HLA-G production and cytokine profile of CD4 HLA-G^pos and CD4 HLA-G^neg T cells. (A) Separation of CD4 T-cell subpopulation by FACS sorting. After exclusion of aggregated cells (SSC-A versus SSC-W), the gate for CD4 T cells was set on FSC/CD4 dot blot to identify the desired subsets. For sorting, the HLA-G–positive population was assessed compared with the control isotype staining; a second gate was set on HLA-G–negative cells. One representative FACS sort experiment is shown. Values indicate the percentage of cells in gates. (B) Ex vivo expression of HLA-G mRNA in FACS-sorted cells. JEG3 mRNA was used as positive control. A representative experiment (1 of 5) is shown. (C) Intracellular expression of HLA-G5 by intracellular staining for the soluble isoform HLA-G5 in sorted HLA-G–positive and negative cells was performed after 4-day stimulation with CD3/CD28 beads using monoclonal antibody 5A6G7. A gate for CD4 T cells was set on FSC/CD4 dot blot to identify the desired subset. HLA-G5 expression (lower panel) was assessed in comparison with the control isotype staining (top panel). A representative staining is shown. Values indicate the percentage of cells in gates. (D) Production of soluble HLA-G. Supernatants of CD3/CD28-stimulated HLA-G–positive and negative subsets were tested by ELISA for soluble HLA-G. Cell lines transfected with either HLA-G1 or HLA-G5 were used as positive controls. One representative experiment is shown. (E) Cytokines assessed by RT-PCR are IFN-γ (left), IL-10 (middle), and TGF-β (right). Graphs show results of 5 experiments. Error bars indicate standard deviation. (F) Cytokine production of purified subsets. CD4 HLA-G^pos and HLA-G^neg T cells were stimulated with CD3/CD28 beads for 24 hours. Supernatants were assessed for cytokine production using the RayBio array. A representative cytokine profile for CD4 HLA-G^neg (left) and for CD4 HLA-G^pos (right) is shown.