BCR targeting of biotin-
-galactosylceramide leads to enhanced presentation on CD1d and requires transport of BCR to CD1d-containing endocytic compartments
Gillian A. Lang1,
Petr A. Illarionov2,
Aharona Glatman-Freedman3,
Gurdyal S. Besra2 and
Mark L. Lang1
1 Department of Microbiology and Immunology, 632W Borwell Building, Dartmouth Medical School, One Medical Center Drive, Lebanon, NH 03756, USA
2 School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
3 Department of Pediatrics, Children's Hospital at Montefiore, Albert Einstein College of Medicine, Bronx, NY 10461, USA
Correspondence to: M. L. Lang; E-mail: mark.l.lang{at}dartmouth.edu
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Abstract
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CD1d is a non-polymorphic MHC class I-related protein that binds and presents glycolipid antigens to T cell antigen receptors expressed by NK-like T (NKT) cells. CD1d-dependent immune responses play critical roles in infectious disease, autoimmunity, allergy and cancer. We tested the hypothesis that B cell antigen receptor (BCR) targeting of a biotin-modified version of the CD1d-binding antigen
-galactosylceramide (biotin-
-GalCer) results in enhanced murine CD1d-mediated presentation as compared with presentation of non-targeted biotin-
-GalCer. Presentation of BCR-targeted antigen to NKT cells was enhanced 100- to 1000-fold compared with non-targeted antigen. CD1d presentation of BCR-targeted antigen was observed after 4 h treatment, consistent with a requirement for endosomal trafficking. Furthermore, unlike non-targeted antigen, BCR-targeted antigen was not loaded directly onto cell-surface CD1d. Blocking BCR signaling with the Syk tyrosine kinase inhibitor piceatannol inhibited presentation of BCR-targeted antigen but not non-targeted antigen. Piceatannol blocked transport of the BCR to CD1d-containing endosomes, showing that intersection of BCR-targeted antigen with endosomes is required for enhanced mCD1d antigen presentation. Our data suggest that the BCR facilitates capture of low quantities of mCD1d antigens to enhance CD1d-dependent immune responses.
Keywords: antigen presentation, BCR, CD1d,
-galactosylceramide, MIIC
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Introduction
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The B cell antigen receptor (BCR) is a multi-chain immunoreceptor specialized for the capture of antigen complexes [reviewed in (1, 2)]. BCR targeting of protein antigens results in a 100- to 1000-fold enhancement of class II antigen presentation as compared with pinocytically acquired antigen (3, 4). Presentation of CD1d-binding glycolipid antigens has been studied in different antigen-presenting cells (APCs) [reviewed in (5, 6)]. However, to date, there have been no reported studies on BCR-mediated uptake and presentation of CD1d antigens. The ability to stimulate production of anti-glycolipid antibodies in mice and other species demonstrates that BCR specificity for glycolipids including CD1d antigens exists in vivo (710). Although the mechanism is unclear, it is evident that CD1d presentation of glycolipid antigens can affect antibody production (1114).
In humans, there are five classes of CD1 protein, comprising group I CD1 (CD1a, b, c), group II CD1 (CD1d) and CD1e which may represent a third group of CD1 molecules (6). In mice, there is a single class of CD1 molecule (mCD1d) which is homologous to the human isoform CD1d (6). The mCD1d molecule is referred to as CD1d hereafter. CD1d proteins are ß2-microglobulin-associated class I-related molecules that present non-protein glycolipid antigens to V
14/J
28-arranged, mCD1d-restricted NK-like T (NKT) cells (1518). CD1d/antigen activation of NKT cells results in the production of IL-4 or IFN
, suggesting the capacity for both Th1 and Th2 responses to CD1d-binding antigens (1518). Human and murine CD1d present galactosylceramides, the ganglioside GD3, mycobacterial phosphatidylinositol mannoside (PIM) and hydrophobic peptides (1921). Human and murine CD1d bind different self-antigens which are typically membrane lipid species [reviewed in (22)]. The factors conferring differential antigen specificity of CD1 isoforms are not fully understood, but include the depth of the lipid-binding pocket of CD1 and the structure of carbohydrates on the antigen head group (23).
Different isoforms of human CD1 are localized to distinct subcellular locations, and are postulated to allow sampling of different antigens in different cellular compartments (24, 25). Several laboratories have studied intracellular trafficking of CD1d molecules to try and identify subcellular compartments responsible for antigen acquisition by CD1d [reviewed in (24)]. Murine CD1d has similar distributions in cells with endogenous CD1d or in CD1d-transfected cell lines, localizing to the plasma membrane and specialized organelles in the endosomal pathway known as MHC class II-containing compartments (MIIC) (26, 27). Recycling of pinocytosed human and murine CD1d between the plasma membrane and MIIC is required for presentation of CD1d-binding antigens (28). It has been suggested that CD1d not associated with invariant chain (Ii) is transported directly from the trans Golgi network to the plasma membrane and preferentially binds self-antigens, while Ii-associated CD1d is transported indirectly to the plasma membrane via the endosomal pathway and preferentially binds foreign antigens (29). However, direct surface loading of CD1d with foreign antigen [
-galactosylceramide (
-GalCer)] can occur without endosomal trafficking of antigen (30, 31), and Ii-deficient mice present foreign CD1d antigens that require endosomal processing (32). CD1d recycling between the plasma membrane and endosomal pathway is proposed to facilitate exchange of self- and foreign CD1d-bound antigens in the endosomal compartments (27, 29). Recently, saposins, enzymes that activate lysosomal hydrolases involved in sphingosine metabolism, were shown to facilitate acquisition of antigen by CD1 in vesicles and subsequent presentation (30, 33, 34).
In this study, we demonstrate that in CD1d-transfected A20 B cells (A20mCD1d) that BCR targeting with antibodyantigen complexes results in enhanced CD1d presentation as compared with non-targeted antigen. We demonstrate that inhibition of the BCR-activated Syk with piceatannol inhibits enhanced presentation on CD1d. We show that piceatannol inhibits BCR delivery to CD1d-containing MIIC-like compartments. Our findings suggest that the BCR facilitates capture and CD1d presentation of limited concentration of CD1d antigens and achieves this by delivery of BCRantigen complexes to CD1d-containing endosomes.
