Parameters of HLA-G1 trogocytosis. (A) Analysis of HLA-G1 transfer to resting T cells after blocking HLA-TCR, CD28-B7, and HLA-G–HLA-G receptor interactions. HLA-TCR interactions were blocked by anti-TCR, anti-CD3, and, to a certain extent, anti–pan–HLA class I blocking antibodies. CD28-B7 interactions were blocked by anti-CD28 antibodies. Interaction of HLA-G1 with its receptors was blocked by anti–HLA-G1, anti–pan–HLA class I, or anti-ILT2 antibodies. Results are expressed as mean ± SD of at least 3 independent experiments for each antibody. (B) HLA-G1 trogocytosis capabilities of activated CD4⁺ and CD8⁺ T cells. T cells were activated by the indicated methods, and their capability to acquire HLA-G1 from LCL–HLA-G1 cells was investigated by flow cytometry. Results shown are representative of more than 10 independent experiments. (C) Kinetics of trogocytosis capability acquisition by T cells upon PHA activation. Resting T cells were stimulated by PHA and then by IL-2 after the second day. The capability of T cells to acquire HLA-G1 from LCL–HLA-G1 cells was investigated by flow cytometry at the indicated times. Results shown are expressed as mean ± SD of more than 10 independent experiments. (D) Capabilities of activated CD4⁺ and CD8⁺ T cells to acquire HLA-G1 from nontransfected HLA-G1–positive autologous monocytes. T cells were activated by PHA+IL-2 treatment (see “Cells and cell lines” under “Materials and methods”), and their capability to acquire HLA-G1 from IFNG-stimulated, HLA-G1–positive autologous monocytes (With IFNG) and their HLA-G1–negative controls (No IFNG) was investigated by flow cytometry. Results shown are representative of 3 independent experiments. (E) Kinetics of HLA-G1 acquisition by resting and activated CD4⁺ and CD8⁺ T cells. Resting and activated T cells from the same donor were incubated with LCL–HLA-G1 cells and their cell-surface expression of HLA-G1 was investigated at the indicated times. Results are expressed as mean ± SD of 4 independent experiments. (F) Lifetime of acquired HLA-G at the T-cell surface. Flow-cytometry analysis of acquired HLA-G1 cell-surface expression by CD4⁺ and CD8⁺ T cells at the indicated times corresponding to culture time after purification. Results shown are representative of 3 independent experiments.