Binding of CD1d dimer molecules loaded with lysophosphatidylcholine (LysoPC) to a subpopulation of CD3⁺ T cells. (A) CD1d dimers loaded with 2 species of LPC (LPC-C16 and LPC-C18:1) stained a unique population of CD3⁺ T cells: FACS dot plots of CD1d dimers loaded with vehicle control (−), 400 μM of 2 species of LPC (C16 and C18:1) or PC, or 20 μM α-GalCer, and used to stain cultured human PBMCs. Numbers in the quadrant correspond to percentage of cells of total CD3⁺ cells, and data shown are gated for live cells. The staining is representative of 4 individual donors. (B) CD1d dimers were loaded with different concentrations of C18:1 LPC before staining of human T cell cultures as described in “Loading CD1d dimers and detection of CD1d-lipid–reactive T cells.” As controls, T cells were also stained with MHC-I tetramer cultured with 400 μM of LPC or PC. Numbers in the quadrant correspond to percentage cells of total CD3⁺ cells, and data shown are gated for live cells. (C) Phenotype of CD1d-LPC-C18:1⁺ cells: FACS dot plots of staining of CD1d-LPC-C181 (as in panel A) but plotted against different markers, TCRαβ, Vα24, and Vβ11 as indicated. Data for the staining of Vα24⁺ cells in these cultures with CD1d-αGalCer are shown as a control. Bottom panel shows FACS dot plot gated for CD1d-LPC-C18:1⁺ T-cell population for the proportion of CD4⁺/CD8⁺/double-negative subpopulations. (D) CD1d dimers were loaded with either 400 μM of LysoPC or PC alone, or with a mixture of LysoPC and PC as indicated, before use for staining T cells as in panel A. (E) Histogram plots of staining of iNKT cells by CD1d dimers loaded with α-GalCer (20 μM), LPC, PC, or the combination of α-GalCer with PC or LPC. Numbers represent mean fluorescent intensity of staining. (F) Competition between LPC and α-GalCer for binding to iNKT cells: Histogram overlay plots comparing mean fluorescence shifts between CD1d dimer loaded with vehicle control (light gray solid), positive control (α-GalCer, dark gray solid), and LPC-C18:1, or PC (open curves). Competition was set up by first loading CD1d dimer with vehicle control, LPC-C18:1, or PC, for 4 hours, then loading with vehicle control or α-GalCer. Ligand-loaded CD1d dimers were then used to stain iNKT cells. Data are representative of 4 similar experiments. (G) Differential iNKT stimulation by ligand-loaded and immobilized CD1d dimer. Polyclonal populations of Vα24⁺ T cells were isolated using magnetic beads from cultures expanded in vitro using α-GalCer. Purified Vα24⁺ T cells were cultured for 40 to 48 hours in plates with immobilized CD1d dimer, loaded with vehicle control or α-GalCer with or without preincubation with control, PC, or LPC as competing ligands as described in “Loading CD1d dimers and detection of CD1d-lipid–reactive T cells.” PHA was used as positive control. The level of IFN-γ in the supernatant was monitored using ELISA. Data are representative of 3 similar experiments (*P < .05). Error bars represent SD.