Evidence for a role of ganglioside GM1 in antigen presentation: binding enhances presentation of Escherichia coli enterotoxin B subunit (EtxB) to CD4+ T cells

Toufic O. Nashar, Zoe E. Betteridge and Richard N. Mitchell1,

Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
1 Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA

Correspondence to: T. O. Nashar


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Successful antigen presentation by antigen-presenting cells is governed by a number of factors including the efficiency of antigen capture by cell-surface receptors, targeting to compartments of antigen processing, surface expression of MHC II–peptide complexes and presence of co-stimulatory signals. Ganglioside GM1 is an important component of membrane glycosphingolipids, and has been implicated in cell differentiation, apoptosis and signal transduction pathways. Using the B subunit of Escherichia coli enterotoxin (EtxB), a potent immunogen that binds GM1 with high affinity, and a non-binding mutant of EtxB, EtxB(G33D), we demonstrate that GM1 is intimately involved in several aspects of antigen presentation. Thus, GM1-mediated presentation of EtxB by B cells and CD11c+ dendritic cells (DC) significantly enhanced the proliferation and cytokine expression of EtxB-specific CD4+ T cells. Investigation regarding potential mechanisms revealed that EtxB binding directly augments the expression of MHC class II on B cells, and fractionation of B cells demonstrated that EtxB binding to GM1 results in rapid internalization and targeting to class II-rich compartments. GM1-mediated uptake of antigens and access to class II compartments in B cells can be exploited to significantly enhance the presentation of ovalbumin-conjugated to EtxB. These results demonstrate that GM1 can play an important role in antigen presentation via the MHC II pathway.

Keywords: antigen binding, antigen presentation, CtxB, enterotoxin, EtxB, GM1


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The efficiency of the presentation of exogenous antigen by MHC II+ antigen-presenting cells (APC) is determined by the presence of specific cell receptors, the endocytic activity, targeting of antigen to compartments of antigen processing and the initial form of the antigen (1,2). Moreover, the simple binding of antigens to selected cell-surface molecules may also affect the outcome of antigen presentation or the generation of the subsequent immune response. Understanding the pathway of antigen processing and the mechanisms by which antigen binding enhances processing will be useful in designing vaccination strategies or to modulate autoimmune diseases.

A number of cell surface receptors including, Ig, transferrin receptor, Fc{gamma}R and DEC-205 can mediate endocytosis and effective antigen presentation (35). More recently, glycosphingolipids including GM1 have also been implicated (6). In association with sphingomyelin and cholesterol, GM1 is concentrated in cell membranes in caveolae-like structures in lymphocytes and in caveolae in other cells (6,7). Cross-linking of GM1 by antibodies or by proteins such as cholera toxin induces the formation of membrane rafts and capping (8,9); these membrane domains appear to play roles in membrane sorting, signal transduction and cell adhesion (6).

Ganglioside GM1 is the main receptor for Escherichia coli enterotoxin (Etx) and the structurally and functionally related cholera toxin (Ctx) from Vibrio cholerae. These are oligomeric A–B toxins composed of an enzymatic part (A subunit) with ADP-ribosyltransferase activity and a pentameric binding domain (B subunit) responsible for binding the toxins to mammalian cells (10). Etx binds with high affinity to GM1 (Kd = 7.3x10–10), but has also been reported to bind weakly to asialo-GM1 and GD1b (11). Binding of the toxins to cells involves the exposed carbohydrate and sialic acid moieties of GM1; GM1 is anchored to the membrane by its ceramide tail (12).

Binding to GM1 on immune cells may explain some of the previously reported immunological properties of Etx and Ctx. Both toxins are potent immunogens in vivo (13,14). The B subunits alone mediate several of the immunological properties of Etx and Ctx (1517), but, importantly, are devoid of any toxic activity. Thus, administration of small amounts of EtxB or CtxB in the absence of any additional adjuvant elicits strong antibody responses in both mice (18,19) and humans (20). In vitro, Ctx and CtxB are strongly inhibitory for lymphocyte proliferation (21,22), although this effect is not seen with EtxB (18). Both Ctx and Etx can activate APC in vitro. For example, Ctx increases MHC class II expression and promotes isotype-switch differentiation in B cells (23,24). Prior treatment of peritoneal macrophages with either Ctx or CtxB enhanced subsequent presentation of native hen egg lysosyme (HEL) and HEL peptides to 3A9 T cell hybridomas in vitro (25). Ctx increases the expression of B7-2 on bone marrow-derived macrophages (26) and peritoneal macrophages respond with increased secretion of IL-1 (27). Although it is likely that some of these effects are related to the ability of these toxins to bind GM1 on the APC, no direct evidence has been presented. Interpretation of some of these results is also complicated by the presence of the A subunit that causes an increase of intracellular cyclic AMP.

Recent experiments compared effects exerted by recombinant EtxB and those by a non-binding mutant of EtxB different only by a single amino acid, EtxB(G33D). Binding of EtxB significantly enhanced the level of the antibody response in vivo (~160-fold) (18), and addition of purified recombinant EtxB to splenic cell cultures proliferating in response to ovalbumin (OVA) (28) resulted in increased numbers of activated (CD25+) B and CD4+ T cells, but the complete depletion of CD8+ T cells by apoptosis (18,28). Purified recombinant EtxB also induced the up-regulation of B7-2, MHC II, CD40 and CD25 on B cells (29). In contrast, EtxB(G33D) had no such stimulating effects on these molecules.