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Methods
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Antibodies and fluorochromes
Unless specified, biotin-, Cy3-, FITC- and HRP-conjugated anti-antibodies were purchased from Jackson Immunoresearch Ltd (West Grove, PA, USA). Anti-biotin mouse IgG and rabbit anti-mouse IgG were also purchased from Jackson Immunoresearch Ltd. Anti-Lamp1, CD1d and IL-2 antibodies were purchased from BD PharMingen (San Diego, CA, USA). Anti-Rab 4 and Cathepsin B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Fc
RIIb mAb (2.4G2) was purified from hybridoma culture supernatants by T-Gel affinity chromatography (Pierce, Rockford, IL, USA). Anti-LAM mAb CS35 was obtained through the Tuberculosis Research Materials and Vaccine Testing contract NO1-AI-75320 (Colorado State University, Fort Collins, CO, USA). The anti-LAM 5c11 IgM mAb was purified from hybridoma supernatant as described previously (35).
Cell lines
A20 murine B cell lines transfected with CD1d cDNA (A20mCD1d) and CD1d/
-GalCer-reactive murine V
14 NKT.3C3 cells were gifts from M. Kronenberg (La Jolla Institute for Allergy and Immunology, San Diego, CA, USA) and are described elsewhere (20). A20mCD1d cells were cultured in 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, 40 µg ml1 gentamicin and 0.8 mg ml1 G418 (GIBCO BRL Life Technologies, Grand Island, NY, USA). NKT cells were cultured in RPMI containing 10% heat-inactivated FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, 1 mM MEM, 2 µM ß-mercaptoethanol and 40 µg ml1 gentamicin.
Glycolipid antigens
Biotin-
-galactosylceramide (2S,3S,4R)-1-O-(
-D-galactopyranosyl)-2-[N-12-(6-(biotinoylamino)hexanoylamino)dodecanoylamino]-1,3,4-octadecanetriol (AGL-592, biotin-
-GalCer) was prepared starting from commercially available D-lyxose according to a modification of the procedures reported by Sakai and co-workers (36). Structural analysis of biotin-
-GalCer was performed by electrospray mass spectrometry using a Micromass LCT spectrometer (Micromass, Manchester, UK) (Supplementary Figure 1, available at International Immunology Online). The previously published spectroscopic data are identical to those reported herein. Biotin-
-GalCer contains a biotin moiety on the terminal end of one of the acyl chains and is functionally equivalent to
-GalCer in in vitro assays (Supplementary Figure 2, available at International Immunology Online). Non-mannose-capped lipoarabinomannan (AraLAM) was obtained through the Tuberculosis Research Materials and Vaccine Testing contract NO1-AI-75320 (Colorado State University).
Antigen presentation
This procedure was performed by the method of Tangri and colleagues with modifications (20). Aliquots of biotin-
-GalCer dissolved in 2 : 1 chloroform : methanol were evaporated to dryness under sterile conditions and reconstituted in culture media by vortexing immediately prior to use. To control for effects of the solvent, equal volumes of chloroform : methanol were evaporated before addition of media and used as a no-antigen control. A20mCD1d cells (105 per well in 100 µl volume) were washed twice in serum-free media, and then seeded into 96-well culture plates prior to the addition of biotin-
-GalCer (50 µl volume at 3x final concentration) and incubation at 37°C for 4 h. Alternatively, biotin-
-GalCer was mixed with mouse IgG (anti-biotin) and rabbit anti-mouse IgG to form complexes. Antibodies were added at a 10- µg ml1 final concentration and cells cultured as for non-targeted antigen. Where appropriate, Fc
RIIb-blocking 2.4G2 mAb was added to a final concentration of 10 µg ml1 prior to stimulation. Samples were washed four times (in serum-containing media) to remove biotin-
-GalCer and antibodies. NKT.3C3 cells were then added as appropriate (5 x 104 cells per well in 50 µl volume) and incubated at 37°C for 20 h. Supernatants were transferred to an empty culture plate and stored at 20°C until required. Cells were used between 5 and 10 days in culture for experiments reported here. Where appropriate, supernatants were carefully aspirated and cells were fixed by the addition of 100 µl of 0.15% w/v PFA in PBS for 30 min at room temperature. Following fixation, cells were washed four times for 5 min at 22°C in NKT cell media to remove PFA.
Measurement of IL-2 by ELISA
A standard sandwich ELISA protocol was used in this assay. Briefly, 50 µl of a 5- µg ml1 solution of anti-IL-2 mAb (BD PharMingen) in binding buffer (0.1 M Na2HPO4, pH 9.0) was added to ELISA plates and incubated overnight at 4°C. Plates were then washed four times with wash buffer (PBS, 0.05% v/v Tween 20), before the addition of 200 µl of blocking solution (PBS, 1% w/v BSA, 0.05% v/v Tween 20) and incubation for 1 h at 22°C. After washing, 75 µl of IL-2 standards (BD PharMingen) or 75 µl of culture supernatant (from antigen-presentation assays) was added to plates and incubated overnight at 4°C. After washing, 100 µl of biotin-conjugated anti-IL-2 mAb was added and incubated for a further 2 h at 22°C. This was followed by washing and addition of 100 µl of a 2- µg ml1 solution of HRP-conjugated streptavidin. Plates were incubated for 1 h at room temperature before washing and addition of 100 µl of ABTS peroxidase substrate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA). After incubation for 15 min at 22°C, reactions were stopped by the addition of 100 µl of a 10% w/v solution of SDS. Absorbance at 405 nm was then measured using a Dynex plate reader (Dynex, Chantilly, VA, USA). Data were analyzed using Graphpad PrismTM software (San Diego, CA, USA).
BCR internalization and intracellular staining
Cells were fixed with 1.0% w/v PFA in PBS for 30 min at 4°C, or chilled to 4°C and incubated with anti-mouse IgG (1 µg per 106 cells) for 1 h, before washing twice in ice-cold RPMI and further incubation with a Cy3-conjugated cross-linking secondary antibody. Cells were washed and incubated at 37°C for required times, before fixing in 1% w/v PFA in PBS. Following BCR internalization, fixed cells were permeablized with 0.5% w/v saponin, 0.1% w/v BSA and 0.04% w/v NaN3 in PBS and counterstained with a FITC-conjugated anti-mCD1d mAb. Steady-state distribution of mCD1d was assessed following fixation, permeabilization and staining of un-stimulated cells for mCD1d and Lamp1, Rab 4 or Cathepsin B. Background staining for these experiments was determined using FITC-conjugated isotype control antibodies and Cy3streptavidin alone.
