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Keywords:

  • Antigen processing/presentation;
  • CD1 molecules;
  • NKT cell;
  • Niemann-Pick type C disease

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

Invariant natural killer T (iNKT) cells are a specialised subset of T cells that are restricted to the MHC class I like molecule, CD1d. The ligands for iNKT cells are lipids, with the canonical superagonist being α-galactosylceramide, a non-mammalian glycosphingolipid. Trafficking of CD1d through the lysosome is required for the development of murine iNKT cells. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by dysfunction in either of two lysosomal proteins, NPC1 or NPC2, resulting in the storage of multiple lipids, including glycosphingolipids. In the NPC1 mouse model, iNKT cells are virtually undetectable, which is likely due to the inability of CD1d to be loaded with the selecting ligand due to defective lysosomal function and/or CD1d trafficking. However, in this study we have found that in NPC1 patients iNKT cells are present at normal frequencies, with no phenotypic or functional differences. In addi-tion, antigen-presenting cells derived from NPC1 patients are functionally competent to present several different CD1d/iNKT-cell ligands. This further supports the hypothesis that there are different trafficking requirements for the development of murine and human iNKT cells, and a functional lysosomal/late-endosomal compartment is not required for human iNKT-cell development.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

Invariant natural killer T (iNKT) cells are defined by their invariant T-cell receptor and restriction to the MHC class I like molecule, CD1d. iNKT cells express multiple markers associated with NK cells and have the ability to rapidly release both TH1 (e.g. IFN-γ) and TH2 (e.g. IL-4) cytokines after engagement, acting as a bridge between innate and adaptive immunity [1]. iNKT cells play important roles in host protection against pathogens, cancer and auto-immunity. iNKT cells are lipid-reactive, with the canonical superagonist being α-galactosylceramide (α-GalCer) a non-mammalian glycosphingolipid. Mammalian glycosphingolipids (GD3 and iGb3), mammalian phospholipids and pathogen-derived glycolipids (α-galactosyl diacylglycerol, α-glyucuronsyl ceramides) have also been shown to activate iNKT cells [2]. iNKT cells develop in the thymus, where they undergo a process of positive selection with double positive thymocytes presenting selecting ligand(s) on CD1d [3].

Rodents only have one member of the CD1 family, CD1d, whereas humans have five members, CD1a to CD1e [4], that have differential intracellular trafficking patterns [5]. Murine CD1d exhibits a broad intracellular trafficking pattern, transiting through early and late endosomes, and also the lysosome, which is necessary for successful thymic selection [6, 7]. In addition, functional lysosomes are required for the presentation of activating ligands to murine iNKT cells [8]. In contrast, lysosomal trafficking in human antigen presenting cells does not appear to be required to present self-derived iNKT-cell stimulating ligands but may be required for exogenous ligands [9].

Lysosomal storage disorders result from inherited defects in lysosomal proteins [10]. These disorders can be caused either by a primary defect in a catabolic enzyme (e.g. Tay-Sachs and Sandhoff disease) or a defect in a transporter, channel or regulatory protein (e.g. Niemann-Pick type C (NPC1) disease). Lysosomal storage caused by a deficient lysosomal enzyme has been shown to lead to reduced iNKT cells in murine models of Sandhoff disease [11, 12], Tay-Sachs disease [11], GM1 gangliosidosis [11-13] and Fabry disease [14, 15]. In the NPC1 mouse the numbers of iNKT cells also are greatly reduced but this is associated with impaired late-endosome/lysosome fusion in addition to the lysosomal lipid storage [11, 16]. NPC disease can be caused by mutations in one of two genes NPC1 or NPC2 [17]. Dysfunction of the NPC1 protein leads to decreased lysosomal calcium content which accounts for the failure of endocytic vesicle fusion and the complex pattern of lipid storage observed [18].

