Journal: The Journal of Experimental Medicine

Article Title: Identification of α-galactosylceramide as an endogenous mammalian antigen for iNKT cells

doi: 10.1084/jem.20240728

Figure Lengend Snippet: Serum contains antigens for iNKT cells. (A) DN32.D3 cells were co-cultured with 5.5 × 10 2 , 1.66 × 10 3 , 5.0 × 10 3 and 1.5 × 10 4 parental or CD1d-transduced MC38, LLC1, B16F10, 2B4, EL4, DN32.D3, HEK293T, HeLa, A549, MDA-MB-231, and PANC-1 cells for 16 h and analyzed for CD69 expression. (B) 5 × 10 5 CD1d −/− or CD1d-transduced B16F10 cells were injected subcutaneously into the right flank of C57BL/6J mice ( n = 8). Tumor volume was measured every 3–4 days. (C) DN32.D3 cells were co-cultured with the indicated cell number of WT or Ugcg −/− Ugt8a −/− CD1d-transduced B16F10 cells for 16 h and analyzed as in A (left). Concentrations of IL-2 in the supernatants were measured (right). (D) CD1d-transduced B16F10 cells were cultured in RPMI 1640 supplemented with 10% FCS or in RPMI 1640 with 0.6% FCS and 9.4% animal component-free cell culture supplement for 7 days. DN32.D3 cells were then co-cultured with those B16F10 cells for 16 h and analyzed as in A. (E) DN32.D3 cells were co-cultured with CD1d-transduced B16F10 cells that were cultured in RPMI 1640 supplemented with 0.6% FCS and 9.4% animal component-free cell culture supplement for 7 days as in D in the absence or presence of α-GalCer (t18:0/26:0) (KRN7000) for 16 h and analyzed as in A. (F) Lipids extracted from serum were separated into seven fractions by open column chromatography and analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol) followed by staining with copper acetate reagent. Commercial β-GlcCer was used as a reference (right lanes). Open and closed arrowheads denote the origin and solvent front, respectively. (G) CD1d −/− or CD1d-transduced DN32.D3 cells were stimulated with each fraction separated from serum lipids in F for 16 h and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. (H) CD1d-transduced DN32.D3 cells were stimulated with the C:M = 19:1 fraction of serum lipids with or without hydrolysis treatment for 16 h and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. (I) CD1d-transduced DN32.D3 cells were stimulated with the C:M = 19:1 fraction of serum lipids treated with Gba (left) or Galc (right) for 16 h and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. Data are presented as mean ± SD (A–E and G–I) and are representative of three independent experiments (A–I). Statistical significance was determined by Student’s t test. *, P < 0.05. Source data are available for this figure: .

Article Snippet: N-stearoyl-D-erythro-sphingosine was from Katayama Chemical Industries Co. Recombinant human glucosylceramidase and recombinant human Galc were from R&D Systems.

Techniques: Cell Culture, Expressing, Injection, Column Chromatography, High Performance Thin Layer Chromatography, Staining, Solvent, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: Identification of α-galactosylceramide as an endogenous mammalian antigen for iNKT cells

doi: 10.1084/jem.20240728

Figure Lengend Snippet: Serum contains antigens for iNKT cells. (A) Surface expression of CD1d on CD1d-transduced cell lines. Filled histogram, anti-mouse CD1d antibody; open histogram, isotype control antibody. (B) WT or TCRα −/− DN32.D3 cells were co-cultured with CD1d-transduced B16F10 cells for 16 h and analyzed for CD69 expression. (C) CD1d −/− or CD1d-transduced B16F10 cells were seeded onto 24-well plates. Growth curves were generated using cell counting by flow cytometer every 24 h. (D) 5 × 10 5 CD1d −/− or CD1d-transduced B16F10 cells were injected subcutaneously into the right flank of Jα18-deficient mice ( n = 7). Tumor volume was measured every 3–4 days. (E) The crude lipids extracted from WT, Ugcg −/− , Ugt8a −/− , and Ugcg −/− Ugt8a −/− B16F10 cells were analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol) and stained with copper acetate reagent. (F) Lipid extracts from B16F10 cells (5 × 10 6 ) were separated into 84 fractions in a 96-well plate by LC-FRC system and evaporated. DN32.D3 cells were stimulated in the 96-well plate for 16 h and analyzed for CD69 expression. Fractionation was performed in triplicate. (G) The C:M = 19:1 fraction of serum lipids before and after hydrolysis treatment was analyzed by HPTLC as in E. (H) Commercial α- and β-GalCer (d18:1/16:0) (left) and α- and β-GalCer (d18:1/24:1) (right) were treated with Galc and analyzed by HPTLC as in E. (I) The C:M = 19:1 fraction of serum lipids and commercial β-GlcCer or β-GalCer were treated with Gba (left) or Galc (right) and analyzed by HPTLC as in E. (J) Screening of columns to separate three diastereomers of synthesized HexCer (d18:1/16:0). MRM chromatograms of SFC/MRM analysis using the columns in are shown. The MRM transition was set to 700.57 > 264.27 (precursor ions selected as [M+H] + ). The SFC analysis conditions for 1-AA, 2-PC, BEH 2-EP, BEH, DEA, Diol, P4VP (PEEK), and PTZ (PEEK) (left) were as follows: column temperature, 50°C; mobile phase A, supercritical carbon dioxide; mobile phase B, M:W (95:5, vol/vol) with 0.1% (wt/vol) ammonium acetate; flow rate of mobile phase, 1.0 ml min −1 ; flow rate of make-up pump, 0.1 ml min −1 ; back-pressure regulator, 10 MPa. The gradient conditions were as follows: 1% B, 0–1 min; 1–75% B, 1–24 min; 75% B, 24–26 min; and 1% B, 26–30 min. The SFC analytical conditions for other columns (center and right) were as described above with modification as follows: column temperature, 40°C; gradient conditions, 1% B, 0–1 min; 1–50% B, 1–17 min; 50% B, 17–26 min; and 1% B, 26–30 min. The MRM operating conditions were identical to those of the SFC/MRM analysis method. The colored shadows indicate the peaks coincident with the RT of synthesized α-GalCer (red), α-GlcCer (blue), β-GlcCer (green), and β-GalCer (yellow), respectively. Open and close arrowheads denote the origin and solvent front, respectively (E and G–I). Data are presented as mean ± SD (B–D and F) and are representative of three independent experiments (B–E and G–J). Statistical significance was determined by Student’s t test (C and D). NS, not significant. Source data are available for this figure: .

Article Snippet: N-stearoyl-D-erythro-sphingosine was from Katayama Chemical Industries Co. Recombinant human glucosylceramidase and recombinant human Galc were from R&D Systems.

Techniques: Expressing, Control, Cell Culture, Generated, Cell Counting, Flow Cytometry, Injection, High Performance Thin Layer Chromatography, Staining, Fractionation, Synthesized, Modification, Solvent