Cutting edge: V alpha 14-J alpha 281 NKT cells naturally regulate experimental autoimmune encephalomyelitis in nonobese diabetic mice

Although deficiencies in the NKT cell population have been observed in multiple sclerosis and mouse strains susceptible to experimental autoimmune encephalomyelitis (EAE), little is known about the function of these cells in CNS autoimmunity. In this work we report that TCR Valpha14-Jalpha281 transgenic nonobese diabetic mice, which are enriched in CD1d-restricted NKT cells, are protected from EAE. The protection is associated with a striking inhibition of Ag-specific IFN-gamma production in the spleen, implying modulation of the encephalitogenic Th1 response. This modulation is independent of IL-4 because IL-4-deficient Valpha14-Jalpha281 mice are still protected against EAE and independent of NKT cell-driven Th1 to Th2 deviation, because no increased autoantigen-specific Th2 response was observed in immunized Valpha14-Jalpha281 transgenic mice. Our findings indicate that enrichment and/or stimulation of CD1d-dependent NKT cells may be used as a novel strategy to treat CNS autoimmunity.     

Invariant V alpha 14+ NKT cells participate in the early response to enteric Listeria monocytogenes infection

Invariant Valpha14(+) NKT cells are a specialized CD1-reactive T cell subset implicated in innate and adaptive immunity. We assessed whether Valpha14(+) NKT cells participated in the immune response against enteric Listeria monocytogenes infection in vivo. Using CD1d tetramers loaded with the synthetic lipid alpha-galactosylceramide (CD1d/alphaGC), we found that splenic and hepatic Valpha14(+) NKT cells in C57BL/6 mice were early producers of IFN-gamma (but not IL-4) after L. monocytogenes infection. Adoptive transfer of Valpha14(+) NKT cells derived from TCRalpha degrees Valpha14-Jalpha18 transgenic (TCRalpha degrees Valpha14Tg) mice into alymphoid Rag(null) gamma(c)(null) mice demonstrated that Valpha14(+) NKT cells were capable of providing early protection against enteric L. monocytogenes infection with systemic production of IFN-gamma and reduction of the bacterial burden in the liver and spleen. Rechallenge experiments demonstrated that previously immunized wild-type and Jalpha18null mice, but not TCRalpha(null) or TCRalpha(null) Valpha14Tg mice, were able to mount adaptive responses to L. monocytogenes. These data demonstrate that Valpha14(+) NKT cells are able to participate in the early response against enteric L. monocytogenes through amplification of IFN-gamma production, but are not essential for, nor capable of, mediating memory responses required to sterilize the host.     

A novel mouse model for invariant NKT cell study

We have generated a novel mouse model harboring the in-frame rearranged TCRValpha specific for invariant NKT (iNKT) cells (Valpha14-Jalpha18) on one allele by crossing the mouse cloned from NKT cells with wild-type mice. This genomic configuration would ensure further rearrangement and expression of TCRValpha14-Jalpha18 under the endogenous promoters and enhancers. Mice harboring such an in-frame rearranged TCRValpha (Valpha14-Jalpha18 mouse) possessed an increase in iNKT cells in the thymus, liver, spleen, and bone marrow. Intriguingly, both Th1- and Th2-type cytokines were produced upon stimulation with alphaGalactosylceramide, an agonist of iNKT cells, and the IgE level in the serum remained unaffected in the Valpha14-Jalpha18 mouse. These features markedly distinguish the nature of iNKT cells present in the Valpha14-Jalpha18 mouse from that of iNKT cells found in the Valpha14-Jalpha18 transgenic mouse. Besides these, the expression of TCRVgammadelta cells remained intact, and the use of the TCRVbeta repertoire in iNKT cells was highly biased to TCRVbeta8 in the Valpha14-Jalpha18 mouse. Furthermore, alphaGalactosylceramide-CD1d dimer-reactive immature iNKT cells expressed less Rag2 as compared with the conventional immature T cells at the positive selection stage. Cell cycle analysis on the thymocytes revealed that no particular subset proliferated more vigorously than the others. Crossing the Valpha14-Jalpha18 mouse with the CD1d knockout mouse revealed a novel population of iNKT cells whose coreceptor expression profile was similar to that assigned to iNKT precursor cells. These mice will be useful for the study on the development of iNKT cells as well as on their functions in the immune system.     

