SAP^−/− mice selectively lack Vα14 NKT cells. (A) Vα14 NKT cells, identified as positive for CD1d-αGC tetramer (y axis) and negative for empty CD1d tetramer and CD8 (x axis) by flow cytometry of thymocytes, splenocytes, and liver lymphocytes, are indicated in the boxed areas with their corresponding % values. Data representative of four individual adult FVB.SAP^−/− mice compared to wild-type (WT) littermates and B6.CD1^−/−. (B) Histograms of CD1d surface thymocyte and splenocyte expression in WT and mutant mice. (C) T cell subsets identified by CD4 and CD8 or CD4 and CD44 in the indicated tissues, with corresponding % values as indicated. (D) Vα14 NKT cells in fetal thymuses after 14 days of culture. Data representative of two individual B6.SAP^−/− fetal thymuses and their WT littermate controls.

Colocalization of CD1d, saposin, and Lamp1. (A) Tumor necrosis factor–α–activated DC stained with antibodies against CD1d, saposin, and Lamp1, as indicated. Direct transmission image is shown underneath to delineate the outer cell membrane. Left, WT; right, SAP^−/−. (B) Same experiment as in (A) with fresh thymocytes. Scale bars indicate 10 μm.

Schematic representation of the hypothetical mechanism of lipid editing by LTPs. LTPs are presented in two conformations reflecting the structural changes associated with lipid extraction (20).

Antigen presentation by SAP^−/− APCs. (A) Failure to stimulate CD1d-autoreactive Vα14 NKT hybridomas. Fresh thymocytes from SAP^−/− and WT FVB littermates or CD1d^−/− mice were used to stimulate CD1d-autoreactive NKT hybridomas expressing a Vα14 invariant TCR α chain (DN32.D3) or other Vα (TCB11 and 1C8DC1). Data representative of four separate sets of mice. (B) Presentation of exogenous glycolipid antigens. Thymocytes, DCs, and splenocytes from WT, SAP^−/−, and CD1d^−/− mice were pulsed with various concentrations of αGC or Gal-α-1,2 αGal-Cer, as indicated, before coculture with the Vα14 DN32.D3 NKT hybridoma cells.

LTP-mediated lipid transfer to or extraction from CD1d. (A) Saposins transfer lipids from liposomes onto CD1d molecules. CD1-GT (2 μM) incubated at pH = 5.0 with 1 mM PS (prepared as 400 nm liposomes) and 15 μM saposin. Lipid loading is visualized by native IEF. The migration of the CD1d band toward the cathode indicates loading with PS. (B) Association of saposin A with CD1d. CD1-GT, CD1-SFT, and CD1-αGC complexes were purified to homogeneity by anion exchange, incubated at equimolar ratio with saposin A (4 μM) for 1 hour at room temperature, and analyzed by native IEF. After transfer, blots were hybridized with the CD1 antibody 20H2 and a rabbit anti-saposin A antiserum. Lane 1, saposin alone; lane 2, saposin plus CD1-GT; lane 3, saposin plus CD1d-αGC; and lane 4, saposin plus CD1-SFT. The presence of CD1-saposin complexes is marked by the appearance of bands stained by both anti–saposin A and anti-CD1d (arrows). Controls including saposin A alone (lane 5), saposin plus G_T1b (lane 6), saposin plus αGC (lane 7), saposin plus sulfatide (lane 8), and saposin plus H-2K^b (lane 9) were hybridized with the anti-saposin serum. Note that the anti-saposin serum reacts to different levels when incubated with various lipids. (C) G_M2A unloads glycolipids bound to CD1d. Purified CD1-GT complexes (2.5 μM) were incubated with increasing concentrations of G_M2A. Coomassie Blue staining reveals the appearance of a neutral band stained by anti-CD1 and corresponding to the migration of empty CD1d. G_M2A (not seen on Coomassie Blue staining) appears in the Western blot by itself (top of gel) and in an intermediate band that corresponds to the G_M2A-G_T1b complex. (Far right) Anti-G_M2A Western blot of G_M2A by itself (lane 1) and G_M2A incubated with 2.5 μM G_T1b (lane 2). In the composite overlay, CD1 species are in green, whereas G_M2A species are in blue.