CD4 HLA-G^pos T cells have suppressor activity and they do not express CD25 or FoxP3. (A) Suppression assay using CD4 HLA-G^pos T cells. CD4 HLA-G^pos T cells were titrated into CFDA-SE–labeled CD4 HLA-G^neg T cells cultured in the presence of allogeneic PBMCs and soluble anti-CD3. Cell proliferation was quantified by flow cytometric assessment of CSFE dilution histograms on day 4. The left panel compiles suppression capacity of CD4 HLA-G^pos T cells at different ratios in 3 independent experiments. The right panel shows a representative suppression assay with percentages of proliferation stated in histogram. (B) FACS staining for CD25/CD4/HLA-G at the cell surface (left) and intracellular flow cytometry for FOXP3/CD4/HLA-G (right). The dot plots show the staining on gated CD4 T cells; values indicate the percentage of cells in gates. One representative experiment is shown. (C) Expression of FoxP3 mRNA by CD4 HLA-G^pos/neg cells. FACS-sorted cells (HLA-G^pos, HLA-G^neg, CD4⁺CD25⁻, and CD4⁺CD25⁺⁺) from 2 different healthy donors (light and dark bars) were analyzed for mRNA levels of Foxp3. Expression of Foxp3 in PBMCs from one of the donors was used as calibrator (= 1).