With the differential trafficking of murine and human CD1d for iNKT-cell ligand presentation ex vivo and the requirement of normal lysosomal CD1d trafficking/function for murine iNKT-cell development in vivo, we reasoned that examining iNKT cells in NPC patients would reveal whether the findings in the murine model extends to humans. It has been reported that iNKT cells are present at normal frequencies in the peripheral blood of Fabry disease patients [19] and are slightly increased in Gaucher disease patients [20]. Here, we have studied iNKT-cell frequencies and functional responses in NPC1 disease patients and the ability of patient-derived EBV-B-cell lines to stimulate iNKT cells. In contrast to the murine model of NPC1, we found unchanged iNKT-cell frequencies in NPC1 patients. In addition, the functional response of NPC1 iNKT cells to stimulation was normal, as was the ability of NPC1 antigen presenting cells to present a variety of iNKT cells ligands to control iNKT cells.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

Human NPC1 patients do not have an alteration in iNKT-cell number of phenotype

We analysed the frequency of iNKT cells in the peripheral blood of controls, NPC1 patients and NPC1 heterozygote carriers by flow cytometry (gating strategy, Supporting Information Fig. 1). As previously reported [21], the frequencies of iNKT cells are very low in normal human peripheral blood, typically in the range of 0.1–1% of total T cells (Fig. 1A). In contrast to the NPC1 mouse where iNKT cells are undetectable, iNKT cells could be identified and were present at normal frequencies in the peripheral blood of NPC1 patients and heterozygotes (Fig. 1A). This indicates that fusion of late endosomes and lysosomes is not required for the generation, delivery or loading of iNKT-cell selecting ligand(s) in the thymus or for their maintenance in the periphery. The percentage of iNKT cells expressing the NK cell marker, CD161, was determined and no difference between the groups was observed (Fig. 1B). In addition the CD4 and CD8 status of the iNKT cells was investigated and there was no difference between the groups (Fig. 1C).

image

Figure 1. Frequencies and phenotype of iNKT cells from NPC1 patients and controls. (A) iNKT-cell frequencies, expressed as the percent of viable T cells, were determined by CD1d-αGalCer tetramers and antibody (6B11) staining. For each sample at least one determination of frequency by tetramer staining and at least one by 6B11 antibody was performed and the average value is shown. Each symbol represents an individual donor, controls (n = 32), NPC1 heterozygotes (n = 9) and NPC1 patients (n = 30). (B) Percentage of CD161+ iNKT cells was determined only on samples that had greater than 500 iNKT cells (controls (n = 12), NPC1 heterozygotes (n = 4) and NPC1 patients (n = 15)). (C) Percentage of CD4+, CD8+ or CD4CD8 iNKT cells were determined on samples which had greater than 500 iNKT cells (controls (n = 12), NPC1 heterozygotes (n = 4) and NPC1 patients (n = 15)). Data shown are pooled across multiple different experiments for each individual donor. Gating strategy is shown in Supporting Information Fig. 1.

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Lysosomal storage does not cause increased surface CD1d expression

It has previously been reported that the expression of CD1d on peripheral blood monocytes is increased in Gaucher disease and this was suggested to be due to lysosomal glycosphingolipid storage [20]. We analysed the expression of CD1d on monocytes (CD14+) and B cells (CD19+) (gating strategy, Supporting Information Fig. 2) and found no differences between the groups (Fig. 2) suggesting that in NPC1 patients and heterozygote carriers there is no alteration in cell surface CD1d expression.

image

Figure 2. Cell surface expression of CD1d on blood monocytes and B cells from NPC1 patients, NPC1 heterozygotes and controls. (A) Intensity of CD1d staining on peripheral blood monocytes and (B) B cells was determined. Shown is the MFI of CD1d after gating on the appropriate cell subset. Each symbol represents an individual donor controls (n = 32), NPC1 heterozygotes (n = 12) and NPC1 patients (n = 30). Data shown are pooled across multiple different experiments for each individual donor. Gating strategy is shown in Supporting Information Fig. 2.

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NPC1 iNKT cells and antigen presenting cells are functionally competent

In order to test the function of iNKT cells derived from NPC1 patients we generated iNKT cells lines from three patients that were co-cultured with human CD1d expressing THP1 cells that had been pulsed with three different exogenous antigens or treated with the TLR 7/8 ligand R848 [22]. The response of the iNKT cells was determined by measuring IFN-γ, IL-4 and GM-CSF production in the supernatant. The three NPC1 iNKT-cell lines responded to both exogenous and endogenous ligands and produced comparable levels of the cytokines compared to a control iNKT-cell line (Fig. 3A).