Confocal microscopy
Cells were mounted on poly-L-lysine-coated chamber slides using ProlongTM anti-fade reagent (Molecular Probes, Eugene, OR, USA) and analyzed using either a BioRad MRC1024 (Hercules, CA, USA) or a Zeiss LSM Meta (Thornwood, NY, USA) laser-scanning confocal microscope in conjunction with a Zeiss x40 objective lens. For each condition, 50100 cells were analyzed. Co-localization of BCR with CD1d was assessed using NIH Image J software (http://rsb.info.nih.gov/ij). Briefly, co-localized BCR (red) and mCD1d (green) pixels were determined and subtracted from the total BCR-associated signal (integral pixel intensity). Non-co-localized integral pixel intensities were then taken as a percentage of the total BCR-associated integral pixel intensity and used to determine the percent co-localization of the total BCR-associated signal with mCD1d. Data were then expressed using Graphpad PrismTM software.
Flow cytometry
Cells were surface stained with FITC-conjugated antibodies for 1 h at room temperature before washing three times in 0.1% w/v BSA in PBS. Where necessary, 10 µg ml1 final concentration of anti-Fc
R mAb (2.4G2) or 3% mouse serum was used to block potential Fc
R binding. Cells were fixed in 1.0% w/v PFA in PBS and analyzed using a Becton-Dickinson FACScan in combination with CellQuest software (Becton-Dickinson, Palo Alto, CA, USA).
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Results
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Presentation of CD1d antigen to NKT cells is enhanced by BCR targeting
We tested the hypothesis that CD1d-binding glycolipid antigens targeted to the BCR would be presented by CD1d with a higher efficiency than non-targeted antigen. APCs when pulsed with non-targeted
-GalCer present the antigen on CD1d to NKT cell hybridomas (20). We synthesized a modified
-GalCer structure containing a biotin moiety on the terminal end of one acyl chain to facilitate targeting to the BCR as described in Methods. APCs were pulsed with biotin-
-GalCer for 4 h before removal by washing and the addition of NKT cells. Biotin-
-GalCerCD1d complexes stimulated IL-2 production by NKT cells, suggesting that either only one acyl chain of
-GalCer is required to bind in the groove of CD1d to activate NKT cells or the biotin-acyl binds the groove of CD1d. Similar biotin constructs bind CD1d and activate NKT cells (36). As reported previously (37), in the absence of antigen, APCs or NKT cells, there was no production of IL-2, and the response was blocked specifically by anti-CD1d antibodies (data not shown). Further characterization of biotin-
-GalCer is provided in Supplementary Figures 1 and 2 (available at International Immunology Online) showing the structure as compared with
-GalCer and the equivalent activation of NKT cells by both antigens.
We then incubated cells with different concentrations of biotin-
-GalCer alone or biotin-
-GalCeranti-biotin mouse IgGrabbit anti-mouse IgG complexes to target transmembrane IgG (BCR). Cross-linking the BCR with anti-Ig antibodies is the standard method used to stimulate BCR signaling, trafficking and antigen presentation in vitro (3840). In the absence of APCs, antigen or NKT cells, there was no IL-2 production by NKT cells (Fig. 1A). We observed that while IL-2 production was not detected in cells treated with 110 ng ml1 free biotin-
-GalCer, at these concentrations, BCR-targeted antigen triggered IL-2 production similar to that observed for 100 or 1000 ng ml1 free antigen. These data show that BCR targeting results in a 100-fold greater efficiency of presentation of CD1d-binding antigens. An anti-CD1d antibody, but not an anti-CD1b antibody, blocked presentation of BCR-targeted
-GalCer (Fig. 1B), confirming that BCR-enhanced IL-2 production is CD1d dependent.
A20mCD1d cells express CD1d and the BCR, but not Fc
R (Supplementary Figure 3, available at International Immunology Online). Furthermore, 2.4G2 does not affect binding of anti-BCR antibodies. Thus, binding of anti-mouse IgG immune complexes is consistent with targeting the BCR, but not Fc
R. To confirm that Fc
Rs were not involved in immune complex-mediated antigen presentation, we pre-blocked with the 2.4G2 mAb (does not cause receptor activation) prior to performing the antigen-presentation assay. We observed that 2.4G2 mAb had no effect on CD1d presentation of BCR-targeted or non-targeted antigen (Fig. 1B, inset). This shows that Fc
Rs were not targeted by the antibody complexes, and provides further evidence that our methods lead to BCR targeting of biotin-
-GalCer.
Kinetics of CD1d presentation of BCR-targeted antigen are consistent with endosomal trafficking
We examined the kinetics of CD1d presentation of BCR-targeted
-GalCer (Fig. 2A). IL-2 production by NKT cells was triggered by cells following a 3- to 4-h pulse with low concentrations (50 ng ml1) of BCR-targeted antigen. When cells were pulsed with similarly low concentrations of non-targeted antigen, IL-2 production was not detected (data not shown). When we used high concentrations of non-targeted antigen (2 µg ml1), we detected maximal IL-2 production within 1 h (Fig. 2B). The IL-2 titers were lower in these experiments with fixed APCs as was the relative efficiency of presentation of BCR-targeted and non-targeted antigen. This is likely attributable to cell fixation which results in lower IL-2 titers (37). Our kinetic data are consistent with reports of direct surface loading of CD1d with non-targeted
-GalCer (30, 31). However, this approach does not account for endosomal acquisition of antigen by CD1d recycling between the plasma membrane and endosomes. To independently confirm our observations and determine if BCR-targeted
-GalCer was loaded directly, we compared presentation of BCR-targeted antigen (2550 ng ml1) when cells were pulsed for 4 h and then fixed or fixed and then pulsed for 4 h (Fig. 2C). Minimal IL-2 production was observed when cells were fixed prior to the antigen pulse, suggesting a requirement for internalization and trafficking of antigen to CD1d-containing compartments and not for direct surface loading onto CD1d. In contrast, with free antigens at higher concentrations (1.02.0 µg ml1), surface-loaded antigens accounted for most of the IL-2 produced (Fig. 2D).

