Monocytes can differentiate into macrophages or dendritic cells (DCs). The processes that promote their differentiation along one pathway rather than the other remain unknown. NKT cells are regulatory T cells that respond functionally to self and foreign antigens presented by CD1d molecules. Hence, in addition to contributing to antimicrobial responses, they may carry out autoreactively activated functions when there is no infectious challenge. However, the immunological consequences of NKT cell autoreactivity remain poorly understood. We show here that human NKT cells direct monocytes to differentiate into immature DCs. The ability to induce monocyte differentiation was CD1d-dependent and appeared specific to NKT cells. Addition of exogenous antigens or costimulation from IL-2 was not required but could enhance the effect. DC differentiation was a result of NKT cell secretion of GM-CSF and IL-13, cytokines that were produced by the NKT cells upon autoreactive activation by monocytes. NKT cells within PBMC samples produced GM-CSF and IL-13 upon exposure to autologous monocytes directly ex vivo, providing evidence that such NKT cell-autoreactive responses can occur in vivo. These results show that when NKT cells are activated by autologous monocytes, they are capable of providing factors that specifically direct monocyte differentiation into immature DCs. Thus, autoreactively activated NKT cells may contribute to the maintenance of the immature DC population, and microbial infection or inflammatory conditions that activate NKT cells further could stimulate them to promote an increased rate of DC differentiation.

CD1d molecules present both self Ags and microbial lipids to NKT cells. Previous studies have established that CD1d lysosomal trafficking is required for presentation of autoantigens to murine invariant NKT cells. We show in this study that this is not necessary for autoantigen presentation by human CD1d, but significantly affects the presentation of exogenous Ags. Wild-type and tail-deleted CD1d molecules stimulated similar autoreactive responses by human NKT clones, whereas presentation of exogenous lipids by tail-deleted CD1d was highly inefficient. Chloroquine treatment markedly inhibited exogenous Ag presentation by wild-type CD1d transfectants, but did not affect NKT autoreactive responses. Conversely, APC expression of HLA-DRαβ and the invariant chain (Ii) was associated with faster internalization of CD1d into the endocytic system and enhanced CD1d-mediated presentation of exogenous Ags, but did not appear to augment NKT autoreactivity. Knockdown of the Ii by small interfering RNA resulted in reduced CD1d surface expression and slower internalization in HLA-DR+ APCs, but not HLA-DR− APCs, demonstrating a direct effect of MHC/Ii complexes on CD1d trafficking. CD1d-mediated presentation of exogenous Ags was much more efficient in immature dendritic cells, which actively recycle MHC class II molecules through the endocytic system, than in mature dendritic cells that have stabilized MHC class II expression at the cell surface, suggesting a physiological role for MHC/Ii complexes in modulating CD1d function. These results indicate that autoantigens and exogenous lipids are acquired by human CD1d at distinct cellular locations, and that Ii trafficking selectively regulates CD1d-mediated presentation of extracellular Ags.

We show that inactivation of the protein kinase Cdk1/Cyclin B (Cdc28/Clb 2 in the budding yeast Saccharomyces cerevisiae) is not only necessary for cells to leave mitosis, as is well known, but also sufficient to trigger mitotic exit. Cells carrying the mutation cdc28-as1, which makes Cdc28 (Cdk1) uniquely sensitive to the ATP analog 1NM-PP1, were arrested with spindle poisons and then treated with 1NM-PP1 to inhibit Cdk1. This treatment caused the cells to exit mitosis and enter G1-phase as shown by initiation of rebudding (without cytokinesis), production of “shmoos” (when α-factor was present), stabilization of Sic1, and degradation of Clb2. This result provides a system in which to test whether particular gene products are required downstream from Cdk1 inactivation in exit from mitosis. In this system, the mutation cdc28-as1 is combined with a conditional mutation in the gene of interest. Using this approach, we demonstrate that Protein Phosphatase 1 (PPase1; Glc7 in S. cerevisiae) is required for reestablishment of G1-phase following Cdk1 inactivation. This system could be used to test whether other protein phosphatases are also needed downstream from Cdk1 inactivation, and it could be combined with phosphoproteomics to gain information about the substrates those phosphatases act on during mitotic exit.