image

Figure 3. Functional response of NPC1 iNKT-cell lines and NPC1 and NPC1 heterozygote-derived antigen presenting cells. (A) IFN-γ, IL-4 and GM-CSF produced by iNKT-cell lines derived from three different NPC1 patients in response to exogenous ligands and endogenous ligands (R848) presented by THP1 cells were determined by ELISA. (B) IFN-γ produced by a control iNKT-cell line in response to three different exogenous ligands (αGalCer at 50 ng/mL, Gal(α1-2)GalCer at 150 ng/mL and C20:2 at 15 ng/mL) presented by EBV-B-cell lines derived from NPC1 patients, NPC1 heterozygotes or control C1R cells transfected with human CD1d. (C) IFN-γ produced by a control iNKT-cell lines in response to three different exogenous ligands (αGalCer at 50 ng/mL, Gal(α1-2)GalCer at 150 ng/mL and C20:2 at 15 ng/mL) presented by EBV-B-cell lines derived from NPC1 patients and NPC1 heterozygotes transfected with mouse CD1d *p < 0.05 and **p < 0.01 as determined by a one way ANOVA (Tukey post-test). (D) Representative histograms of LysoTracker green staining on EBV-B-cell lines; cells were identified on the basis of size (FSC vs. SSC) and the MFI value is indicated from three independent experiments. (A to C) Data are shown as mean ± SEM and are representative of two experiments (n = 2 or 3).

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Finally, we investigated the ability of antigen presenting cells derived from NPC1 patients and heterozygotes to stimulate iNKT-cell lines by generating EBV transformed B-cell lines. Once established these B-cell lines down-regulated endogenous CD1d, and we therefore transduced them with a lentiviral human or mouse CD1d construct before use in antigen presentation assays. Expression of human or mouse CD1d was comparable between NPC1 and heterozygote EBV-B-cell lines but slightly lower than that of C1R, an EBV-B-cell line used as a control (Supporting Information Fig. 3). Using the intensity of LysoTracker green staining, which accumulates in acidic intracellular vesicles, as a measure of lysosomal storage, we confirmed that the enhanced lysosomal storage characteristic of NPC1 peripheral blood B cells [23] was retained in the NPC1 EBV-B-cell lines (Fig. 3D).

We used three different iNKT-cell ligands that have been reported to require different conditions for loading onto CD1d. αGalCer loading has been reported to require access to a functional lysosomal compartment [9], Gal(α1-2)GalCer requires cleavage of the terminal galactose residue by lysosomal α-galactosidase before it can stimulate iNKT cells [15] and C20:2 can be loaded at the cell surface [24]. We found all three iNKT-cell ligands could be presented by the NPC1 and heterozygote EBV-B-cell lines transduced with human CD1d and resulted in similar or greater iNKT-cell activation compared with control C1R cells as determined by IFN-γ in the supernatant (Fig. 3B). This may be due to the fact that the block of lysosomal human CD1d trafficking or function is not complete in these B-cell lines and/or that some α-galactosidase activity may reside in the late endosome. In contrast, when transduced with mouse CD1d presentation of αGalCer and C20:2 was not affected but the two NPC1 lines with the greatest lysosomal storage as defined by LysoTracker green staining (Fig. 3D) exhibited a significant defect in Gal(α1-2)GalCer presentation (Fig. 3C) compared with the other NPC1 EBV-B-cell line.

Concluding remarks

Our study demonstrates that lysosomal dysfunction does not alter iNKT-cell frequencies in the blood of NPC1 patients, implying that this compartment is not required for the generation or loading of iNKT-cell selecting ligand(s) in the human thymus. This is consistent with studies using in vitro models of human CD1d auto-antigen presentation [9], suggesting that loading occurs in the early endosomes. This is in contrast to the murine model of NPC1 in which peripheral iNKT cells are virtually undetectable, most likely because of impaired selection in the thymus [6, 7]. Antigen presentation by human B-cell lines generated from NPC1 patients and heterozygotes and transfected with human CD1d demonstrated normal presentation of three different exogenous antigens, particularly Gal(α1-2)GalCer [9], which needs the terminal sugar to be cleaved before it is recognised by iNKT cells [15], indicating that unimpaired lysosomal trafficking and/or function is not essential for human CD1d ligand loading. In contrast, and in agreement with the reported requirement for normal lysosomal trafficking/function for murine CD1d ligand loading, the two NPC1 B-cell lines that exhibited the greatest lysosomal storage had reduced capacity to stimulate iNKT cells when pulsed with Gal(α1-2)GalCer. The differences between the murine model of NPC1 and human patients may have wider implications for the validity of using mouse models to define human iNKT-cell selecting ligands.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

