HLA-G1 is acquired by T cells from APCs by trogocytosis. (A) Transfer of HLA-G1 from APCs to T cells is cell-to-cell contact dependent. CD4⁺ and CD8⁺ T cells were coincubated with HLA-G1–transfected LCL–HLA-G1 cells (No transwell) or separated by a 0.4-μM–pored membrane (Transwell) for 1 hour, prior to flow-cytometry analysis of HLA-G1 expression. Results are expressed as mean ± SD of n = 8 independent experiments. (B) HLA-G1 that is newly displayed by CD4⁺ and CD8⁺ T cells is of APC origin and not endogenously produced. Biotinylation of LCL-RSV and LCL–HLA-G1 cell surface was performed prior to coincubating them for 1 hour with resting T cells from PBMCs. CD4⁺ and CD8⁺ T cells were then purified and lysed, and the obtained lysates were analyzed by Western blotting. For each sample, protein content was controlled using antitubulin, and the absence of contamination by LCL cells was controlled using anti-ILT3. Results shown are representative of 3 independent experiments. (C) Visualization of trogocytosis of HLA-G from APCs by T cells by confocal microscopy. Red is cytoplasmic label of LCL–HLA-G1 cells with CMTMR. Blue is cytoplasmic labeling of T cells with Bodipy. Green is HLA-G labeling using 4H84 mAb and then FITC-conjugated goat antimouse secondary antibody. Arrows indicate HLA-G transferred from APCs to T cells after the indicated time of coincubation.