T cell recruitment to elicit contact sensitivity (CS) requires a CS-initiating process mediated by B-1 cells that produce IgM, which activates complement to promote T cell passage into the tissues. We now show that Vα14i NKT cells induce B-1 cell activation likely by releasing IL-4 early postimmunization. The CS initiation process is absent in Jα18 −/− and CD1d −/− NKT cell–deficient mice and is reconstituted by populations enriched for Vα14i NKT cells. Transfers are not effective if cells are derived from IL-4 −/− mice. Staining with specific tetramers directly showed that hepatic Vα14i NKT cells increase by 30 min and nearly double by 2 h postimmunization. Transfer of immune B-1 cells also reconstitutes CS responses in NKT cell–deficient mice. The B-1 cells act downstream of the Vα14i NKT cells to restore CS initiation. In addition, IL-4 given systemically to Jα18 −/− or CD1d −/− NKT cell–deficient mice reconstitutes elicitation of CS. Further, splenocytes from immune Jα18 −/− mice produce less antigen (Ag)-specific IgM antibodies compared with sensitized WT mice. Together these findings indicate that very early after skin immunization Vα14i NKT cells are stimulated to produce IL-4, which activates B-1 cells to produce Ag-specific IgM, subsequently needed to recruit effector T cells for elicitation of CS responses.

GD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3 + GM2 − CD1 − ) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of ∼1:2,000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-γ followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1 − tumors.

A defect in RelB, a member of the Rel/nuclear factor (NF)-κB family of transcription factors, affects antigen presenting cells and the formation of lymphoid organs, but its role in T lymphocyte differentiation is not well characterized. Here, we show that RelB deficiency in mice leads to a selective decrease of NKT cells. RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells. Like RelB-deficient mice, aly/aly mice with a mutation for the NF-κB–inducing kinase (NIK), have reduced NKT cell numbers. An analysis of NK1.1 and CD44 expression on NKT cells in the thymus of aly/aly mice reveals a late block in development. In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors. This link between NIK and RelB was further demonstrated in vivo by analyzing RelB +/− × aly / + compound heterozygous mice. After stimulation with α-GalCer, an antigen recognized by NKT cells, these compound heterozygotes had reduced responses compared with either RelB +/− or aly / + mice. These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.