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Fig. 2. Kinetics of CD1d antigen presentation. A20mCD1d cells were pulsed for times indicated with (A) BCR-targeted antigen or (B) free antigen before washing and incubation for 20 h with NKT cells. IL-2 concentrations in the media were measured by ELISA. Data show the mean ± SEM for triplicate samples and are representative of two independent experiments. (C) APCs were pulsed with BCR-targeted antigen at concentrations indicated for 4 h before washing and fixation, or fixed and washed before incubation of cells with antigen for 4 h. Cells were then co-cultured with NKT cells before measurement of IL-2 titers. (D) APCs were treated as in (C), but pulsed with non-targeted antigen. Graphs show the IL-2 titers for cells fixed before antigen pulse (gray bars) compared with cells fixed after antigen pulse (black bars). Data show the mean ± SEM for triplicate samples and are representative of two independent experiments.
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Transport of BCR-targeted
-GalCer to the MIIC is required for presentation on CD1d
Endosomal trafficking of CD1d facilitates CD1dantigen interactions in CD1d-enriched locations. CD1d requires recycling between the plasma membrane and the endosomal pathway for antigen presentation (27, 29). We wished to determine if BCR targeting would enhance antigen trafficking to CD1d-enriched intracellular compartments as compared with that of non-targeted antigen. A20mCD1d cells were fixed, permeabilized and stained for CD1d and early endosome (EE) and late endosome (LE) markers (Fig. 3). Intracellular steady-state CD1d co-localized extensively with Lamp1 and Cathepsin B, but not with Rab 4 (Fig. 3). These data agree with published findings that CD1d is localized mainly to late endocytic compartments including MIIC (29).

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Fig. 3. Subcellular localization of CD1d. A20mCD1d cells were fixed, permeabilized and stained for CD1d (green), and counterstained for the EE markers Rab 4 (red) or LE/MIIC markers Lamp1, Cathepsin B (red). Co-localization of green and red signals is shown in yellow. Images shown are representative of 50100 cells analyzed for each condition.
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The BCR traffics through EE to LE/lysosomes and MIIC following cross-linking (41, 42). As CD1d is localized to the MIIC, it would be expected that cross-linked BCR would internalize to a CD1d-containing compartment. On cross-linking, BCR was rapidly internalized (510 min) and transported to Rab 4 vesicles (Fig. 4A, upper panels). At 15 min, Rab 4/BCR co-localization peaked (Fig. 4A, lower left panel), with
25% of cells displaying this phenotype, which was followed by a lower percentage of cells (15%) showing co-localization over the next 1-h duration. During this time, BCR accumulated in the cell and by 30 min, most cells displayed segregated BCR- and Rab 4-containing vesicles (Fig. 4A, lower middle panel), confirming that BCR localizes transiently with Rab 4 as it proceeds through the endosomal pathway.

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Fig. 4. Cross-linked BCR is delivered sequentially to EE and CD1d-containing LE/MIIC. Cells were chilled to 4°C and BCR cross-linked as described in Methods. Cells were then incubated at 37°C for times indicated before fixation, permeabilization and counterstaining for CD1d. Cells were examined by confocal microscopy and assessed for BCR internalization and co-localization with CD1b or CD1d (shown in yellow). Graphs show percentage of cells demonstrating BCR internalization (closed symbols) and BCR co-localization with CD1d (open symbols). A total of 100 cells were analyzed for each condition.
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When cells were counterstained for CD1d following BCR cross-linking and internalization, we observed that BCR/CD1d co-localization was delayed and was not detectable until 15 min (Fig. 4B, lower left panel). BCR then accumulated in CD1d-containing vesicles over the next 45 min (Fig. 4B, lower middle and right panels). Thus, the BCR proceeds sequentially through Rab 4- and CD1d-containing vesicles. We tested the effect of BCR cross-linking on CD1d co-localization with MIIC-like compartments. BCR cross-linking had no discernable effect on CD1d/Cathepsin B co-localization, although larger aggregated CD1d+ internal structures were detected following 30 min of BCR internalization (data not shown). This suggests that cross-linked BCR is directed to CD1d+ MIIC, but does not change the localization of internal CD1d itself.
Our data suggest that BCR-targeted CD1d antigens could intersect with CD1d in the endosomal pathway and thus explain why BCR targeting causes enhanced antigen presentation. The low concentrations of biotin-
-GalCer that result in high IL-2 titers are not detectable intracellularly by confocal microscopy (data not shown). To demonstrate that the BCR can internalize a CD1-binding non-protein antigen, we targeted Mycobacterium tuberculosis-derived antigen AraLAM to the BCR, using a strategy similar to that adopted for biotin-
-GalCer (Fig. 5). We observed that targeting resulted in enhanced delivery of AraLAM to Lamp1+ (Fig. 5A) and CD1d+ (Fig. 5B) vesicles. AraLAM was barely detectable in cells pulsed with free AraLAM. Although AraLAM is a CD1b-, but not a CD1d-, binding antigen (15), our data show that the BCR can capture glycolipid and lipoglycan antigens, facilitating their transport to CD1-containing vesicles.

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Fig. 5. BCR targeting enhances delivery of AraLAM to CD1d-containing LE/MIIC. A20mCD1d cells were chilled to 4°C and incubated with 2 µg ml1 free AraLAM or BCR-targeted AraLAM (2 µg ml1 AraLAM per 10 µg ml1 biotin-anti-CS35 mAb, 10 µg ml1 biotin anti-mouse IgG per 2.5 µg ml1 streptavidin) for 1 h before washing. Cells were incubated at 37°C for 30 min, before fixation and staining for (A) AraLAM and Lamp1 or (B) AraLAM and CD1d. AraLAM was detected with 5c11 IgM mAb in conjunction with FITC anti-IgM (green). Lamp1 and CD1d were detected with anti-Lamp1 or anti-CD1d antibody in conjunction with Cy3 goat anti-rat IgG (red). Similar data were obtained in two independent experiments. Images shown are representative of at least 50 cells.
