Distinct Endosomal Trafficking Requirements for Presentation of Autoantigens and Exogenous Lipids by Human CD1d Molecules

Abstract

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.

Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation

“…By confocal microscopy we observed that tail-deleted CD1d molecules could still reach the lysosomal Lamp-1 + compartment (Fig. S7), although less efficiently (likely because of association with invariant chain), which is highly expressed in myeloid cells (30,44,45). Consequently, presentation of exogenous synthetic iNKT cell agonists was only moderately affected by tail-deleted CD1d molecules, yet it remained dependent on prosaposin expression.…”

“…To further dissect the role of saposins in lipid-loading in the secretory versus the endolysosomal compartments, we expressed in prosaposin-sufficient and -deficient THP-1 cells CD1d molecules lacking the cytoplasmic motif known to be important for its internalization and endo-lysosomal targeting (TD-CD1d) (30). By confocal microscopy we observed that tail-deleted CD1d molecules could still reach the lysosomal Lamp-1 + compartment (Fig.…”

“…Furthermore, the saposin-dependence of peroxisome-derived lipids (48) or of lysophospholipids (49), which stimulate thymic and peripheral iNKT cells, remains to be determined. It is also possible that in myeloid cells association of the TD-CD1d molecules with the invariant chain in the ER (30,44,45) may alter the kinetics of trafficking to the lysosomes, rendering these CD1d molecules more sensitive to the presence or absence of prosaposin. Our results underscore the important role for prosaposin and CD1d trafficking through the endolysosomal compartment for CD1d-restricted presentation of endogenous lipids by infected human APCs.…”

“…Conversely, although it is established that reactivity of murine iNKT cells to CD1d loaded with endogenous iNKT cell agonists requires trafficking of CD1d molecules through the lysosome (26)(27)(28)(29), the contribution of the endolysosomal environment to human iNKT cell autoreactivity upon infection is unclear. To address this question, we overexpressed in THP-1 cells CD1d molecules deleted of the last 10 amino acids of the cytoplasmic tail [TD-CD1d (30)]. Upon infection with L. monocytogenes, THP-1 cells expressing TD-CD1d elicited lower iNKT cell activation ( Fig.…”

The cellular and biochemical rules of lipid antigen presentation

“…Exogenous microbial antigens are loaded within LE/Ly, whereas endogenous self-lipids stimulating human iNKT cells are loaded in other compartments [50], probably within the ER. This may be the case of antigens, which stimulate non-invariant mouse NKT cells.…”