The findings above highlight the selective and direct effects of EtxB on immune cells following binding to GM1. However, direct involvement of GM1 in antigen presentation by different APC remains unclear. This study investigates the role of GM1 receptors in mediating presentation of EtxB by B cells and DC. The results implicate GM1 in several aspects of antigen presentation including enhanced antigen uptake, efficient targeting of antigen to MHC class II-rich compartments and increased B cell expression of MHC II. Binding of EtxB to GM1 on APC also augments the magnitude of the subsequent T cell proliferation and expression of cytokines. We further show that these GM1-mediated events can be exploited to significantly enhance the presentation of OVA coupled to EtxB. This is the first report that directly implicates GM1 in antigen presentation and investigates the pathways of EtxB presentation by purified APC.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals and immunization
BALB/c (Harlan-Olac, Bicester, UK) and NIH (NIH, Bethesda, MD) mice (8–10 weeks old) were used. For immunization, mice were injected i.p. 100 µg of EtxB emulsified in incomplete Freund's adjuvant. Between 10 and 14 days later mesenteric lymph nodes (MLN) were dissected out.

Antigens
A Gly33 -> Asp substitution was introduced into the GM1 binding site of EtxB (18). Recombinant human EtxB and the mutant protein, EtxB(G33D), were purified as reported previously (30). Identical physical and chemical properties were found for both antigens (18). Briefly, these included stability in SDS gels, retention time as analysed by high-resolution gel filtration chromatography, stability in low pH buffers and binding to a panel of antibodies. However, when compared with EtxB, EtxB(G33D) showed a highly significant reduction in binding to GM1 (>99% reduction in the A450 reading on microtiter plates) and failed to bind a number of cells, including mouse B lymphocytes (31 and this study) and T cells (T. O. Nashar, unpublished). The crystal structure of EtxB(G33D) revealed that it had the same overall three-dimensional structure as wild-type EtxB (32). Two techniques were used to measure lipopolysaccharide (LPS) content in the protein preparations including enhanced silver staining (33) and an enzymatic assay, kinetic QCL lysate kit (Biowhitaker, Walkersville, MD). Using silver staining no LPS was detected. QCL lysate kit detected very small, but comparable amounts of LPS, 2 ng/10 µg of protein in either EtxB or EtxB(G33D) preparations. Protein concentration was determined by UV spectrometry and was calculated using a theoretical exclusion coefficient for EtxB. Biotinylation of EtxB and EtxB(G33D) was performed using sulfosuccimidyl-6-(biotinamido)hexanoate linker (Pierce, Rockford, IL). Methods for the conjugation of the proteins were followed as described by the manufacturer for biotinylation of IgG.

Antibodies
The following antibodies were used (PharMingen, Cambridge, UK): phycoerythrin (PE)–anti-B220/RA3-6B2, FITC-labelled anti-I-Ad/AMS-32.1, biotin–anti-I-Ad/39-10-8, FITC–anti-CD40/HM40-3, FITC–anti-CD54 (ICAM-1)/3E2, biotin-conjugated CD86 (B7-2)/GL1, isotype-matched control FITC rat IgG2a/R35-95, hamster biotin–anti-mouse CD11c/HL3, biotin–polyclonal hamster IgG isotype control/G235–2356 and rat anti-mouse CD16/CD32 (Fc Block)/2.4G2. To detect EtxB or EtxB(G33D) on the surface of B cells, rabbit anti-EtxB and biotin-conjugated affinity pure (Fab')2 goat anti-rabbit IgG (H + L) (Immunotech, Marseille, France) were used. To detect biotinylated antibodies, ExtrAvidin–FITC conjugate (Sigma, Poole, UK) was used. To detect hµ, goat anti-hIgM–HRP conjugate (Southern Biotechnology Associates, Birmingham, AL) was used. For cell fractionation, MACS colloidal super-paramagnetic microbeads conjugated with monoclonal anti-mouse CD4, anti-mouse B220 or anti-mouse CD11c and MACS columns (all Miltenyi Biotec, Bergisch Gladbach, Germany) were used.

Fractionation of various cell populations
CD4+ T cells and B220+ cells were isolated from MLN and spleen respectively. Cell suspensions were recovered and incubated with MACS antibody-conjugated magnetic beads and then fractionated on columns according to instructions supplied by the manufacturer. Splenic B cells were positively selected with anti-B220 antibody. MLN CD4+ T cells were positively selected with anti-CD4 antibody-conjugated beads. The purity of the positively selected cells was >95% as revealed by flow cytometry (Becton Dickinson, Erenbodegem-Aalst, Belgium; CellQuest software) following staining with specific antibodies. Viability of cells was >98% as examined by Trypan blue dye exclusion.

