1 Immunobiology Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata 700 032, India
2 Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India
3 Department of Organic Chemistry and 4 Cell Physiology Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata 700 032, India
5 Department of Pathology, Vivekananda Institute of Medical Sciences, 99 Sarat Bose Road, Kolkata 700 019, India
Correspondence to: C. Mandal; E-mail: cmandal{at}iicb.res.in
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Abstract |
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Keywords: anti-9-OAcSGs, effector function, glycosylation, subclass
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Introduction |
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Sialic acids (originally abbreviated as Neu5Ac), a family of acidic nine-carbon sugars, are typically located at the terminal positions of glycoconjugates of cell membranes and play a significant role in the mediation of many biological phenomena involving cellcell interactions either by reacting with specific surface receptors or via masking of carbohydrate recognition sites (3, 4). Among the diverse multitude of variations of Neu5Ac, the most frequently occurring modification is O-acetylation at positions C-7, -8 and -9 to form N-acetyl-7, -8, -9-O-acetyl neuraminic acid, respectively, thus generating a family of O-acetylated sialoglycoconjugates (O-AcSGs) (5). However, as O-acetyl esters from C-7 and C-8 positions spontaneously migrate to C-9 position, even under physiological conditions, the O-acetylation at C-9 position [9-O-acetylated sialic acid (9-OAcSA)] is considered as the commonest biologically occurring modification (6, 7).
The binding specificity of Achatinin-H, a 9-OAcSA26GalNAc-binding lectin, purified from the hemolymph of the African giant land snail, Achatina fulica (811), allowed us to identify an enhanced expression of five molecules having terminal 9-OAcSA corresponding to 36, 90, 120, 135 and 144 kDa on PBMC of ALL patients; a basal level of expression of only two molecules having terminal 9-OAcSA (36 and 144 kDa) was identified on PBMC from normal individuals (3, 7, 12, 13). An increased antibody (anti-9-OAcSGs) production against these glycoproteins in these children confirmed its immunogenicity (1416). Importantly, no cross-reactivity was observed with patients having other hematological disorders (3, 1216). At disease presentation, high titers of immune-complexed 9-OAcSGs were observed in these patients' sera (17). These circulating anti-9-OAcSGs (free as well as immune complexed) have been successfully utilized for diagnosis and monitoring of the disease status (1317).
A central question in cancer immunology is whether recognition of tumor antigens by the immune system initiates activation (i.e. surveillance) or functional unresponsiveness. Paradoxically, while strong evidence exists that specific immune-surveillance systems operate at early stages of tumorigenesis, immune unresponsiveness is a feature of established tumors (18). It is now recognized that tumors can directly or indirectly impede the development of anti-tumor immune responses through immunosuppressive cytokines (transforming growth factor-ß and IL-10), T cells with immunosuppressive activities (regulatory T cells, 19), inactivation of death receptor signaling pathways (20) or expression of anti-apoptotic signals (21). Tumor escape can also result from changes that occur directly in the tumor, such as loss of antigen expression, loss of MHC components (22) and development of IFN insensitivity (23). This induction of unresponsiveness by tumors can occur either through anergy or through deletion from resistance mechanisms to recognition and killing of tumor cells by activated immunological effectors. In pediatric ALL, the role of antibodies induced against disease-associated glycotopes remains obscure and accordingly, this study aims to explore their role in immune surveillance to understand the disease biology.
The present study has focused on (i) affinity purification of anti-9-OAcSGs, (ii) analysis of their subclass distribution, (iii) determination of their total glycosylation and sialylation profile and (iv) evaluation of the functional activity exerted by anti-9-OAcSGs.
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Methods |
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Cell lines
The REH (human B-ALL) and U937 (human promonocytic) cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). The CEM-C7, a human T-ALL cell line, was a kind gift from Reinherd Schwartz-Albiez, Deutsches Krebsforschungszentrum, Heidelberg, Germany (13). The cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FCS, 0.002 M L-glutamine, antibiotics and antimycotics (medium A).
Chemicals and reagents
All chemicals unless stated otherwise were purchased from Sigma, St. Louis, MO, USA.
Probes
Preparation of bovine submaxillary mucin and its derivatives. Bovine submaxillary glands were used for purification of bovine submaxillary mucin (BSM) (25), and protein content was measured (26). The percentage of 9-OAcSA was demonstrated by fluorimetric HPLC (27) and quantified as 22.5% by fluorimetric estimation (28).
BSM was de-sialylated (asialo BSM) and de-O-acetylated (de-OAc BSM) by incubating with 0.25% H2SO4 (0.045 M) for 1 h at 80°C and by alkaline hydrolysis with NaOH (0.01 M) for 1 h at 4°C, respectively.