Human blood samples

Venous blood was collected in EDTA tubes and maintained at room temperature for a maximum of 60 h prior to cell separation. Control samples were obtained after informed consent/assent and ethical approval from centres in the United Kingdom or United States. NPC1 patient samples were obtained from patients from centres in the United Kingdom, United States, and Germany with informed consent or assent. Heterozygote samples were obtained with informed consent/assent from the parents of affected patients or known carrier siblings.

Cell preparation

Peripheral blood was loaded onto an equal volume of Histopaque 1077 (Sigma-Aldrich) and spun at 400 × g for 30 min at room temperature. The mononuclear cell layer was isolated and washed twice with Dulbecco's PBS (D-PBS), counted and viability determined using Trypan blue.

Cell staining

Antibodies were used according to the manufacturers instructions and the following clones were used CD14 allophycocyanin-H7 (MΦP9), CD1d PE (CD1d42), CD19 PE-Cy™7 (SJ25C1), CD161 allophycocyanin (DX12), CD3 Pacific Blue™ (UCHT1), CD4 allophycocyanin-H7 (SK3), CD8α PerCP-Cy™5.5 (SK1) Invariant NK T-cell PE (6B11) and CD45 FITC (2D1) (all from BD Biosciences). Mononuclear cells (1 million) were stained with antibody cocktails diluted in FACS buffer (BD CellWASH™ supplemented with 2% fetal bovine serum and 5% mouse serum) on ice for 30 min. For iNKT-cell identification 3–5 million mononuclear cells were stained. Human CD1d tetramers loaded with αGalCer were prepared as previously described [25], with staining performed according to standard protocols with the inclusion of a viability stain (Live/Dead® fixable aqua dye Invitrogen). Cells were washed twice with FACS buffer before resuspension in BD FACS™ lysing solution. Isotype control antibodies in combination with fluorescence minus one staining protocols were used to establish gating.

Flow cytometry

Samples were acquired on a three laser BD FACS Canto™ II flow cytometer using BD FACSDiva™ software version 6.1 collecting a minimum of 10 000 gated B cells or monocytes for CD1d expression or a minimum of 1 000 000 viable lymphocytes for iNKT-cell frequencies. Data were analysed using FlowJo software v8.6 (TreeStar Inc).

Preparation of iNKT-cell lines

B-cell depleted mononuclear cells (B cells were collected for biochemical monitoring as part of a biomarker study) were washed three times in complete medium (RPMI) 1640 containing 5% human AB serum (Sigma-Aldrich, Poole, UK), 10 μM beta-mercaptoethanol, 20 μg/mL gentamycin, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 2 mM glutamine (all media from Invitrogen Paisley, UK)). The cells were counted and plated at 3–5 million cells in 2 mL complete medium with 100 ng/mL αGalCer. On day 4 recombinant human IL-2 was added at 20 U/mL (Peprotech EC, London, UK) and on day 6 IL-2 was increased to 500 U/mL. Cells were checked everyday and split as required with fresh medium containing 500 U/mL IL-2 added every 2 days. Once expanded iNKT-cell frequency was checked by FACS using human CD1d/αGalCer tetramers and anti-CD3 as described above. When sufficient numbers of iNKT cells were present in the cultures iNKT cells were sorted by staining with anti-CD3 antibody and CD1d/αGalCer tetramers using a MoFlo sorter (Dako Cytomation). The purified iNKT cells were then re-stimulated and expanded with 1 μg/mL PHA-P (Sigma-Aldrich) and irradiated feeder cells according to standard procedures. Purity of the cell lines was confirmed by FACS staining before use in stimulation assays and was greater than 98% tetramer positive. NPC1 genotypes of the donors used for the generation of the lines are line A not found, line B 3182T>C and 3562G>T, and line C I1061T, I1094T.