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BCR trafficking to CD1d-containing endosomes is required for CD1d antigen presentation
Due to limitations in detecting biotin-
-GalCer directly, we adopted a strategy to block BCR transport to the MIIC and test the effect on CD1d antigen presentation of biotin-
-GalCer. BCR trafficking to antigen-processing compartments including MIIC is dependent on BCR signaling (42). Signaling through the tyrosine kinase Syk is required for BCR-mediated class II antigen presentation (43). We tested the effect of the Syk inhibitor piceatannol on CD1d presentation of BCR-targeted and non-targeted biotin-
-GalCer (Fig. 6). Low levels of piceatannol (08 µM) inhibit Syk, while higher concentrations inhibit Syk and protein kinase C (PKC) (44). Syk and PKC are required for transport of the BCR to MIIC and antigen presentation (43, 45). Piceatannol resulted in a dose-dependent inhibition of CD1d presentation of BCR-targeted
-GalCer (Fig. 6A). Dimethyl sulfoxide (DMSO), the carrier for piceatannol, had no effect on antigen presentation. The 50% inhibitory concentration for inhibition of CD1d antigen presentation was between 2.5 and 5 µM, when piceatannol has the greater selectivity for Syk. Piceatannol had no effect on presentation of non-targeted
-GalCer (Fig. 6B), suggesting that Syk activity was not required for maintaining the CD1d antigen-presentation machinery. To confirm this, we treated cells with piceatannol for 0.5, 4.5 and 24.5 h to replicate the conditions of the antigen-presentation assay. Cells were fixed and stained for CD1d and BCR. Piceatannol had no effect on the expression of CD1d (Supplementary Figure 4A, available at International Immunology Online). There was a small decrease in BCR expression between 4.5 and 24.5 h under control conditions, but neither DMSO nor piceatannol had any further effect on BCR expression (Supplementary Figure 4B, available at International Immunology Online). Our data therefore show that Syk activation by the BCR is necessary for presentation of a BCR-targeted CD1d antigen.
We then tested the effect of piceatannol on transport of BCR to CD1d-containing vesicles (Fig. 7). Under control conditions (or DMSO treatment), BCR was internalized into large vesicles with extensive co-localization with CD1d. At concentrations which inhibited CD1d antigen presentation (10 µM), piceatannol blocked translocation of the BCR to CD1d-containing MIIC, decreasing the extent of BCR/CD1d co-localization by
6070% and preventing the formation of large aggregated MIIC-like structures. Consistent with this observation, presentation of BCR-targeted antigen was inhibited by 6070% following 10 µM piceatannol treatment (Fig. 6A). The incomplete inhibition by piceatannol is likely due to incomplete inhibition of Syk, or activation of other proximal BCR-signaling effectors that facilitate antigen presentation (as limited trafficking of BCR to MIIC occurs under these conditions). Thus, preventing BCR intersection with CD1d blocks presentation of BCR-targeted antigen, and suggests that transport of BCRantigen complexes to MIIC-like vesicles could be required for CD1d antigen presentation.

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Fig. 7. Piceatannol blocks delivery of the BCR to CD1d-containing endosomes. Cells were untreated or treated with DMSO or DMSO plus piceatannol for 30 min, before chilling cells to 4°C and cross-linking the BCR (red). Cells were incubated at 37°C for 30 min to allow BCR internalization. Inhibitors were present throughout the procedure. Cells were then fixed, permeabilized and counterstained for CD1d (green). Cells were analyzed by confocal microscopy. Co-localization of BCR and CD1d is indicated in yellow, and co-localized pixels (white). Images shown are representative of at least 50 cells. Co-localization was quantitated using Image J software. Graph shows the percentage of BCR that is co-localized with CD1d. Each data point represents an individual cell.
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Discussion
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Evidence suggests that production of antibodies reactive to M. tuberculosis-derived glycolipid antigens may contribute to protective immunity and affect the course of infection (7, 9, 10, 46). The M. tuberculosis-derived glycolipids lipoarabinomannan (LAM) and PIM are CD1b- and CD1d-binding antigens, respectively (15, 21). LAM-reactive antibodies decrease splenic deposition of LAM and increase hepatobiliary clearance in mice (7). Epidemiologic studies have shown that low levels of anti-LAM antibodies in children aged 25 years correlates with greater dissemination of M. tuberculosis (46). More compelling is the observation in murine models that PIMs administered as BSA conjugates or in protein-free liposomes trigger production of anti-PIM antibodies, that have protective effects against subsequent challenge with live M. tuberculosis (9, 10).
It is therefore of interest to determine if CD1d-binding M. tuberculosis antigens such as PIM alter the course of M. tuberculosis infection through BCR-mediated CD1d antigen presentation to NKT cells. As a first step to achieving this aim, we tested the hypothesis that the BCR can facilitate presentation of the CD1d-binding antigen biotin-
-GalCer. We have shown for the first time that BCR targeting of biotin-
-GalCer caused similar IL-2 production by NKT hybridomas than that induced by up to 100-fold higher concentrations of non-targeted antigen (Fig. 1A). We assessed the kinetics of antigen presentation at optimal concentrations of BCR-targeted and non-targeted antigen. CD1d antigen presentation was faster for non-targeted antigen than BCR-targeted antigen (Fig. 2A and B). However, this was due to direct surface loading of high concentrations of non-targeted antigen (Fig. 2D). Other investigators have observed direct loading of CD1d with
-GalCer in addition to the presentation of antigen acquired in endosomes (30, 31). Indeed, lower concentrations (100 ng ml1) of non-conjugated
-GalCer than those used in this study result in detectable surface loading of CD1d (31). This may be attributable to differences in efficiency of
-GalCer versus biotin-
-GalCer. Moody and colleagues also demonstrated that shorter chain CD1b antigens (C32 acyl chains) preferentially loaded surface CD1d, while C80 chains required endosomal processing (47). Thus, CD1d antigens with acyl chain of different lengths may differentially load surface versus endosomal CD1d. At low concentrations of BCR-targeted antigen, surface loading did not account for antigen presentation (Fig. 2C). Testing high concentrations of BCR-targeted antigen was not possible because 1000 ng ml1 biotin-
-GalCer would result in a 10-fold molar excess of antigen as compared with targeting antibody, in essence providing a combination of non-targeted and BCR-targeted antigens for the APCs. Taken together, our data suggest that the function of the BCR is not to make CD1d antigen presentation faster, but to increase its efficiency. This is consistent with studies on class II, whereby the kinetics of presentation of non-targeted and BCR-targeted protein antigen are similar (48, 49).