Purification of Achatinin-H. Following activation of Sepharose 4B (Amersham Biosciences, Uppsala, Sweden) (29), activated beads were coupled separately with BSM and asialo BSM (5 mg ml1 gel). Achatinin-H was affinity purified from the hemolymph of A. fulica snails using BSMSepharose 4B (8) as it contains a high proportion of 9-OAcSAs linked with subterminal N-acetyl galactosamine (GalNAc) of the underlying oligosaccharide chain (30). Carbohydrate-binding specificity of purified Achatinin-H toward 9-OAcSA26GalNAc was established (8) and used as a probe in FACS analysis and for affinity purification of 9-OAcSGs.
Purification and characterization of anti-9-OAcSGs. Pooled sera (10 ml) from ALL patients at presentation of the disease, i.e. before any treatment and normal controls, were used to purify anti-9OAcSG fractions with preferential affinity for 9-OAcSA
26GalNAc using the method of Pal et al. (15). Briefly, serum was subjected to a 33% ammonium sulfate fractionation and sequentially passed through asialo BSMSepharose, BSMSepharose and Protein ASepharose column (Amersham Biosciences, Uppsala, Sweden). Different subclasses of IgG were eluted with sodium citrate (0.1 M) using a pH gradient that ranged from pH 6.01.0 followed by immediate neutralization with 2.0 M Tris and extensive dialysis against PBS. Purity was tested by SDSP (10%, 31) and subsequent western blotting using HRP-conjugated goat anti-human IgG (Jackson ImmunoReasearch Laboratories, Inc., West Grove, PA, USA). The specificity of Ig subclasses of all these purified fractions was confirmed by an ELISA.
Specificity of purified anti-9-OAcSGs toward 9-OAcSA26GalNAc
Hemagglutination activity. The biological activities of the purified antibody fractions were checked by hemagglutination (HA) (8). The HA titer was reported as the reciprocal of the highest antibody dilution giving complete agglutination.
The sugar specificity of purified anti-9-OAcSGs was confirmed using different sugars (8), and the concentration of sugar needed for 100% inhibition of HA was determined using a fixed concentration (16 HA units) of purified antibody.
Flow cytometric analysis revealed binding of purified antibodies to 9-OAcSGs on PBMC of ALL patients. Purified anti-9-OAcSGs were conjugated with FITC (32). The binding of FITCanti-9-OAcSGs (0.5 µg per 106 cells) to cell lines, B- (REH) and T-ALL (CEM-C7, MOLT-4) and PBMC from both B- (n = 22) and T-ALL (n = 6) patients along with PE-conjugated anti-CD 19 or anti-CD 7 (known as B- and T-cell marker, respectively, BD, Palo Alto, CA, USA) was examined by staining with respective probes for 1 h at 4°C in the dark (33). The binding was assessed using a FACSCalibur flow cytometer (BD, Palo Alto, CA, USA) and analyzed by CELLQUEST software (BD, Palo Alto, CA, USA). To confirm the epitope specificity of purified Igs, cells were pre-incubated with cold Achatinin-H or pre-treated with O-acetyl esterase, an enzyme capable of cleaving the 9-O-acetyl group from Neu5Ac (27), and binding of FITCanti-9-OAcSGs was determined. Isotype-matched antibodies served as controls. For each analysis, 10 000 events were recorded.
Confocal microscopy demonstrated binding of purified anti-9-OAcSGs to ALL cell surface. To visualize the binding of purified anti-9-OAcSGs to PBMC from both B- and T-ALL patients and T- and B-ALL cell lines (CEM-C7, MOLT-4 and REH), cells were incubated with FITC-antibodies and observed under a confocal microscope. Briefly, cells (1 x 105 cells per 50 µl) were adhered on poly-L-lysine-coated glass cover slips for 1 h at 2025°C, fixed with PFA (1%, 100 µl) for 30 min on ice followed by incubation with ammonium chloride (50 mM, 100 µl) for 5 min on ice. After washing with PBS, they were incubated with FITCanti-9-OAcSGs (0.005 µg) for 20 min on ice in the dark. After washing and mounting, cells were examined under a confocal scanning microscope (Leica SP2, Leica, Wetzlar, Germany); the slides were illuminated with a 488-nm laser, and images were collected using a band-pass filter (505550 nm) with the pinhole set at 100 µm.
