HLA-G1^acq+ T cells evolve from HLA-G1⁺, HLA-G1–dependent regulatory cells into HLA-G1⁻, HLA-G1–independent regulatory cells. The phenotype and regulatory functions of CD4⁺HLA-G1^acq+ T cells after they had lost HLA-G1 expression (CD4⁺[HLA-G1^acq+→HLA-G1⁻] cells) were investigated by flow cytometry and by adding them as irradiated third-party cells in functional assays, respectively. (A) Phenotype of control T cells and T cells after a 1-hour coincubation with LCL and LCL–HLA-G1 cells, analyzed either at the time of trogocytosis assay (Day 0) or 2 days afterward (Day 2). Cell-surface expression of the indicated molecules was investigated by flow cytometry for CD4⁺ T cells, CD4⁺ T cells incubated with LCL-RSV cells, and T cells incubated with LCL–HLA-G1 cells at the time of trogocytosis assay (day 0) and 2 days afterward (day 2). Results shown are representative of 3 independent experiments. Analysis for phenotypical markers unaffected by the coincubation is in Document S2. (B) Irradiated CD4⁺ T cells and CD4⁺HLA-G1^acq− and CD4⁺HLA-G1^acq+ T cells taken immediately after trogocytosis (day 0) or 2 days afterward (day 2) were added to mixed-lymphocyte reactions between autologous PBMCs and allogeneic LCL-RSV cells. When indicated, HLA-G1 was masked at the CD4⁺HLA-G1^acq+ T cells by blocking anti–HLA-G1 mAb, either during the proliferation assay itself or during trogocytosis only. Day 0: CD4⁺HLA-G1^acq+ but not CD4⁺HLA-G1^acq− T cells inhibited resting autologous T-cell allo-proliferation. This inhibition was abrogated by blocking anti–HLA-G1 mAb. Day 2: CD4⁺[HLA-G1^acq+→HLA-G1⁻] but not CD4⁺[HLA-G1^acq−→HLA-G1⁻] T cells inhibited resting autologous T-cell allo-proliferation. This inhibition was not abrogated by blocking anti–HLA-G1 mAb when added during the proliferation assay but was abrogated if HLA-G was blocked during trogocytosis assay itself, showing that HLA-G1 drives CD4⁺[HLA-G1^acq+→HLA-G1⁻] regulatory cell differentiation but does not mediate their function. Results shown are representative of 5 independent experiments.