To isolate fresh CD11c+ DC, MLN were incubated with 1 mg/ml collagenase D (Boehringer Mannheim, Mannheim, Germany) at 37°C for 30 min. Following incubation, cells were washed and resuspended in modified Eagle's medium (Gibco/BRL, Paisley, UK) containing 20 mM HEPES, 100 IU/ml Penicillin, 100 µg/ml streptomycin, 4 mM L-glutamine and 5x10–5 M 2-mercaptoethanol (complete medium) to which 10% FCS was added. Cells were then overlaid on 14.5% (w/v) metrizamide (Nycomed, Oslo, Norway) and separated by centrifugation at 800 g for 20 min. The low-density cells present at the interface were removed, washed and examined under light microscopy. The majority of cells (65–70%) appeared as large mononuclear cells or had DC `veiled' morphology. The remaining cells were small lymphocytes. In order to measure the proportion of CD11c+ in the low-density fraction, Fc receptors were blocked with anti-CD16/CD32 antibody for 10 min on ice and the cells were subsequently labelled with anti-CD11c antibody. Flow cytometry revealed that 40% of cells expressed the CD11c marker. Further enrichment of CD11c+ cells was achieved by using MACS beads conjugated to anti-mouse CD11c antibody. The vast majority of fractionated cells (90%) had DC morphology, high forward versus light scatter profile and expressed MHC class II, as revealed by flow cytometry. Contaminating cells were small lymphocytes (10%). Removal of the latter was further achieved when cells were allowed to adhere to plastic during incubation with antigens and subsequently washed (see below). This procedure ensured that the majority of lymphocytes and other non-adherent cells present in culture were removed before addition of T cells.

Assays for binding of EtxB and EtxB(G33D) on surface of B cells
Splenic B220+ cells were isolated by positive selection, as described above. Cells (6x105) were re-suspended in HBSS (Sigma, St Louis, MO) containing 0.2% azide (to stop internalization) and incubated on ice with increasing doses of either EtxB or EtxB(G33D) for 20 min. Cells were then incubated with rabbit anti-EtxB antibody for 30 min on ice. This was followed by addition of biotinylated (Fab')2 goat anti-rabbit IgG for 30 min and ExtrAvidin–FITC conjugate for an additional 15 min. Cells were then analysed by flow cytometry.

Assays for EtxB internalization and sub-cellular fractionation of B cells
A20µWT cells transfected with phosphorylcholine (PC)-specific human µ heavy chain were cultured as described (34). Biotin–EtxB and PC–OVA conjugates were added to cells on ice at 1 and 100 µg/ml respectively. Cells were incubated initially on ice for 20 min to allow binding, then for an additional 20, 60 or 120 min at 37°C. PC–OVA was added to cells in the last 20 min following incubation of EtxB on ice. When PC–OVA was not used, cells were incubated with EtxB for 20 min at 37°C. The remaining antigens were washed off, and cells were homogenized and ruptured as described (34,35). Briefly, cells were ruptured by nitrogen cavitation. After centrifugation, to remove intact cells and nuclei, subcellular components were overlaid above a Nycodenz discontinuous gradient (Gibco/BRL) made of 8, 16 and 24% to separate plasma membrane and class II vesicles (CIIV) from the MHC class II-enriched compartment (MIIC) (35). Fractions of 0.5 ml were then collected from top to bottom of the gradient. Details of the ELISA method used to detect class II and human µ in the fractions have been described previously (34). Biotin–EtxB was detected with Ultravidine–horseradish peroxidase (Leinco Tech, St Louis, MO).

Antigen presentation by B cells
Splenic B cells were resuspended in complete medium to which 0.5% (v/v) of fresh normal mouse autologous serum (MAS) was added. Cells were cultured at 2x106 viable cells/ml in 2 ml volumes in 24-well plates (Nunc). To investigate the dose effect of EtxB on proliferation, B cells were pulsed with 1, 10, 30, 80 and 160 µg/ml of either EtxB or EtxB(G33D) for 24 h. For each treatment, 6x105 cells were then cultured with 1.25x106/ml of purified CD4+ T cells for 4, 5 and 6 days. Maximal responses were detected on day 4. To investigate cytokine secretion following presentation of EtxB or EtxB(G33D), B220+ cells were pulsed with 80 µg/ml of the antigens as described above. Cells were washed and viable B cells were counted and incubated at increasing ratios with 1.25x106/ml of purified CD4+ T cells. T cell proliferation was measured on day 4 and 5. Maximal responses were detected on day 4, when samples for cytokine analysis were assayed.

In all the experiments above, proliferation was measured following addition of 1 µCi/well of [3H]thymidine (Amersham, Little Chalfont, UK) in the last 6 h before harvesting (Mach III harvesting 96; Tomtec, Orange, CT) and counting by standard liquid scintillation (1450 Microbeta Plus; LKB-Wallac, Turku, Finland).

Antigen presentation by CD11c+ DC
MACS purified CD11c+ cells were resuspended in complete medium to which 0.5% fresh MAS added. To establish cell culture, 105 CD11c+ cells were pulsed with 0.1, 0.5 and 1 µg/ml of either EtxB or EtxB(G33D) for 2 h. Adherent cells were washed inside wells and then incubated with 1x106/ml of purified CD4+ T cells isolated from mice injected with EtxB in incomplete Freund's adjuvant. In similar experiments where kinetics of proliferation in these cultures was determined, the maximum level occurred on day 4. On the latter day, triplicate samples of 0.1 ml of cells were removed from the cultures and pulsed with 1 µCi/well of [3H]thymidine (Amersham) for 6 h before harvesting and counting by standard liquid scintillation.

