monocytes in the preparation. (B) MACS-sorted CD4 HLA-G^neg and HLA-G^pos T cells were cultured without stimulus for 72 hours to assess HLA-G expression over time. Ex vivo expression of CD4 HLA-G^pos T cells was set as reference. Graph shows summary of 3 experiments. (C) After MACS sorting, whole CD4 (▴), CD4 HLA-G^pos (●), and CD4 HLA-G^neg T cells (■) were stimulated with allogeneic mature DCs at different ratios and proliferation was determined by [³H] incorporation after 4 days. (D) Whole CD4 (), CD4 HLA-G^pos (), and CD4 HLA-G^neg () T cells were isolated and stimulated with anti-CD3/CD28 beads. Proliferation was determined after 4 days by [³H] incorporation. (E) CD4 HLA-G^pos () and CD4 HLA-G^neg () T cells were isolated and stimulated with mature allogeneic DCs in the presence or absence of 500 U/mL IL-2. Proliferation was determined after 4 days. (F) CD4 HLA-G^pos () or CD4 HLA-G^neg T cells () were stimulated for 4 days with allogeneic PBMCs and restimulated again with PBMCs (allo MLR, left) or CD3/CD28 beads (right). Proliferation was determined 3 days later. A representative experiment (1 of at least 3) is shown for panels C-F. Error bars indicate standard error of the mean (SEM).