Cutting edge: influence of the TCR Vbeta domain on the selection of semi-invariant NKT cells by endogenous ligands

Invariant Valpha14 (Valpha14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a Valpha14-Jalpha18 rearrangement and expression of mostly Vbeta8.2 and Vbeta7. Whereas the TCR Vbeta domain influences the binding avidity of the Valpha14i TCR for CD1d-alpha-galactosylceramide complexes, with Vbeta8.2 conferring higher avidity binding than Vbeta7, a possible impact of the TCR Vbeta domain on Valpha14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vbeta7(+), but not Vbeta8.2(+), Valpha14i NKT cells is favored in situations where endogenous ligand concentration or TCRalpha-chain avidity are suboptimal. Furthermore, thymic Vbeta7(+) Valpha14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vbeta domain influences the selection of Valpha14i NKT cells by endogenous ligands, presumably because Vbeta7 confers higher avidity binding.     

Targeted expression of human CD1d in transgenic mice reveals independent roles for thymocytes and thymic APCs in positive and negative selection of Valpha14i NKT cells

CD1d-dependent invariant Valpha14 (Valpha14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Valpha14-Jalpha18 TCR alpha-chain paired mostly with Vbeta8.2 and Vbeta7. The cellular requirements for thymic positive and negative selection of Valpha14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Valpha14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Valpha14i NKT cells that proved functional when activated ex vivo with the Ag alpha-galactosyl ceramide. Valpha14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vbeta8.2. The low number of Vbeta8.2+ Valpha14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vbeta7+ Valpha14i NKT cells. Vbeta8.2+, but not Vbeta7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Valpha14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.     

Prevention of diabetes in nonobese diabetic mice mediated by CD1d-restricted nonclassical NKT cells

A role for regulatory lymphocytes has been demonstrated in the pathogenesis of type 1 diabetes in the NOD mouse but the nature of these cells is debated. CD1d-restricted NKT lymphocytes have been implicated in this process. Previous reports of reduced diabetes incidence in NOD mice in which the numbers of NKT cells are artificially increased have been attributed to the enhanced production of IL-4 by these cells and a role for classical NKT cells, using the Valpha14-Jalpha18 rearrangement. We now show that overexpression in NOD mice of CD1d-restricted TCR Valpha3.2(+)Vbeta9(+) NKT cells producing high levels of IFN-gamma but low amounts of IL-4 leads to prevention of type 1 diabetes, demonstrating a role for nonclassical CD1d-restricted NKT cells in the regulation of autoimmune diabetes.     

Repeated alpha-galactosylceramide administration results in expansion of NK T cells and alleviates inflammatory dermatitis in MRL-lpr/lpr mice

NK T (NKT) cells expressing the invariant Valpha14-Jalpha18 TCR alpha-chain recognize glycolipid Ags such as alpha-galactosylceramide (alpha-GalCer) presented by the MHC class I-like molecule CD1d. Upon activation by alpha-GalCer, invariant NKT cells secrete multiple cytokines and confer protection in certain immune-mediated disorders. Here we have investigated the role of NKT cells in the development of inflammatory dermatitis in MRL-lpr/lpr mice, which shares features with lupus in humans. Our results show that the numbers Sand functions of NKT (TCRbeta(+)CD1d/alpha-GalCer tetramer(+)) cells, particularly of the NK1.1(-) subset, are reduced in MRL-lpr/lpr mice compared with MRL-fas/fas and/or nonautoimmune C3H/Hej and BALB/c mice. Repeated treatments with alpha-GalCer result in the expansion of NKT cells and alleviate dermatitis in MRL-lpr/lpr mice. Our results indicate that NKT cell deficiency can be corrected by repeated alpha-GalCer treatment and that NKT cells may play a protective role in inflammatory dermatitis of lupus-prone mice.     