For the analysis of cytokines duplicate samples were isolated on day 4, and expression of IL-4 and IFN-{gamma} was determined.

Assay for secreted cytokines
A cell-based sandwich ELISA method for measuring cytokine levels of IL-2, IL-4, IL-5 and IFN-{gamma} was used, as described previously (36). Briefly, duplicate 0.1 ml samples of cells were transferred under sterile conditions to wells coated with anti-cytokine-specific antibody. Cells were incubated overnight at 37°C and then washed. Plates included wells to which a serial dilution of standard cytokines was added. To detect secreted cytokines, biotinylated anti-cytokine antibodies followed by addition of Extravidine–peroxidase and tetramethylbenzidine as substrate were used. The level of each cytokine in the test samples was determined by PROBIT regression analysis from standard cytokines.

Labelling of isolated B cells for cell-surface markers
Splenic B220+ cells were isolated as described above. Cultures were established at 2x106/ml in complete medium to which 0.5% MAS was added. Cells were cultured in the presence or absence of 40 µg/ml of EtxB or EtxB(G33D) for 48 h. They were washed, counted, and then labelled on ice with PE–anti-B220 and FITC-conjugated antibodies directed against MHC class II, B7-2, CD40 and ICAM-1. Controls included unlabelled cells from cultures incubated with EtxB or cells incubated with rat isotype-matched antibodies. Following washing, cells were analysed by flow cytometry by gating on B220+ cells.

Conjugation of OVA
OVA (Sigma) was reduced with DTT to expose SH groups and run on a Sephacryl 300 column to separate out non-monomeric fractions. Fractions were pooled and dialysed against PBS until the OVA:SH ratio was 2.0. The number of SH groups was determined by the Ellman assay. To conjugate OVA to EtxB, EtxB was reacted with GMBS (Pierce) at a 1:4 molar ratio and loaded on G25 column to separate out excess of GMBS. Subsequently, OVA was mixed with EtxB–GMBS at 1:1 molar ratio. The reaction was stopped with 50 mM ß-mercaptoethanol. The mixture was run on Sephacryl 300 column. Fractions were analysed on a Western blot using anti–OVA or anti-EtxB to examine the presence of conjugated material. To determine the concentration of the conjugated material, fractions were pooled and run on a Sephadex 200 column to obtain the retention volume that was used to determine the molecular weight. The ratio of EtxB:OVA was estimated to be 1:2.7.

OVA was conjugated to PC as described (34).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
GM1-mediated antigen presentation by B cells enhanced efficiency of presentation of EtxB to CD4+ T cells
The priming with EtxB in vivo induces an EtxB-specific recall response in lymph node cell culture (18). Therefore, we investigated whether GM1-mediated presentation by purified B cells was essential for the EtxB-induced proliferation.

In order to examine the binding properties of various doses of EtxB, MACS purified splenic B220+ cells were incubated with 0.5, 1, 10, 30, 80, 160 and 320 (not shown) µg/ml of either EtxB or EtxB(G33D), in the presence of sodium azide and on ice. Following washing, the presence of bound EtxB or EtxB(G33D) was determined by flow cytometry. In comparison with EtxB, little or no binding of EtxB(G33D) was detected in the range of 0.5–10 µg/ml (Fig. 1Go). This agrees with previous reports on the inability of EtxB(G33D) to bind GM1 on B cells (31), to plastic dishes coated with GM1 (18) or to other cells including T cells (T. O. Nashar, unpublished). Within this dose range, EtxB binding demonstrates two populations with different densities of GM1 and representing precursor versus mature B cells.



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Fig. 1. Specificity of binding of increasing doses of EtxB to GM1 on B cells. Pure splenic B220+ cells (6x105 cells) were incubated on ice for 20 min with increasing doses of either EtxB or EtxB(G33D) in the presence of 0.2% sodium azide. Cells were then washed and incubated with rabbit anti-EtxB antibody for 30 min, followed by addition of biotinylated (Fab')2 goat anti-rabbit IgG and ExtrAvidin–FITC conjugate for an additional 30 and 15 min respectively. Cells were analysed by flow cytometry.

 
In contrast to the specific binding activity of EtxB in the dose range of 0.5–10 µg/ml, higher doses (30–320 µg/ml) resulted in the binding of both EtxB and EtxB(G33D). The non-specific adherence occurring at that concentration is likely due to increased hydrophobicity of these antigens. Similar results were obtained in independent experiments using biotinylated EtxB and EtxB(G33D) and B cell lines (not shown).