Generation of EBV transformed B-cell lines

Total blood lymphocytes (1–3 million cells) were washed twice with RPMI 1640 medium with 20 μg/mL gentamycin and then resuspended in 1 mL EBV containing supernatant in a T25 flask. After 24 h 9 mL of RPMI 1640 containing 15% fetal bovine serum (Biosera UK), 2 mM glutamine and 2 μg/mL cyclosporin A (New England Biolabs) was added and the cells were passaged as required once the transformed B cells started to grow out. As transformed B cells down-regulate surface CD1d, the cells were transduced with a lentiviral construct encoding cyan fluorescent protein tagged to human or untagged mouse CD1d. After transduction, CD1d expression and lysosomal storage (using the fluorescent dye LysoTracker® green DND-26 (Invitrogen), 200 nM in D-PBS for 10 min at room temperature) was assessed by FACS staining and EBV-B-cell lines were sorted for CD1d positive cells using a MoFlo sorter. NPC1 genotypes of the donors used for the generation of the lines are NPC1 1920delG, IVS9-1009G>A and data unavailable and for NPC1 heterozygote 1920delG and data unavailable.

iNKT-cell stimulation

NPC1 patient-derived iNKT-cell lines were used at least 14 days after re-stimulation. Antigen presenting cells (human CD1d cherry lentiviral transfected THP1 cells) were left untreated, pulsed with αGalCer (100 ng/mL), Gal(α1-2)GalCer (150 ng/mL) or C20:2 (15 ng/mL) or matured with the Toll like receptor 7/8 agonist R848 (5 μg/mL Invivogen). THP1 cells were co-cultured with iNKT cells at a 2:1 THP1 to iNKT-cell ratio in 96 U bottom wells and supernatant was harvested after 36 h. IFN-γ (MabTech), IL-4 (BD Pharmingen) and GM-CSF (eBioscience) levels in the supernatant were measured by ELISA according to manufacturers protocols.

Antigen presentation function

NPC1 patient or NPC1 heterozygote human or mouse CD1d lentiviral transduced EBV transformed B-cell lines were left untreated or pulsed with αGalCer (50 ng/mL), Gal(α1-2)GalCer (150 ng/mL) or C20:2 (15 ng/mL) before being used as antigen presenting cells in iNKT-cell stimulation assays as described above using iNKT cells prepared from a healthy donor. As we were unable to transduce control blood due to the donors working within the department the control B-cell line C1R was transfected with human CD1d cyan fluorescent protein and used.

Statistical analysis

Statistical significance was tested by a one-way ANOVA with a Tukey post-test using Prism v4 (GraphPad Software Inc, La Jolla, CA, USA) with *p < 0.05 and **p < 0.01 considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

A.O.S. was funded by the MRC (G0700851), N.P. is funded by the MRC (G0800158), D.t.V. by Action Medical Research (SP4023) and Niemann-Pick Disease Group UK and D.A.S. by SOAR-NPC. M.S. is supported by Cancer Research UK (grant C399/A2291 to V.C.). This work was supported in part by the intramural research program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development and a Bench to Bedside grant from the Office of Rare Diseases (F.D.P.). N.M.Y. was supported by APMRF and DART.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information
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Abbreviations
iNKT cell

invariant natural killer T cell

NPC

Niemann-Pick type C disease

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

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eji2289-sup-0001-s1.pdf622K

Figure S1. Gating Doublets were excluded by FSC-H versus FSC-A and lymphocytes identified by size and granularity (FSC-A versus SSC-A). Viable lymphocytes were selected on the basis of exclusion of live/dead aqua stain. Total T cells were identified as CD3+ viable lymphocytes and iNKT cells as either 6B11+CD3+ or tetramer+CD3+ cells. Fluorescence minus one controls with isotype control antibodies were used to establish gating for iNKT cells and CD161, CD4 and CD8.

Figure S2. Gating strategy for identification of B cells and monocytes. Doublets were excluded by FSC-H versus FSC-A and viable cells were selected on the basis of exclusion of live/dead aqua. B cells were identified as CD19+SSClow and monocytes as CD14+SSCmid. CD1d expression was determined by MFI of the PE channel and specificity determined by fluorescence minus one with isotype control antibody controls.

Figure S3. Example of human CD1d expression on EBV-B cells after transfection. EBV-B cells were identified on the basis of size (FSC vs SSC) and dead cell excluded with the use of a viability dye, human CD1d was detected at the cell surface with a fluorescent antibody.

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