Natural killer T (NKT) cells have been implicated in diverse immune responses ranging from suppression of autoimmunity to tumor rejection. Thymus-dependent NKT cells are positively selected by the major histocompatibility complex class I–like molecule CD1d, but the molecular events downstream of CD1d are still poorly understood. Here, we show that distinct members of the Rel/nuclear factor (NF)-κB family of transcription factors were required in both hematopoietic and nonhematopoietic cells for normal development of thymic NKT cells. Activation of NF-κB via the classical IκBα-regulated pathway was required in a cell autonomous manner for the transition of NK-1.1–negative precursors that express the TCR Vα14-Jα18 chain to mature NK-1.1–positive NKT cells. The Rel/NF-κB family member RelB, on the other hand, had to be expressed in radiation resistant thymic stromal cells for the generation of early NK-1.1–negative NKT precursors. Moreover, NF-κB–inducing kinase (NIK) was required for both constitutive thymic DNA binding of RelB and the specific induction of RelB complexes in vitro. Thus, distinct Rel/NF-κB family members in hematopoietic and nonhematopoietic cells regulate NKT cell development with a unique requirement for NIK-mediated activation of RelB in thymic stroma.

The development of CD1d-dependent natural killer T (NKT) cells is poorly understood. We have used both CD1d/α-galactosylceramide (CD1d/αGC) tetramers and anti-NK1.1 to investigate NKT cell development in vitro and in vivo. Confirming the thymus-dependence of these cells, we show that CD1d/αGC tetramer-binding NKT cells, including NK1.1 + and NK1.1 − subsets, develop in fetal thymus organ culture (FTOC) and are completely absent in nude mice. Ontogenically, CD1d/αGC tetramer-binding NKT cells first appear in the thymus, at day 5 after birth, as CD4 + CD8 − NK1.1 − cells. NK1.1 + NKT cells, including CD4 + and CD4 − CD8 − subsets, appeared at days 7–8 but remained a minor subset until at least 3 wk of age. Using intrathymic transfer experiments, CD4 + NK1.1 − NKT cells gave rise to NK1.1 + NKT cells (including CD4 + and CD4 − subsets), but not vice-versa. This maturation step was not required for NKT cells to migrate to other tissues, as NK1.1 − NKT cells were detected in liver and spleen as early as day 8 after birth, and the majority of NKT cells among recent thymic emigrants (RTE) were NK1.1 − . Further elucidation of this NKT cell developmental pathway should prove to be invaluable for studying the mechanisms that regulate the development of these cells.

We have previously shown that nonobese diabetic (NOD) mice are selectively deficient in α/β-T cell receptor (TCR) + CD4 − CD8 − NKT cells, a defect that may contribute to their susceptibility to the spontaneous development of insulin-dependent diabetes mellitus (IDDM). The role of NKT cells in protection from IDDM in NOD mice was studied by the infusion of thymocyte subsets into young female NOD mice. A single intravenous injection of 10 6 CD4 −/low CD8 − or CD4 − CD8 − thymocytes from female (BALB/c × NOD)F1 donors protected intact NOD mice from the spontaneous onset of clinical IDDM. Insulitis was still present in some recipient mice, although the cell infiltrates were principally periductal and periislet, rather than the intraislet pattern characteristic of insulitis in unmanipulated NOD mice. Protection was not associated with the induction of “allogenic tolerance” or systemic autoimmunity. Accelerated IDDM occurs after injection of splenocytes from NOD donors into irradiated adult NOD recipients. When α/β-TCR + and α/β-TCR − subsets of CD4 − CD8 − thymocytes were transferred with diabetogenic splenocytes and compared for their ability to prevent the development of IDDM in irradiated adult recipients, only the α/β-TCR + population was protective, confirming that NKT cells were responsible for this activity. The protective effect in the induced model of IDDM was neutralized by anti–IL-4 and anti–IL-10 monoclonal antibodies in vivo, indicating a role for at least one of these cytokines in NKT cell-mediated protection. These results have significant implications for the pathogenesis and potential prevention of IDDM in humans.