To investigate GM1-mediated antigen presentation of EtxB, splenic B220+ cells were pulsed with 1, 10, 30, 80 or 160 µg/ml of either EtxB or EtxB(G33D) for 24 h. Following washing, 6x105 pulsed B cells were incubated with 1.25x106 EtxB-primed CD4+ T cells, and the T cell proliferation and cytokine response was measured after 4 days. At all doses examined, presentation of EtxB was more effective than EtxB(G33D) (Fig. 2AGo) even at high doses where non-specific binding occurred. EtxB resulted in significantly greater proliferation than EtxB(G33D) suggesting that specific binding of EtxB to GM1 results in improved antigen processing and/or presentation. The experiment was also performed with a fixed concentration of antigen and increasing cell number. Splenic B cells were isolated and incubated with 80 µg/ml of either EtxB or EtxB(G33D). Cells were washed and then incubated at increasing concentration with EtxB-primed CD4+ T cells. Presentation of pulsed B cells was significantly more effective than that of EtxB(G33D), with increasing T cell proliferation correlating with increasing B cell number (Fig. 2BGo).



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Fig. 2. Binding to GM1 enhances presentation of EtxB by primary B cells. Pure splenic B220+ cells (6x105) or increasing numbers of these cells were incubated with increasing doses (A) or with 80 µg/ml (B) of either EtxB or EtxB(G33D). Cells were washed and incubated with 1.25x106 pure MLN CD4+ T cells, isolated from mice previously immunized with EtxB. Representative proliferation results from these cultures are shown on day 4, when maximum response was detected. Cytokine secretion was examined under conditions where proliferation was maximum, i.e. at higher doses of 80 µg/ml of EtxB and EtxB(G33D), and on day 4 (C). All cells we pulsed with 1 µCi/well of [3H]thymidine in the last 6 h before harvesting and counting by standard liquid scintillation. Data on proliferation represent mean of triplicate samples ± SEM. Data on cytokines represent mean values of duplicate samples ± variance extrapolated from a standard curve generated for each of the cytokines.

 
Analysis of the cytokine profile in these cultures demonstrated that presentation of each of the antigens induced secretion of IL-2, IL-4 and IL-5 without detectable IFN-{gamma} (Fig. 2CGo). Cytokine production was 4-, 7- and 2-fold greater (IL-2, IL-4 and IL-5 respectively) after incubation with EtxB compared with EtxB(G33D).

It is noteworthy that the results in Fig. 2Go(A and B) represent direct effects of EtxB on B cells and were not complicated by EtxB effects on T cells as the APC were thoroughly washed following incubation with the antigens. Further, the detectable higher levels of IL-2 in cultures containing EtxB supports that T cells were responsible for the observed proliferation in these cultures. We conclude that specific binding of GM1 by EtxB on primary B cells enhances the presentation of EtxB to CD4+ T cells, resulting in greater T cell proliferation and cytokine production.

Binding of EtxB up-regulates expression of MHC class II
To investigate possible mechanisms responsible for the enhanced GM1-mediated presentation of EtxB in primary B cells, the effects of EtxB and EtxB(G33D) on the expression of co-stimulatory molecules including MHC class II, B7-2, CD40 and ICAM-1 were examined. Incubation with EtxB, but not EtxB(G33D) resulted in increased expression of MHC class II (Fig. 3Go). However, there were no effects of EtxB or EtxB(G33D) on the other B cell co-stimulatory molecules (not shown). Similar results were found regardless of whether splenic B cells were purified by either positive or negative selection. Despite the lack of effects on co-stimulatory markers other than class II, the addition of EtxB to lymphocyte cultures resulted in increased expression of CD25 on B cells (28,29). Increased expression of CD25 by EtxB would result in activation of B cells following binding its ligand IL-2. Similarly, expression of higher level of class II would result in expression of higher density of T cell ligands. Thus EtxB–GM1 interaction results in the activation of B cells including their higher expression of class II that contribute to EtxB enhanced presentation.



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Fig. 3. Binding to GM1 up-regulates expression of MHC class II on primary B cells. Pure splenic B220+ cells were incubated at 2x106/ml with 40 µg/ml of either EtxB or EtxB(G33D). After 48 h cells were harvested, washed, counted, and then labelled with PE–anti-B220 and FITC–anti-class II (I-Ad). Class II expression was analysed by gating on B220+ cells. The control sample (small window) represents unlabelled B cells incubated in the presence of EtxB.

 
Binding to GM1 targets EtxB to MHC class II-rich compartments
While EtxB-induced up-regulation of MHC class II can enhance antigen presentation, we also examined whether binding to GM1 results in improved internalization and targeting to class II-rich compartments.

In murine A20 B cells containing a transfected PC-specific human µ surface Ig, internalization and processing of BCR bound antigens occurs in class II-rich early and late endosomal compartments of CIIV and MIIC respectively (34,35). To examine whether GM1-mediated uptake targets efficiently EtxB to the same class II-rich compartments, A20µWT (A20 cells transfected with IgM anti-PC) were incubated with 1 µg/ml of biotin-labelled EtxB initially for 20 min on ice, and then for a further 20, 60 and 120 min. Using biotin-labelled EtxB(G33D) no non-specific binding of antigen to A20µWT occurred (not shown). Internalization of EtxB was examined in these cells either under conditions where the hµ was cross-linked with 100 µg/ml of PC–OVA conjugate (Fig. 4a–eGo) or in the absence of the latter (Fig. 4fGo). The conjugate was added in the last 20 min of incubation of B cells with EtxB on ice. Cells were then homogenized and total cell membranes were fractionated on continuous Nycodenz gradients as previously described (35). PC-specific hµ, class II and EtxB were subsequently detected by incubating subcellular fractions with anti-hµ–HRP and biotinylated anti-I-Ad specific antibodies and with Ultravidin–HRP (class II and EtxB).