NKT cells play a limited role in the neutrophilic inflammatory responses and host defense to pulmonary infection with Pseudomonas aeruginosa

CD1d-restricted NKT cells are reported to play a critical role in the host defense to pulmonary infection with Pseudomonas aeruginosa. However, the contribution of a major subset expressing a Valpha14-Jalpha18 gene segment remains unclear. In the present study, we re-evaluated the role of NKT cells in the neutrophilic inflammatory responses and host defense to this infection using mice genetically lacking Jalpha18 or CD1d (Jalpha18KO or CD1dKO mice). These mice cleared the bacteria in lungs at a comparable level to wild-type (WT) mice. There was no significant difference in the local neutrophilic responses, as shown by neutrophil counts and synthesis of MIP-2 and TNF-alpha, in either KO mice from those in WT mice. Administration of alpha-galactosylceramide, a specific activator of Valpha14+ NKT cells, failed to promote the bacterial clearance and neutrophilic responses, although the same treatment increased the synthesis of IFN-gamma, suggesting the involvement of this cytokine downstream of NKT cells. In agreement against this notion, these responses were not further enhanced by administration of recombinant IFN-gamma in the infected Jalpha18KO mice. Our data indicate that NKT cells play a limited role in the development of neutrophilic inflammatory responses and host defense to pulmonary infection with P. aeruginosa.     

Monocyte chemoattractant protein-1-dependent increase of V alpha 14 NKT cells in lungs and their roles in Th1 response and host defense in cryptococcal infection.

To elucidate the role of NKT cells in the host defense to cryptococcal infection, we examined the proportion of these cells, identified by the expression of CD3 and NK1.1, in lungs after intratracheal infection with Cryptococcus neoformans. This population increased on day 3 after infection, reached a peak level on days 6-7, and decreased thereafter. In Valpha14 NKT cell-deficient mice, such increase was significantly attenuated. The proportion of Valpha14 NKT cells, detected by binding to alpha-galactosylceramide-loaded CD1d tetramer, and the expression of Valpha14 mRNA increased after infection with a similar kinetics. The delayed-type hypersensitivity response and differentiation of the fungus-specific Th1 cells was reduced in Valpha14 NKT cell-deficient mice, compared with control mice. Additionally, elimination of this fungal pathogen from lungs was significantly delayed in Valpha14 NKT cell-deficient mice. Production of monocyte chemoattractant protein (MCP)-1 in lungs, detected at both mRNA and protein levels, increased on day 1, reached a peak level on day 3, and decreased thereafter, which preceded the increase in NKT cells. Finally, the increase of total and Valpha14(+) subset of NKT cells after infection was significantly reduced in MCP-1-deficient mice. Our results demonstrated that NKT cells, especially Valpha14(+) subset, accumulated in a MCP-1-dependent manner in the lungs after infection with C. neoformans and played an important role in the development of Th1 response and host resistance to this fungal pathogen.     

Cutting edge: influence of the TCR V beta domain on the avidity of CD1d:alpha-galactosylceramide binding by invariant V alpha 14 NKT cells

CD1d tetramers loaded with alpha-galactosylceramide (alpha-GalCer) bind selectively to mouse invariant Valpha14 (Valpha14i) NKT cells and their human counterparts. Whereas tetramer binding strictly depends on the expression of a Valpha14-Jalpha18 chain in murine NKT cells, the associated beta-chain (typically expressing Vbeta8.2 or Vbeta7) appears not to influence tetramer binding. In this study, we describe novel alpha-GalCer-loaded mouse and human CD1d-IgG1 dimers, which revealed an unexpected influence of the TCR-beta chain on the avidity of CD1d:alpha-GalCer binding. A subset of Valpha14i NKT cells clearly discriminated alpha-GalCer bound to mouse or human CD1d on the basis of avidity differences conferred by the Vbeta domain of the TCR-beta chain, with Vbeta8.2 conferring higher avidity binding than Vbeta7.     

