Serum contains antigens for iNKT cells. (A) DN32.D3 cells were co-cultured with 5.5 × 102, 1.66 × 103, 5.0 × 103 and 1.5 × 104 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 × 105 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: SourceData F1.
Figure 1.

Serum contains antigens for iNKT cells. (A) DN32.D3 cells were co-cultured with 5.5 × 102, 1.66 × 103, 5.0 × 103 and 1.5 × 104 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 × 105 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: SourceData F1.

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