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Fig. 4. Binding to GM1 targets EtxB to MHC class II-rich compartments. A20µWT B cells transfected with PC-specific hµ antibody were incubated with 1 µg/ml of biotin-labelled EtxB for 20 min on ice to allow binding followed by incubation at 37°C for additional 20, 60 or 120 min. Internalization of EtxB was examined either under conditions where hµ was cross-linked with PC–OVA conjugate (a–d) or in its absence (f). PC–OVA (100 µg/ml) was added to cells in the last 20 min following incubation of EtxB on ice. To examine internalization of EtxB in the absence of hµ effects (f) cells were incubated with EtxB for 20 min at 37°C, as described above. Non-internalized antigen was washed off and the cells were homogenized and ruptured by nitrogen cavitation as described (34). Subcellular components were then separated from nuclei and intact cells by centrifugation, and subsequently overlaid on a discontinuous Nycodenz gradient made of 8, 16 and 24%. Aliquots of 50 µl from collected fractions were dried onto ELISA plates and probed with biotin–anti-class II (I-Ad) and goat anti-µ–peroxidase conjugate. Class II and EtxB were subsequently detected with Ultravidine–peroxidase. Data are expressed as percentage of total activity in all fractions. Fractions represent class II-rich early endosomes (CIIV), plasma membrane (PM) and late endosomes (MIIC), as previously described (35).

 
As shown in Fig. 4Go, class II activity was detected within two major compartments between fractions 4–9 (late endosomes) and 14–20 (plasma membrane and CIIV) as described (35). The distribution of class II in fractions 5–9 represents the class II that is rich in cathepsin B and contains low levels of the enzymatically active form of the lysosomal enzyme ß hexoaminidase (35). The position of the plasma membrane coincides with that of hµ [(35) fractions 14–17 in this study (see below)]. On the other hand, CIIV is an early endosomal compartment (34,35) that displays lower class II activity and is identified by its putative marker protein (i.e. p50Iga). Fractions 18–20 in this study coincide with the position of CIIV.

After 20 min internalization at 37°C a portion of hµ was present in CIIV and late endosomes, with the remainder on the cell surface (Fig. 4bGo) similar to that previously reported (34,35).

Following initial incubation on ice to allow for binding, internalization of biotinylated EtxB was examined in A20 cells, in the presence of PC–OVA, 20, 60 and 120 min at 37°C (Fig. 4c–eGo). As early as 20 min after incubation, EtxB was detected in fractions 2–12 and 15–19 corresponding to late endosomes and plasma membrane/CIIV respectively. After 60 min, fractions 4–9 and 14–20 still had detectable activity of EtxB, but with the appearance of high level of activity in fraction 11. The appearance of the latter peak was not consistent in two out of three similar experiments. After 120 min, the bulk of the remaining EtxB activity was present in late endosomes. The activity in fractions collected at `0' min (37°C) followed similar distribution as described for 20 min incubation of the cells with EtxB (not shown). This rapid internalization of EtxB is likely induced by its capping surface GM1.

The results demonstrate that binding of EtxB to GM1 enables it to enter effectively MHC class II-rich compartments. Further, the pattern and the kinetics by which EtxB was distributed in the various fractions suggests a progressive access of EtxB from an early to a late compartment that represent CIIV and MIIC respectively.

As hµ internalization may influence uptake and targeting of EtxB, we examined whether EtxB could access the class II-rich compartment in the absence of any effects from the latter (Fig. 4fGo). A20µWT cells were incubated with 1 µg/ml of biotin-labelled EtxB initially for 20 min on ice and then for an additional 20 min at 37°C. Fractionation of the cells and detection of EtxB activity in the subcellular fractions were performed as described above. Consistent with the results in Fig. 4Go(a and c), high levels of activities of EtxB and MHC class II were detected in the late endosome and plasma membrane/CIIV as early as 20 min after incubation at 37°C. These were detected in fractions 4–6 and 17–20 respectively. The results suggest that effective access of EtxB into the class II-rich compartments is independent of cross-linking hµ.

GM1-mediated antigen presentation by CD11c+ DC
Having shown above that GM1-mediated presentation of EtxB by B cells effectively enhanced its presentation, we examined its presentation by DC that are known to be potent APC.

Binding to GM1 on DC significantly enhances presentation of EtxB to CD4+ T cells
To investigate the role of GM1 in presentation by DC, CD11c+ cells were isolated from MLN and incubated with 0.5, 0.1 or 1 µg/ml of EtxB or EtxB(G33D). Adherent cells were subsequently washed to remove non-internalized antigens and were mixed with CD4+ T cells isolated from mice injected with EtxB in incomplete Freund's adjuvant. As shown in Fig. 5Go, presentation of EtxB by DC resulted in a significant increase in the level of T cell proliferation compared with equivalent doses of EtxB(G33D). In concert with increased T cell proliferation, EtxB presentation resulted in augmented IFN-{gamma} and IL-4 production.