In the present study, we have not determined if the formation of an aggregated CD1d+ MIIC or if the degree of BCR cross-linking influences CD1d antigen presentation. BCR cross-linking is necessary for the formation of aggregated MIIC and enhances class II presentation of BCR-internalized antigen [reviewed in (42)]. However, in the absence of BCR cross-linking, soluble protein antigen accesses the MIIC (via the same endocytic pathway as the cross-linked BCR) and presentation on class II is also stimulated (50). The method used herein to target biotin-
-GalCer to the BCR likely results in variable multiplicity of BCR ligation (including cross-linking). In future experiments, we will determine how CD1d antigen presentation is influenced by the degree of BCR cross-linking.
Cross-linked BCR was transported to CD1d-containing MIIC-like endosomes (Figs 3 and 4). We cannot detect endosome-localized biotin-
-GalCer directly by conventional microscopy methods. This is likely because optimal CD1d antigen presentation occurs at biotin-
-GalCer concentrations too low to be detected by microscopy and/or because the biotin moiety is bound in the groove of CD1d. However, the BCR facilitated enhanced intracellular trafficking of M. tuberculosis-derived AraLAM to CD1d-containing endosomes that were detected with anti-LAM antibodies (Fig. 5). BCR targeting of AraLAM to CD1-containing compartments is consistent with the observations of Prigozy and co-workers who showed that macrophage mannose receptors facilitated the uptake and transport of mannosylated LAM to CD1b-containing compartments (15). This shows that receptors expressed on APCs including the BCR can direct different CD1 antigens to endosomes containing the appropriate CD1 molecules for presentation to CD1-restricted T cells.
Loading of not only endosomal CD1d but also CD1b with antigen is facilitated by a group of proteins called saposins (30, 33, 34). Saposins are endosome-localized glycoproteins that activate lysosomal hydrolases involved in sphingolipid metabolism (51). CD1d presentation of
-GalCer internalized to endosomal compartments was enhanced by saposin expression (30). Several studies show that endosome-localized CD1d is required for antigen presentation (2830, 32). Kronenberg and co-workers showed that the addition of an additional terminal galactose residue to
-GalCer resulted in a requirement for endosomal trafficking and processing, showing importantly that the endosomal pathway may be required for limiting clipping of different antigen structures (52). Jayawardena-Wolf and co-workers showed that a tail-truncated CD1d was deficient in antigen presentation and this was linked to an inability to traffic to the MIIC (28). Intracellular CD1d localizes to MIIC-like compartments (20, 2729, 32, 53), making the MIIC a good candidate destination for free or BCR-targeted antigen. The BCR requires trafficking to the MIIC for class II presentation [reviewed in (42)]. Our studies show that cross-linked BCR is rapidly internalized to CD1d-containing MIIC-like compartments (Figs 3 and 4). Blocking BCR trafficking with the Syk inhibitor piceatannol also inhibited presentation of
-GalCer by CD1d (Fig. 5A). However, Syk inhibition had no effect on presentation of non-targeted antigen (Fig. 5B). Although we suggested that incomplete inhibition of antigen presentation with piceatannol may be caused by incomplete Syk inhibition (as evidenced by reduced, but not eliminated, BCR delivery to MIIC), it remains formally possible that other mechanisms could contribute to enhanced presentation. At present it is unclear what these would be as FcR did not contribute to presentation in our experiments. Non-specific pinocytic uptake of antigenantibody complexes is highly unlikely, as this would not lead to such rapid MIIC delivery of antigen or such efficient presentation (1, 2). Our evidence points to BCR being the major point of entry for our antigenantibody complexes.
In summary, both cell-surface loading and internalization of antigen to CD1d-containing compartments appear to be real mechanisms for CD1d antigen presentation, with the mechanism utilized being determined by the structure of the antigen and its concentration. The BCR may represent a specialized mechanism for capture of low glycolipid concentrations early in infection when bacterial lesions are small and antigen quantities are limited and utilize the endosomal pathway. Conversely, during acute or more disseminated infection, higher antigen concentrations may result in direct surface loading of CD1d.
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Supplementary data
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Supplementary data are available at International Immunology Online at www.intimm.oxfordjournals.org.
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Acknowledgements
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This research was funded by NIH Grant P20 RR16437 from the COBRE Program of the National Center for Research Resources. G.S.B., Lister Jenner Research Fellow, acknowledges support from the Medical Research Council (UK) and the Wellcome Trust. The authors thank J. T. Belisle (Colorado State University), for providing CS35 mAb and AraLAM under the NIH, NIAID Contract NO1-AI-75320, M. Kronenberg (La Jolla Institute for Allergy and Immunology, San Diego, CA, USA) for providing A20mCD1d and NKT.3C3 cells and for critical review of the manuscript and L. Brossay (Brown University, Providence, RI, USA) for helpful discussions.
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Abbreviations
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APC | antigen-presenting cell |
AraLAM | non mannose-capped lipoarabinomannan |
-GalCer | -galactosylceramide |
BCR | B cell antigen receptor |
EE | early endosome |
DMSO | dimethyl sulfoxide |
LAM | lipoarabinomannan |
LE | late endosome |
MIIC | MHC class II-containing compartment |
NKT | NK-like T |
PIM | phosphatidylinositol mannoside |
PKC | protein kinase C |
Syk | spleen tyrosine kinase |
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Notes
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Transmitting editor: I. Pecht
Received 5 September 2004,
accepted 20 April 2005.