Glycolipids with nonreducing end alpha-mannosyl residues that have the potential to activate invariant Valpha19 NKT cells

We have previously demonstrated that alpha-mannosyl ceramide and its derivatives promote immune responses of NK1.1(+) invariant Valpha19-Jalpha33 T cell receptor (TCR) alpha(+) T cells (Valpha19 NKT cells). In this study, attempts were made to determine the structural requirements for natural ligands for Valpha19 NKT cells. Naturally occurring and synthetic glycolipids were analyzed for their ability to stimulate the cells prepared from invariant Valpha19-Jalpha33 TCR transgenic mice, in which development of Valpha19 NKT cells is facilitated. As a result, alpha-mannosyl phosphatidylinositols such as 2,6-di-alpha-mannosyl phosphatidylinositol and alpha-mannosyl-4alpha-glucosaminyl-6-phosphatidylinositol (alpha-Man-GlcNH(2)-PtdIns) as well as alpha-mannosyl ceramide derivatives were found to activate the cells from the transgenic mouse liver, gut lamina propria and spleen in vivo and in vitro. Thus, glycolipids with nonreducing end alpha-mannosyl residues are suggested to be potent antigens for Valpha19 NKT cells. Next, a series of invariant Valpha19-Jalpha33 TCR(+) hybridomas, each with variations in the sequence of the Valpha-Jalpha junction and the TCR beta chain, were tested for responsiveness toward the alpha-mannosyl glycolipids. A loose correlation between the primary structure of the TCR and the reactive glycolipids was observed. For instance, hybridomas expressing TCRs consisting of an alpha chain with a variation in the Valpha19-Jalpha33 junction and a Vbeta6(+)beta chain showed affinity towards alpha-mannosyl ceramide and alpha-Man-GlcNH(2)-PtdIns, whereas those expressing TCRs with an invariant Valpha19-Jalpha33 alpha chain and a Vbeta8(+)beta chain responded to 2,6-di-alpha-mannosyl phosphatidylinositol. Thus, it is suggested that Valpha19 NKT cells with microheterogeneity in the TCR structure have been generated for defense against various antigens expressing alpha-mannosyl glycolipids.     

Niemann-Pick C1 functions in regulating lysosomal amine content

Mutations in the late endosomal/lysosomal membrane protein Niemann-Pick C1 (NPC1) are known to cause a generalized block in retrograde vesicle-mediated transport, resulting in the hyper-accumulation of multiple lysosomal cargos. An important, yet often overlooked, category of lysosomal cargo includes the vast array of small molecular weight amine-containing molecules that are substrates for ion trapping in the highly acidic organelle lumen. We show here that the introduction of amine-containing molecules in lysosomes can significantly stimulate NPC1-mediated late endosome/lysosome fusion, and subsequently the secretion of lysosomal cargo. To illustrate the physiological importance of this NPC1-mediated transport pathway, we show that NPC1-deficient cells are more susceptible to the toxic effects of a lysosomotropic polyamine metabolite 3-aminopropanal. Moreover, NPC fibroblasts are shown to have higher levels of polyamine oxidase, an enzyme involved in the formation of 3-aminopropanal. Collectively, these findings provide strong support for a novel functional role for NPC1 and may also provide clues toward understanding NPC disease progression.     

DOCK2 is required in T cell precursors for development of Valpha14 NK T cells

Mouse CD1d-restricted Valpha14 NKT cells are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance and host defense against pathogens. DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster myoblast city, is critical for lymphocyte migration and regulates T cell responsiveness through immunological synapse formation, yet its role in Valpha14 NKT cells remains unknown. We found that DOCK2 deficiency causes marked reduction of Valpha14 NKT cells in the thymus, liver, and spleen. When alpha-galactosylceramide (alpha-GalCer), a ligand for Valpha14 NKT cells, was administrated, cytokine production was scarcely detected in DOCK2-deficient mice, suggesting that DOCK2 deficiency primarily affects generation of Valpha14 NKT cells. Supporting this idea, staining with CD1d/alpha-GalCer tetramers revealed that CD44- NK1.1- Valpha14 NKT cell precursors are severely reduced in the thymuses of DOCK2-deficient mice. In addition, studies using bone marrow chimeras indicated that development of Valpha14 NKT cells requires DOCK2 expression in T cell precursors, but not in APCs. These results indicate that DOCK2 is required for positive selection of Valpha14 NKT cells in a cell-autonomous manner, thereby suggesting that avidity-based selection also governs development of this unique subset of lymphocytes in the thymus.     

