Natural killer T cells from interleukin-4-deficient mice are defective in early interferon-gamma production in response to alpha-galactosylceramide

Discovery of the natural killer (NK) T cell-specific ligand, alpha-galactosylceramide (alpha-GalCer) has enabled us to investigate the functional regulation of NKT cells. However, the detailed mechanism of cytokine production by NKT cells remains to be elucidated. Here we evaluated the role of interleukin (IL)-4 in the production of interferon (IFN)-gamma from NKT cells using IL-4-deficient C57BL/6 mice (IL-4(-/-) mice). Administration of alpha-GalCer into wild-type C57BL/6 mice caused the production of both IFN-gamma and IL-4 in serum or cytoplasm within 4 h of the injection. Unexpectedly, however, IL-4(-/-) mice-derived NKT cells did not produce any IFN-gamma at early phase after primary stimulation with alpha-GalCer. Because NKT cells from IL-4(-/-) mice produced IFN-gamma when they were stimulated secondarily with alpha-GalCer in vitro for 72 h, NKT cells from IL-4(-/-) mice were not completely genetically deficient for IFN-gamma production. To elucidate which cells, NKT cells or dendritic cells (DC), were responsible for the deficiency in IFN-gamma production in IL-4(-/-) mice, we carried out an add-back experiment using purified NKT cells and DC, which were prepared from either wild-type mice or IL-4(-/-) mice. NKT cells from wild-type mice produced IFN-gamma when they were cocultured with DC prepared from either wild-type or IL-4(-/-) mice, whereas NKT cells from IL-4(-/-) mice did not produce IFN-gamma by coculturing with DC from either wild-type or IL-4(-/-) mice. These results indicate that NKT cells, not DC, were responsible for the deficiency in IFN-gamma production in IL-4(-/-) mice. Thus, IL-4 is required for the activation of NKT cells to produce IFN-gamma in response to alpha-GalCer.