Figure 4. Peripheral iNKT Cells Show Signs of Chronic Exposure to Self Antigens in Gla^−/− Mice.

(A) WT and Gla^−/− mice received i.v. a fluorescent poly-caspase substrate (Flivo). After 45 min, mice were sacrificed, and substrate fluorescence was measured in αGalCer-loaded CD1d-tetramer⁺ TCRβ⁺ iNKT cells.

(B) Dot plots show the percentages of liver, spleen, and thymus iNKT cells expressing surface Ly49C/I in WT and Gla^−/− mice. iNKT cells were identified as in (A).

(C) Peripheral iNKT cells in Gla^−/− mice are tolerant. WT and Gla^−/− mice received i.v. αGalCer in PBS containing 0.5% polysorbate 20 or PBS/0.5% polysorbate 20 alone (vehicle). After 3 hr, mice were sacrificed and liver mononuclear cells were isolated and stained with αGalCer-loaded CD1d-tetramers and TCRβ mAb. Cells were fixed and permeabilized prior to intracellular IFN-γ staining analyzed by flow cytometry. (B and C) Numbers in dot plots represent percentages of Ly49C/I⁺ or IFN-γ⁺ iNKT cells in the respective quadrants.

(D) Serum concentrations of IFN-γ in WT and Gla^−/− mice were determined by ELISA 16 hr following i.p. injection of αGalCer in vehicle or vehicle alone. Error bars depicted in graphs represent mean values ± SEM (A–D). Results are representative of at least three independent experiments with similar results. At least three mice were used per group.