Non-reducing end alpha-mannosylated glycolipids as potent activators for invariant Valpha19 TCR-bearing natural killer T cells

A novel invariant Valpha19-Jalpha33 T cell receptor alpha chain, first found in mammalian blood cells, was primarily expressed by natural killer T cell repertoire (Valpha19 NKT cell). Attempts have been made to find specific antigens for Valpha19 NKT cells. A series of alpha- and beta-glycosyl ceramides were synthesized and tested whether they had potential to stimulate the cells isolated from invariant Valpha19-Jalpha33 TCR transgenic mice (where the development of Valpha19 NKT cells is facilitated). Comprehensive examinations revealed substantial antigenic activity in alpha-ManCer that was presented by MR1, one of the MHC class Ib molecules. Next, naturally occurring and synthetic alpha-mannosyl glycolipids were further analyzed to determine structural requirements for natural ligands for Valpha19 NKT cells. As a result, alpha-mannosyl phosphatidyl inositols (PI) such as (alpha-Man)(2)-PI and alpha-Man-alpha-GlcNH(2)-PI (a partial structure of mycobacterial lipoarabinomannan and GPI-anchors) as well as alpha-ManCer derivatives were found to activate Valpha19 NKT cells in vivo and in vitro. Thus, Valpha19 NKT cells are possibly responsive to certain alpha-mannosyl glycolipids and may have roles in the innate and adaptative immune systems to protect against various antigens expressing alpha-mannosyl glycolipids and to regulate the adaptive immune system responding to the intracellular ligands.     

The novel lipid raft adaptor p18 controls endosome dynamics by anchoring the MEK–ERK pathway to late endosomes

The regulation of endosome dynamics is crucial for fundamental cellular functions, such as nutrient intake/digestion, membrane protein cycling, cell migration and intracellular signalling. Here, we show that a novel lipid raft adaptor protein, p18, is involved in controlling endosome dynamics by anchoring the MEK1–ERK pathway to late endosomes. p18 is anchored to lipid rafts of late endosomes through its N‐terminal unique region. p18^?/? mice are embryonic lethal and have severe defects in endosome/lysosome organization and membrane protein transport in the visceral endoderm. p18^?/? cells exhibit apparent defects in endosome dynamics through perinuclear compartment, such as aberrant distribution and/or processing of lysosomes and impaired cycling of Rab11‐positive recycling endosomes. p18 specifically binds to the p14–MP1 complex, a scaffold for MEK1. Loss of p18 function excludes the p14–MP1 complex from late endosomes, resulting in a downregulation of the MEK–ERK activity. These results indicate that the lipid raft adaptor p18 is essential for anchoring the MEK–ERK pathway to late endosomes, and shed new light on a role of endosomal MEK–ERK pathway in controlling endosome dynamics.     

Differential regulation of Th1 and Th2 functions of NKT cells by CD28 and CD40 costimulatory pathways

