iNKT and NK cell–mediated antitumor effects in mice immunized with α-GalCer–loaded allogeneic fibroblasts. (A) C57BL/6 mice were immunized with α-GalCer–loaded, mRNA-transfected allogeneic fibroblasts, and spleen cells were collected 16 hours after immunization. Cells were stained with CD3–fluorescein isothiocyanate (FITC) and NK1.1-allophycocyanin and either CD69–phycoerythrin (PE) or intracellular IFN-γ–PE and evaluated by FACS (Document S1). Data of CD69 and IFN-γ shown have been gated on CD3⁻NK1.1⁺ cells. (B) To evaluate the function of iNKT cells, mice were injected intravenously with either 5 × 10⁵ parental CD1d^hi-NIH3T3/Gal cells or CD1d^hi-B16/Gal cells. Spleens were removed 2 days later, and suspended cells were restimulated with or without α-GalCer (100 ng/mL) for 16 hours in IFN-γ ELISPOT assay plates (Document S1).^53,54  Data are means from 3 mice per group. (C) Antitumor immunity generated by innate lymphocytes in response to injected allogeneic fibroblasts was evaluated using a B16 lung metastasis model. Mice were injected with 2 × 10⁵ B16 melanoma cells intravenously, then 3 hours later given 5 × 10⁵ NIH3T3, NIH3T3/Gal, CD1d^hi-NIH3T3, or CD1d^hi-NIH3T3/Gal. The number of lung metastases was counted 14 days later through the stereomicroscope (Leica MZ7.5) (n = 5 per group). Similar results were obtained in 2 independent experiments. *P < .025 (NIH3T3/Gal, CD1d^hi-NIH3T3/Gal vs other groups; ie, NIH3T3, CD1d^hi-NIH3T3, and control).