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Fig. 5. Binding to GM1 enhances presentation of EtxB by DC to CD4+ T cells. Freshly isolated CD11c+ DC were incubated for 2 h with 0.1, 0.5 and 1 µg/ml of either EtxB or EtxB(G33D) and washed. MACS pure MLN CD4+ T cells (1x106) isolated from EtxB-primed mice were added to each well. Proliferation and IL-4 and IFN-{gamma} secretion into supernatants was measured on day 4. Data are presented as mean of triplicate ± SEM (proliferation) or average cytokine value of duplicate samples ± variance extrapolated from a standard cytokine curve.

 
Improved GM1-mediated presentation of EtxB can be exploited to enhance presentation of other antigens.
To examine whether the improved presentation of EtxB could be exploited to enhance uptake and targeting of other soluble proteins into the processing compartments, the presentation of OVA coupled to EtxB was investigated. A20 cells were incubated with increased concentrations of EtxB–OVA, EtxB(G33D)–OVA or an equimolar dose of non-conjugated OVA. Incubation was initially on ice after which cells were washed with medium and incubated for a further 2 h at 37°C. After overnight incubation of the APC with OVA-responsive DO.11, proliferation of IL-2-responsive HT-2 cells was determined. Results from these experiments (Fig. 6Go) show that the presentation of OVA was significantly enhanced when conjugated to EtxB. The activation of OVA-responsive T cells following presentation of OVA conjugated to EtxB(G33D) or free OVA was similar to those of the medium control. Under these experimental conditions (2 h incubation at 37°C, up to 60 µg/ml of OVA) a response to OVA above background was not detected. It is noteworthy that the addition of EtxB with non-conjugated OVA had no effects on its kinetics of processing and presentation (not shown). Thus, GM1-meditated uptake of proteins conjugated to EtxB results in their efficient delivery into the pathways of antigen processing and presentation.



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Fig. 6. Binding of EtxB to GM1 enhances presentation of conjugated OVA. A20 cells were incubated with increasing concentration of EtxB–OVA, EtxB(G33D)–OVA or an equimolar amount of free OVA. Incubation was initially for 1 h on ice after which cells were washed with medium and incubated for a further 2 h at 37°C. The APC were then incubated with OVA-responsive DO.11, and proliferation of IL-2-responsive HT-2 cells was determined. Data represent mean of triplicate samples ± SEM. Under these experimental conditions no response to free OVA above background was detected.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The role of GM1 in antigen presentation was examined by comparing the effects of EtxB, a protein with high affinity for GM1, versus those of a non-binding variant, EtxB(G33D). We demonstrated that binding of EtxB to APC is intimately involved in several aspects of antigen presentation, including uptake and internalization of antigen into MHC class II-rich compartments, and expression of MHC II on APC, with a resulting augmentation in T cell proliferation and cytokine production. The involvement of GM1 in antigen processing and presentation was further demonstrated by efficient presentation of additional antigens such as OVA when coupled to EtxB.

How does antigen uptake through GM1 lead to more effective presentation in B cells? First, binding to GM1 results in enhanced rate of uptake. B cells can internalize any soluble antigen by pinocytosis, subsequently processing and presenting them as peptide–MHC complexes to T cells (37). However, pinocytic antigen uptake is inefficient and large amounts of antigen are ultimately required. Specific receptors on B cells, macrophages and DC (BCR, Fc receptor, complement receptor, scavenger or mannose/fucose family of receptors) bind and internalize antigens much more efficiently (1,2). Presumably antigen uptake by these receptors is essential when the dose of antigen is limited. However, while binding to surface receptors mediates efficient antigen presentation, ligation of some receptors interferes with APC activation. Thus, antibodies that cross-link sIg and Fc{gamma}RII receptors on B cells also recruit tyrosine phosphatases, and thereby inhibit normal BCR signalling (38,39). Similarly, other receptors such as CD19 and CD22 either amplify or restrict T-dependent responses respectively (40). Although binding to surface receptors can lead to improved antigen internalization, this alone is not sufficient to ensure efficient antigen presentation. Thus, binding of CtxB to GM1 on B cells did not necessarily result in its internalization into class II-rich late endosomal compartments (41). Moreover, mutation of a single transmembrane residue that disrupted normal BCR sorting resulted in efficient uptake, but unproductive antigen degradation (42), indicating that internalized antigens do not necessarily access class II MHC compartments following endocytosis.

An important role of GM1 in antigen uptake is suggested by the finding that small doses of EtxB (0.5–10 µg/ml) bound to B cell surfaces were efficiently presented while comparable concentrations of the non-binding mutant EtxB(G33D) were not (Figs 1 and 2GoGo). Moreover, GM1-mediated events occurring at higher doses of EtxB (30–160 µg/ml) may also contribute to effective antigen presentation. Thus, B cells exhibited increased MHC II expression, and augmented T cell responses at high doses of EtxB (Figs 1–3GoGoGo). The effects of high doses are unlikely to be attributable to the detectable small amounts of LPS in EtxB preparations. This is because both EtxB and EtxB(G33D) samples contained comparable amounts of this mitogen, and optimal activation of B cells was achieved by much greater concentration of LPS (50 µg/ml) (29).