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References
|
---|
- Watts, C. 1997. Capture and processing of exogenous antigens for presentation on MHC molecules. Annu. Rev. Immunol. 15:821.[CrossRef][ISI][Medline]
- Lanzavecchia, A. 1990. Receptor-mediated antigen uptake and its effect on antigen presentation to class II-restricted T lymphocytes. Annu. Rev. Immunol. 8:773.[CrossRef][ISI][Medline]
- Rock, K. L., Benacerraf, B. and Abbas, A. K. 1984. Antigen presentation by hapten-specific B lymphocytes. I. Role of surface immunoglobulin receptors. J. Exp. Med. 160:1102.[Abstract/Free Full Text]
- Rock, K. L., Gamble, S. and Rothstein, L. 1990. Presentation of exogenous antigen with class I major histocompatibility complex molecules. Science 249:918.[ISI][Medline]
- Brigl, M. and Brenner, M. B. 2004. CD1: antigen presentation and T cell function. Annu. Rev. Immunol. 22:817.[CrossRef][ISI][Medline]
- Porcelli, S. A. and Modlin, R. L. 1999. The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids. Annu. Rev. Immunol. 17:297.[CrossRef][ISI][Medline]
- Glatman-Freedman, A., Mednick, A. J., Lendvai, N. and Casadevall, A. 2000. Clearance and organ distribution of Mycobacterium tuberculosis lipoarabinomannan (LAM) in the presence and absence of LAM-binding immunoglobulin M. Infect. Immun. 68:335.[Abstract/Free Full Text]
- Livingston, P. O., Ritter, G. and Calves, M. J. 1989. Antibody response after immunization with the gangliosides GM1, GM2, GM3, GD2 and GD3 in the mouse. Cancer Immunol. Immunother. 29:179.[ISI][Medline]
- Mehta, P. K. and Khuller, G. K. 1988. Protective immunity to experimental tuberculosis by mannophosphoinositides of mycobacteria. Med. Microbiol. Immunol. (Berl.) 177:265.[ISI][Medline]
- Singh, A. P. and Khuller, G. K. 1994. Induction of immunity against experimental tuberculosis with mycobacterial mannophosphoinositides encapsulated in liposomes containing lipid A. FEMS Immunol. Med. Microbiol. 8:119.[CrossRef][ISI][Medline]
- Schofield, L., McConville, M. J., Hansen, D. et al. 1999. CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells. Science 283:225.[Abstract/Free Full Text]
- Galli, G., Nuti, S., Tavarini, S. et al. 2003. CD1d-restricted help to B cells by human invariant natural killer T lymphocytes. J. Exp. Med. 197:1051.[Abstract/Free Full Text]
- Campos, R. A., Szczepanik, M., Itakura, A. et al. 2003. Cutaneous immunization rapidly activates liver invariant V{alpha}14 NKT cells stimulating B-1 B cells to initiate T cell recruitment for elicitation of contact sensitivity. J. Exp. Med. 198:1785.[Abstract/Free Full Text]
- Lisbonne, M., Diem, S., de Castro Keller, A. et al. 2003. Cutting edge: invariant V alpha 14 NKT cells are required for allergen-induced airway inflammation and hyperreactivity in an experimental asthma model. J. Immunol. 171:1637.[Abstract/Free Full Text]
- Prigozy, T. I., Sieling, P. A., Clemens, D. et al. 1997. The mannose receptor delivers lipoglycan antigens to endosomes for presentation to T cells by CD1b molecules. Immunity 6:187.[CrossRef][ISI][Medline]
- Burdin, N., Brossay, L. and Kronenberg, M. 1999. Immunization with alpha-galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis. Eur. J. Immunol. 29:2014.[CrossRef][ISI][Medline]
- Moody, D. B., Ulrichs, T., Muhlecker, W. et al. 2000. CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection. Nature 404:884.[CrossRef][ISI][Medline]
- Shamshiev, A., Gober, H. J., Donda, A., Mazorra, Z., Mori, L. and De Libero, G. 2002. Presentation of the same glycolipid by different CD1 molecules. J. Exp. Med. 195:1013.[Abstract/Free Full Text]
- Naidenko, O. V., Maher, J. K., Ernst, W. A., Sakai, T., Modlin, R. L. and Kronenberg, M. 1999. Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules. J. Exp. Med. 190:1069.[Abstract/Free Full Text]
- Tangri, S., Brossay, L., Burdin, N., Lee, D. J., Corr, M. and Kronenberg, M. 1998. Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing. Proc. Natl Acad. Sci. USA 95:14314.[Abstract/Free Full Text]
- Fischer, K., Scotet, E., Niemeyer, M. et al. 2004. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells. Proc. Natl Acad. Sci. USA 101:10685.[Abstract/Free Full Text]
- Moody, D. B. and Besra, G. S. 2001. Glycolipid targets of CD1-mediated T-cell responses. Immunology 104:243.[CrossRef][ISI][Medline]
- Burdin, N., Brossay, L., Degano, M. et al. 2000. Structural requirements for antigen presentation by mouse CD1. Proc. Natl Acad. Sci. USA 97:10156.[Abstract/Free Full Text]
- Sugita, M., Peters, P. J. and Brenner, M. B. 2000. Pathways for lipid antigen presentation by CD1 molecules: nowhere for intracellular pathogens to hide. Traffic 1:295.[CrossRef][ISI][Medline]
- Schaible, U. E., Hagens, K., Fischer, K., Collins, H. L. and Kaufmann, S. H. 2000. Intersection of group I CD1 molecules and mycobacteria in different intracellular compartments of dendritic cells. J. Immunol. 164:4843.[Abstract/Free Full Text]
- Neefjes, J. 1999. CIIV, MIIC and other compartments for MHC class II loading. Eur. J. Immunol. 29:1421.[CrossRef][ISI][Medline]
- Chiu, Y. H., Jayawardena, J., Weiss, A. et al. 1999. Distinct subsets of CD1d-restricted T cells recognize self-antigens loaded in different cellular compartments. J. Exp. Med. 189:103.[Abstract/Free Full Text]
