Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells

Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by alpha-galactosylceramide (alpha-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (T-reg), such as CD4(+)CD25(+) T-reg, for this protection. We found that CD4(+)CD25(+) T cells from NOD.CD1d(-/-) mice deficient in iNKT cell function similarly in vitro to CD4(+)CD25(+) T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon alpha-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4(+)CD25(+) T-regs from NOD mice pretreated with alpha-GalCer demonstrated that activated iNKT cells do not influence the ability of T-regs to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4(+)CD25(+) T cells, because splenocytes pretreated with a-GalCer and then inactivated by anti-CD25 of CD25(+) cells did not protect from T1D. Similarly, mice inactivated of CD4(+)CD25(+) T cells before alpha-GalCer treatment were also not protected from T1D. Our data suggest that CD4(+)CD25(+) T cells retain their function during iNKT cell activation, and that the activity of CD4(+)CD25(+) T-regs is required for iNKT cells to transfer protection from T1D.