Valpha14 NKT cells produce large amounts of IFN-gamma and IL-4 upon recognition of their specific ligand alpha-galactosylceramide (alpha-GalCer) by their invariant TCR. We show here that NKT cells constitutively express CD28, and that blockade of CD28-CD80/CD86 interactions by anti-CD80 and anti-CD86 mAbs inhibits the alpha-GalCer-induced IFN-gamma and IL-4 production by splenic Valpha14 NKT cells. On the other, the blockade of CD40-CD154 interactions by anti-CD154 mAb inhibited alpha-GalCer-induced IFN-gamma production, but not IL-4 production. Consistent with these findings, CD28-deficient mice showed impaired IFN-gamma and IL-4 production in response to alpha-GalCer stimulation in vitro and in vivo, whereas production of IFN-gamma but not IL-4 was impaired in CD40-deficient mice. Moreover, alpha-GalCer-induced Th1-type responses, represented by enhanced cytotoxic activity of splenic or hepatic mononuclear cells and antimetastatic effect, were impaired in both CD28-deficient mice and CD40-deficient mice. In contrast, alpha-GalCer-induced Th2-type responses, represented by serum IgE and IgG1 elevation, were impaired in the absence of the CD28 costimulatory pathway but not in the absence of the CD40 costimulatory pathway. These results indicate that CD28-CD80/CD86 and CD40-CD154 costimulatory pathways differentially contribute to the regulation of Th1 and Th2 functions of Valpha14 NKT cells in vivo.     

Distinct endosomal compartments in early trafficking of low density lipoprotein-derived cholesterol

We previously studied the early trafficking of low density lipoprotein (LDL)-derived cholesterol in mutant Chinese hamster ovary cells defective in Niemann-Pick type C1 (NPC1) using cyclodextrin (CD) to monitor the arrival of cholesterol from the cell interior to the plasma membrane (PM) (Cruz, J. C., Sugii, S., Yu, C., and Chang, T.-Y. (2000) J. Biol. Chem. 275, 4013-4021). We found that newly hydrolyzed cholesterol derived from LDL first appears in certain CD-accessible pool(s), which we assumed to be the PM, before accumulating in the late endosome/lysosome, where NPC1 resides. To determine the identity of the early CD-accessible pool(s), in this study, we performed additional experiments, including the use of revised CD incubation protocols. We found that prolonged incubation with CD (>30 min) caused cholesterol in internal membrane compartment(s) to redistribute to the PM, where it became accessible to CD. In contrast, a short incubation with CD (5-10 min) did not cause such an effect. We also show that one of the early compartments contains acid lipase (AL), the enzyme required for liberating cholesterol from cholesteryl ester in LDL. Biochemical and microscopic evidence indicates that most of the AL is present in endocytic compartment(s) distinct from the late endosome/lysosome. Our results suggest that cholesterol is liberated from LDL cholesteryl ester in the hydrolytic compartment containing AL and then moves to the NPC1-containing late endosome/lysosome before reaching the PM or the endoplasmic reticulum.     

CD1d-specific NK1.1+ T cells with a transgenic variant TCR

The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Valpha14-Jalpha281 rearrangement and a limited set of TCR Vbeta segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Valpha3. 2/Vbeta9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-gamma, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the "non-Valpha 14" diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics.     

Impaired dynamics of the late endosome/lysosome compartment in human Niemann-Pick type C skin fibroblasts carrying mutation in NPC1 gene

The Niemann-Pick type C (NPC) disease is characterized by accumulation of lipids within the late endosome/lysosome (LE/LY) compartment as a result of dysfunctions of the NPC1 or NPC2 proteins and an altered distribution and/or functioning of proteins involved in the regulation of membrane dynamics. In our previous report we isolated membranes of the LE/LY compartment from NPC L1 skin fibroblasts with a mutation in the NPC1 gene (exon 8, R348X) and showed that annexin A6 (AnxA6) may contribute to the impaired dynamics of these membranes in a cholesterol-dependent manner and therefore to the overnormative storage of cholesterol. In this report we show that the LE/LY fraction isolated from NPC L1 cells is characterized by a 4-fold enrichment in cholesterol, 2.5-fold in sphingomyelin and 2-fold in saturated fatty acids. As a result, the fluidity of LE/LY membranes isolated from NPC L1 cells is greatly reduced in comparison to control ones. We conclude that modified lipid composition and properties of this compartment may affect distribution and function of proteins implicated in cellular membrane dynamics. As a consequence, the backward vesicular transport of cholesterol from the LE/LY compartment to the Golgi apparatus, endoplasmic reticulum and finally to plasma membrane is impaired.