- Chiu, Y. H., Park, S. H., Benlagha, K. et al. 2002. Multiple defects in antigen presentation and T cell development by mice expressing cytoplasmic tail-truncated CD1d. Nat. Immunol. 3:55.[CrossRef][ISI][Medline]
- Jayawardena-Wolf, J., Benlagha, K., Chiu, Y. H., Mehr, R. and Bendelac, A. 2001. CD1d endosomal trafficking is independently regulated by an intrinsic CD1d-encoded tyrosine motif and by the invariant chain. Immunity 15:897.[CrossRef][ISI][Medline]
- Kang, S. J. and Cresswell, P. 2004. Saposins facilitate CD1d-restricted presentation of an exogenous lipid antigen to T cells. Nat. Immunol. 5:175.[CrossRef][ISI][Medline]
- Burdin, N., Brossay, L., Koezuka, Y. et al. 1998. Selective ability of mouse CD1 to present glycolipids: alpha-galactosylceramide specifically stimulates V alpha 14+ NK T lymphocytes. J. Immunol. 161:3271.[Abstract/Free Full Text]
- Elewaut, D., Lawton, A. P., Nagarajan, N. A. et al. 2003. The adaptor protein AP-3 is required for CD1d-mediated antigen presentation of glycosphingolipids and development of Valpha14i NKT cells. J. Exp. Med. 198:1133.[Abstract/Free Full Text]
- Zhou, D., Cantu, C., III, Sagiv, Y. et al. 2004. Editing of CD1d-bound lipid antigens by endosomal lipid transfer proteins. Science 303:523.[Abstract/Free Full Text]
- Winau, F., Schwierzeck, V., Hurwitz, R. et al. 2004. Saposin C is required for lipid presentation by human CD1b. Nat. Immunol. 5:169.[CrossRef][ISI][Medline]
- Glatman-Freedman, A., Martin, J. M., Riska, P. F., Bloom, B. R. and Casadevall, A. 1996. Monoclonal antibodies to surface antigens of Mycobacterium tuberculosis and their use in a modified enzyme-linked immunosorbent spot assay for detection of mycobacteria. J. Clin. Microbiol. 34:2795.[Abstract]
- Sakai, T., Naidenko, O. V., Iijima, H., Kronenberg, M. and Koezuka, Y. 1999. Syntheses of biotinylated alpha-galactosylceramides and their effects on the immune system and CD1 molecules. J. Med. Chem. 42:1836.[CrossRef][ISI][Medline]
- Lang, G. A., Maltsev, S. D., Besra, G. S. and Lang, M. L. 2004. Presentation of alpha-galactosylceramide by murine CD1d to natural killer T cells is facilitated by plasma membrane glycolipid rafts. Immunology 112:386.[CrossRef][ISI][Medline]
- Herrin, B. R., Groeger, A. L. and Justement, L. B. 2005. The adaptor protein HSH2 attenuates apoptosis in response to ligation of the B cell antigen receptor complex on the B lymphoma cell line, WEHI-231. J. Biol. Chem. 280:3507.[Abstract/Free Full Text]
- Siemasko, K., Skaggs, B. J., Kabak, S. et al. 2002. Receptor-facilitated antigen presentation requires the recruitment of B cell linker protein to Igalpha. J. Immunol. 168:2127.[Abstract/Free Full Text]
- Cheng, P. C., Dykstra, M. L., Mitchell, R. N. and Pierce, S. K. 1999. A role for lipid rafts in B cell antigen receptor signaling and antigen targeting. J. Exp. Med. 190:1549.[Abstract/Free Full Text]
- Drake, J. R., Lewis, T. A., Condon, K. B., Mitchell, R. N. and Webster, P. 1999. Involvement of MIIC-like late endosomes in B cell receptor-mediated antigen processing in murine B cells. J. Immunol. 162:1150.[Abstract/Free Full Text]
- Clark, M. R., Massenburg, D., Zhang, M. and Siemasko, K. 2003. Molecular mechanisms of B cell antigen receptor trafficking. Ann. NY Acad. Sci. 987:26.[Abstract/Free Full Text]
- Lankar, D., Briken, V., Adler, K. et al. 1998. Syk tyrosine kinase and B cell antigen receptor (BCR) immunoglobulin-alpha subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation. J. Exp. Med. 188:819.[Abstract/Free Full Text]
- Wang, B. H., Lu, Z. X. and Polya, G. M. 1998. Inhibition of eukaryote serine/threonine-specific protein kinases by piceatannol. Planta Med. 64:195.[ISI][Medline]
- Siemasko, K., Eisfelder, B. J., Williamson, E., Kabak, S. and Clark, M. R. 1998. Cutting edge: signals from the B lymphocyte antigen receptor regulate MHC class II containing late endosomes. J. Immunol. 160:5203.[Abstract/Free Full Text]
- Costello, A. M., Kumar, A., Narayan, V. et al. 1992. Does antibody to mycobacterial antigens, including lipoarabinomannan, limit dissemination in childhood tuberculosis? Trans. R. Soc. Trop. Med. Hyg. 86:686.[CrossRef][ISI][Medline]
- Moody, D. B., Briken, V., Cheng, T. Y. et al. 2002. Lipid length controls antigen entry into endosomal and nonendosomal pathways for CD1b presentation. Nat. Immunol. 3:435.[ISI][Medline]
- Singer, D. F. and Linderman, J. J. 1990. The relationship between antigen concentration, antigen internalization, and antigenic complexes: modeling insights into antigen processing and presentation. J. Cell Biol. 111:55.[Abstract]
- Lakey, E. K., Casten, L. A., Niebling, W. L., Margoliash, E. and Pierce, S. K. 1988. Time dependence of B cell processing and presentation of peptide and native protein antigens. J. Immunol. 140:3309.[Abstract/Free Full Text]
- Song, W., Cho, H., Cheng, P. and Pierce, S. K. 1995. Entry of B cell antigen receptor and antigen into class II peptide-loading compartment is independent of receptor cross-linking. J. Immunol. 155:4255.[Abstract]
- Kishimoto, Y., Hiraiwa, M. and O'Brien, J. S. 1992. Saposins: structure, function, distribution, and molecular genetics. J. Lipid Res. 33:1255.[Abstract]
- Prigozy, T. I., Naidenko, O., Qasba, P. et al. 2001. Glycolipid antigen processing for presentation by CD1d molecules. Science 291:664.[Abstract/Free Full Text]
- Kang, S. J. and Cresswell, P. 2002. Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules. EMBO J. 21:1650.[Abstract/Free Full Text]