Involvement of Secretory and Endosomal Compartments in Presentation of an Exogenous Self-Glycolipid to Type II NKT Cells

Abstract

Natural Killer T (NKT) cells recognize both self and foreign lipid Ags presented by CD1 molecules. Although presentation of the marine sponge-derived lipid αGalCer to type I NKT cells has been well studied, little is known about self-glycolipid presentation to either type I or type II NKT cells. Here we have investigated presentation of the self-glycolipid sulfatide to a type II NKT cell that specifically recognizes a single species of sulfatide, namely lyso-sulfatide but not other sulfatides containing additional acyl chains. In comparison to other sulfatides or αGalCer, lyso-sulfatide binds with lower affinity to CD1d. Although plate-bound CD1d is inefficient in presenting lyso-sulfatide at neutral pH, it is efficiently presented at acidic pH and in the presence of saposin C. The lysosomal trafficking of mCD1d is required for αGalCer presentation to type I NKT cells, it is not important for presentation of lyso-sulfatide to type II NKT cells. Consistently, APCs deficient in a lysosomal lipid-transfer protein effectively present lyso-sulfatide. Presentation of lyso-sulfatide is inhibited in the presence of primaquine, concanamycin A, monensin, cycloheximide, and an inhibitor of microsomal triglyceride transfer protein but remains unchanged following treatment with brefeldin A. Wortmannin-mediated inhibition of lipid presentation indicates an important role for the PI-3kinase in mCD1d trafficking. Our data collectively suggest that weak CD1d-binding self-glycolipid ligands such as lyso-sulfatide can be presented via the secretory and endosomal compartments. Thus this study provides important insights into the exogenous self-glycolipid presentation to CD1d-restricted T cells.

Oligoclonality and innate-like features in the TCR repertoire of type II NKT cells reactive to a β-linked self-glycolipid

“…Two findings are consistent: (i) cells stained with tetramers loaded with an immunodominant synthetic cis-tetracosenoyl sulfatide also are predominantly Vβ8 + , Vα3 + , and Vβ3 + (Fig. S4); and (ii) antigen fine specificity of a sulfatide-reactive hybridoma, Hy19.3, showed that it is only strongly reactive to lyso-sulfatide (17,18,29) and utilizes the TCR Vα1-Jα26 and Vβ16-Jβ2.1 gene segments with similar CDR1α, CDR3α, and CDR2β sequences to the TCRs of sulfatide/CD1d-tetramer + cells (Fig. S5).…”

“…Consistent with this idea, a number of CD1d-reactive T cell hybrids isolated from MHC class II-deficient mice (15,16,28) are Vα3 + , Vα8 + , and Vα4 + and do not show any reactivity to sulfatide (17,18). Accordingly, none of them expressed the TCR Vα-Jα and Vβ-Jβ gene segments predominantly used by sulfatide/CD1d-tetramer + cells.…”

“…Earlier studies of non-Vα14, CD1d-reactive T cell hybrids in MHC class II-deficient mice revealed a predominant Vβ8 and Vα3.2/Vα8 gene segment usage with diverse CDR3 regions (16). We have identified a major subset of type II NKT cells reactive to the self-glycolipid 3′-sulfated galactosyl ceramide, sulfatide (17,18). The crystal structure of a sulfatide/murine CD1d complex shows how the β-linked galactose head group of sulfatide projects out and away from the binding pocket of CD1d, resulting in a conformational change associated with greater exposure of CD1d residues (19) compared with the conformation of an α-linked galactose in the αGalCer/CD1d complex.…”

The cellular and biochemical rules of lipid antigen presentation

“…Furthermore, sulphatide with a nervonoyl acyl chain forms stable complexes with human CD1a and CD1d and mouse CD1d and preferentially stimulates sulphatide-specific human T-cell clones [17] (and our unpublished results) and a mouse T-cell hybridoma [18]. Lysosulphatide can also form stimulatory complexes with CD1d and activate- specific T cells [19]. CD1d-lysosulphatide complexes are probably formed in a pre-lysosomal compartment and do not require egression from the ER of newly synthesized CD1d molecules [19].…”

“…Lysosulphatide can also form stimulatory complexes with CD1d and activate- specific T cells [19]. CD1d-lysosulphatide complexes are probably formed in a pre-lysosomal compartment and do not require egression from the ER of newly synthesized CD1d molecules [19]. Recently, lysophosphatidylcholine has been found to stimulate human CD1d-restricted type II NKT cells [20].…”