Enhanced expression of MHC class II has also been reported following incubation of B cells with the closely related proteins, CtxB (43) and Ctx (23). These secondary effects of GM1 ligation may be explained by recent findings that GM1-containing glycolipid microdomains in the membrane are associated with signalling molecules such as Lck, Fyn and Lyn, and may mediate signalling events in B and T cells (4446).

The finding that EtxB presentation by B cells induces the secretion of IL-2, IL-4 and IL-5, but not IFN-{gamma} (Fig. 2CGo), is consistent with previous observations that antigen presentation by B cells results in the secretion of predominatly Th2 cytokines (47). The expression of higher densities of peptide–MHC complexes on B cell surfaces has been shown to result in augmented IL-4 production relative to IFN-{gamma} (48).

Events subsequent to the binding of EtxB and its related toxins are not fully elucidated. However, GM1 is involved in mediating internalization of Ctx through cholesterol and glycolipid-rich microdomains in the cell membrane (8). Moreover, internalization of CtxB and Ctx involves an actin-based cytoskeleton and is mediated through non-coated pits (9,49). Using subcellular fractionation techniques, we showed here that binding of EtxB to GM1 results in its rapid internalization and targeting to MHC class II-rich compartments MIIC and CIIV, previously shown to be involved in the processing of exogenous antigen (35,50).

The results in Fig. 4Go regarding the distribution of class II, internalized hµ and EtxB suggest that both CIIV and MIIC are efficiently accessed by EtxB, and that EtxB eventually accumulates in the latter. It is noteworthy that although internalization of EtxB into the class II-rich compartments was found to be independent of hµ cross-linking, internalization of hµ appears to enhance GM1 access into the class II compartments (41). This raises the interesting possibility that EtxB and CtxB (which was used as a marker in the previous study) may differ in the way they are internalized. In this regard, it is well documented that EtxB, unlike CtxB, not only binds to GM1 but also binds weakly to glycoprotein receptors (11). The contribution of these receptors in EtxB internalization remains to be determined.

Further evidence implicating a role for GM1 in antigen presentation is provided by the experiments involving the presentation of EtxB by CD11c+ DC (Fig. 5Go). Thus, the differences in the level of T cell proliferation between EtxB and EtxB(G33D) again suggests more effective antigen presentation when bound to GM1. Potential mechanisms for the enhanced antigen presentation include augmented targeting of bound antigen, or effects on the expression of surface molecules [e.g. MHC class II on B cells (Fig. 3Go)]. However, CD11c+ cells isolated from lymph nodes express intrinsically high levels of MHC I and II, and abundant accessory molecules such as CD54, CD40 and CD86 (51). The findings therefore suggest that the enhanced presentation of EtxB results from more effective targeting and access to compartments of antigen processing and MHC loading.

The findings above implicate GM1 in immune cellular events including uptake and presentation, cytokine secretion, and generation of downstream cell signals. In this study we used a mutant protein of EtxB that is unable to bind to GM1 on a variety of cells or in a GM1 ELISA. However, as a specific antibody to GM1 is currently unavailable we cannot rule out binding of EtxB to other receptors, albeit with much lower affinity (11). Further, due to the wide distribution of GM1 on various type of cells involved in an immune response in vivo, it is likely that the outcome of presentation of bound antigens is determined not only by the APC but also by their regulatory effects on other cells and their products. For example, binding of EtxB to CD8+ T cells induces their depletion in proliferating cultures by apoptosis (18,28). Nevertheless, the experiments involving pre-incubation of APC with EtxB strongly suggest that the efficacy of EtxB-augmented antigen presentation involves APC and not the responding T cells.

The findings above also indicate that targeting GM1 on the APC may be exploited to enhance responses to other antigens. Evidence in support of this includes in vivo studies on the administration of soluble antigens chemically linked to EtxB or its related toxins. In this case, conjugation of antigens to the toxins resulted in augmented immune responses (52,53). Whether a similar strategy can be used to modulate the outcome of immunological responses to auto-antigens remains unclear. Nevertheless, it is promising that administration of CtxB conjugated to either myelin basic protein or to insulin ameliorated experimental autoimmune encephalomyelitis (54) and clinical diabetes (55) respectively.


    Acknowledgments
 
The excellent technical assistance and advice of Mrs Jill Tarlton and Miss Tamera Jones are greatly appreciated. We also would like to thank Professor Tim Hirst and Dr N. A. Williams for discussions during the performance of some of these experiments. This work is supported by a Research Career Development fellowship awarded to T. O. N. by the Wellcome Trust, UK and NIH RO1 GM 47726 to R. N. M.


    Abbreviations
 
APC antigen-presenting cell
BCR B cell receptor
CIIV class II vesicles
DC dendritic cell
hµ human IgM
HEL hen egg lysosyme
LPS lipopolysaccharide
MIIC MHC class II-enriched compartment
MAS mouse autologous serum
MLN mesenteric lymph node
OVA ovalbumin
PC phosphorylcholine
PE phycoerythrin

    Notes
 
Transmitting editor: D. T. Fearon

Received 7 September 2000, accepted 8 January 2001.


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