Nicolas Schrantz

Lysosomal Glycosphingolipid Recognition by NKT Cells

NKT cells represent a distinct lineage of T cells that coexpress a conserved αβ T cell receptor (TCR) and natural killer (NK) receptors. Although the TCR of NKT cells is characteristically autoreactive to CD1d, a lipid-presenting molecule, endogenous ligands for these cells have not been identified. We show that a lysosomal glycosphingolipid of previously unknown function, isoglobotrihexosylceramide (iGb3), is recognized both by mouse and human NKT cells. Impaired generation of lysosomal iGb3 in mice lacking β-hexosaminidase b results in severe NKT cell deficiency, suggesting that this lipid also mediates development of NKT cells in the mouse. We suggest that expression of iGb3 in peripheral tissues may be involved in controlling NKT cell responses to infections and malignancy and in autoimmunity.

The Niemann-Pick type C2 protein loads isoglobotrihexosylceramide onto CD1d molecules and contributes to the thymic selection of NKT cells

The Niemann-Pick type C2 (NPC2) protein is a small, soluble, lysosomal protein important for cholesterol and sphingolipid transport in the lysosome. The immunological phenotype of NPC2-deficient mice was limited to an impaired thymic selection of Vα14 natural killer T cells (NKT cells) and a subsequent reduction of NKT cells in the periphery. The remaining NKT cells failed to produce measurable quantities of interferon-γ in vivo and in vitro after activation with α-galactosylceramide. In addition, thymocytes and splenocytes from NPC2-deficient mice were poor presenters of endogenous and exogenous lipids to CD1d-restricted Vα14 hybridoma cells. Importantly, we determined that similar to saposins, recombinant NPC2 was able to unload lipids from and load lipids into CD1d. This transfer activity was associated with a dimeric form of NPC2, suggesting a unique mechanism of glycosphingolipid transfer by NPC2. Similar to saposin B, NPC2 dimers were able to load isoglobotrihexosylceramide (iGb3), the natural selecting ligand of NKT cells in the thymus, into CD1d. These observations strongly suggested that the phenotype observed in NPC2-deficient animals was directly linked to the efficiency of the loading of iGb3 into CD1d molecules expressed by thymocytes. This conclusion was supported by the rescue of endogenous and exogenous iGb3 presentation by recombinant NPC2. Thus, the loading of endogenous and exogenous lipids and glycolipids onto CD1d is dependent on various small, soluble lipid transfer proteins present in the lysosome.

Cutting Edge: Impaired Glycosphingolipid Trafficking and NKT Cell Development in Mice Lacking Niemann-Pick Type C1 Protein

Niemann-Pick Type C1 (NPC1) is a late endosomal/lysosomal transmembrane protein involved in the cellular transport of glycosphingolipids and cholesterol that is mutated in a majority of patients with Niemann-Pick C neurodegenerative disease. We found that NPC1-deficient mice lacked Vα14-Jα18 NKT cells, a major population of CD1d-restricted T cells that is conserved in humans. NPC1-deficient mice also exhibited marked defects in the presentation of Sphingomonas cell wall Ags to NKT cells and in bacterial clearance in vivo. A synthetic fluorescent α-glycosylceramide analog of the Sphingomonas Ag trafficked to the lysosome of wild-type cells but accumulated in the late endosome of NPC1-deficient cells. These findings reveal a blockade of lipid trafficking between endosome and lysosome as a consequence of NPC1 deficiency and suggest a common mechanism for the defects in lipid presentation and development of Vα14-Jα18 NKT cells.

B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt’s lymphoma cells. Whereas soluble anti-μ Ab triggers caspase-independent mitochondrial activation, cross-linked anti-μ Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt’s lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.

Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T‐cell ligand

GD3, a ganglioside expressed on melanoma, is the only tumour‐associated glycolipid described to date that can induce a CD1d‐restricted natural killer T (NKT)‐cell response. We analysed the fine specificity of GD3‐reactive NKT cells and discovered that immunization with GD3 induced two populations of GD3‐reactive NKT cells. One population was CD4+ CD8− and was specific for GD3; the other population was CD4− CD8− and cross‐reacted with GM3 in a CD1d‐restricted manner, but did not cross‐react with GM2, GD2, or lactosylceramide. This indicated that the T‐cell receptors reacting with GD3 recognize glucose‐galactose linked to at least one N‐acetyl‐neuraminic acid but will not accommodate a terminal N‐acetylgalactosamine. Immunization with GM2, GM3, GD2, or lactosylceramide did not induce an NKT‐cell response. Coimmunization of GM3‐loaded antigen‐presenting cells (APCs) with GD3‐loaded APCs suppressed the NKT‐cell response to GD3 in a CD1d‐restricted manner. This suppressive effect was specific for GM3 and was a local effect lasting 2–4 days. In vitro, GM3‐loaded APCs also suppressed the interleukin‐4 response, but not the interferon‐γ response, of NKT cells to α‐galactosylceramide. However, there was no effect on the T helper type 2 responses of conventional T cells. We found that this suppression was not mediated by soluble factors. We hypothesize that GM3 induces changes to the APC that lead to suppression of T helper type 2‐like NKT‐cell responses.

Fatty acid amide hydrolase shapes NKT cell responses by influencing the serum transport of lipid antigen in mice

The potent regulatory properties of NKT cells render this subset of lipid-specific T cells a promising target for immunotherapeutic interventions. The marine sponge glycolipid alpha-galactosylceramide (alphaGalCer) is the proto-typic NKT cell agonist, which elicits this function when bound to CD1d. However, our understanding of the in vivo properties of NKT cell agonists and the host factors that control their bioactivity remains very limited. In this report, we isolated the enzyme fatty acid amide hydrolase (FAAH) from mouse serum as an alphaGalCer-binding protein that modulates the induction of key effector functions of NKT cells in vivo. FAAH bound alphaGalCer in vivo and in vitro and was required for the efficient targeting of lipid antigens for CD1d presentation. Immunization of Faah-deficient mice with alphaGalCer resulted in a reduced systemic cytokine production, but enhanced expansion of splenic NKT cells. This distinct NKT response conferred a drastically increased adjuvant effect and strongly promoted protective CTL responses. Thus, our findings identify not only the presence of FAAH in normal mouse serum, but also its critical role in the tuning of immune responses to lipid antigens by orchestrating their transport and targeting for NKT cell activation. Our results suggest that the serum transport of lipid antigens directly shapes the quality of NKT cell responses, which could potentially be modulated in support of novel vaccination strategies.