Quantification of circulating anti-9-OAcSGs. Microtiter plates were coated with purified BSM (1.0 µg per 100 µl per well) in 0.02 M phosphate buffer, pH 7.4, overnight at 4°C. Sera (1 : 10 dilution) were added to the BSM-coated wells, whose non-specific binding sites had been previously blocked with PBS containing 2% BSA, and incubated overnight at 4°C. The plates were washed and incubated with different subclasses of murine anti-human IgG (diluted 1 : 3000, 100 µl per well, Dianova, Hamburg, Germany), and specific bindings were captured using HRP-conjugated anti-murine IgG (diluted 1 : 10 000, 100 µl per well, (Jackson ImmunoReasearch Laboratories, Inc., West Grove, PA, USA) and detected at 405 nm in a Multiskan MS Lab Systems ELISA reader using azino-bis thio-sulfonic acid (Roche Molecular Biochemicals, Mannheim, Germany) as the substrate. Standard curves generated using increasing amounts of affinity-purified anti-9-OAcSGs (020 µg per 100 µl per well) were applied for quantitation of anti-9-OAcSGs present in sera.
Analysis of glycosylation of affinity-purified anti-9-OAcSGs
Detection of total glycosylation and sialylation by digoxigenin enzyme assay. The degree of glycosylation and sialylation was evaluated by placing an equal amount (1.0 µg per 5.0 µl) of IgGALL and IgGN by a digoxigenin (DIG) enzyme assay using a DIG glycan detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to manufacturer's instructions. DIG-labeled glycoconjugates, present on nitrocellulose paper, were detected using alkaline phosphatase-conjugated anti-DIG antibody and visualized with 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate as substrate. The developed spots were scanned and quantified in arbitrary units using Image Master Totallab Software, version 1.11 (Amersham Pharmacia Biotech, Sweden). Equal amounts (5.0 µg per 5.0 µl) of transferrin and creatinase were used as positive and negative controls, respectively.
Quantitation of total Neu5Ac and 9-OAcSA by fluorimetric estimation. Quantitation of total Neu5Ac and 9-OAcSA was performed fluorimetrically (28) by oxidizing purified antibodies (3.0 µg) and processed using acetyl acetone method with or without saponification of the O-acetyl groups of Neu5Acs. The relative fluorescence intensity [max(excitation=410nm)/
max(emission=510nm)] of each sample was measured against reagent blanks on a Hitachi F-4010 spectrofluorimeter (Tokyo, Japan). The Neu5Ac content was determined from standard curves obtained using pure Neu5Ac. The values obtained for the de-O-acetylated samples indicated total Neu5Ac content, while the percentage of (8)9-O-acetylated Neu5Ac was determined by subtracting the respective unsubstituted Neu5Acs from that obtained after de-O-acetylation.
Detection of neutral sugars by gasliquid chromatography. Neutral sugars present in the anti-9-OAcSGs were detected by gasliquid chromatography (GLC) as their alditol acetates (34). The 9-OAcSA-specific IgGs (100 µg each) were hydrolyzed with trifluoroacetic acid (2.0 M) at 120°C for 90 min, followed by reduction with sodium borohydride (10 mg). Resulting alditols were acetylated with acetic anhydride in distilled pyridine [1 ml, 1 : 1 (v/v)] at 2225°C for 16 h and analyzed by GLC using a Hewlett-Packard 6890 plus gas chromatograph equipped with a flame ionization detector. The constituent sugars were identified from the retention time of authentic sugars. For the detection and quantification of the peaks, a Hewlett-Packard 3380A chemstation was used. For resolution, a fused-silica capillary column HP-5 (30 m, 0.32 mm, 0.25 µm) and nitrogen as carrier gas were used with a temperature program of 150°C (5 min), 2°C (1 min) and 200°C (10 min) at splitless mode.
Detection of terminal sugar by DIG glycan differentiation kit. The presence of terminal sugars along with their specific linkages was analyzed by DIG glycan differentiation kit (Roche Molecular Biochemicals, Mannheim, Germany) using several plant lectins, namely Galanthus nivalis agglutinin (GNA), Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), peanut agglutinin (PNA) and Datura stramonium agglutinin (DSA), as per the manufacturer's protocol.
Lectin bead-binding assay. The purified 9-OAcSA-specific IgG1 and IgG2 from ALL patients and normal individuals were iodinated with 125I-Na using the chloramine T method (35). Fixed concentrations (0.2 µg) of 125I-IgG1 and/or IgG2 were incubated separately with Sepharose/agarose-bound lectins (20 µl) of different linkage and specificity, e.g. Con A, Ricinus communis agglutinin (RCA), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), wheat germ agglutinin (WGA), Ulex europaeus agglutinin (UEA), Limulus polyphemus agglutinin (LPA), MAA, SNA, and Achatinin-H, overnight at 4°C. The beads were initially incubated with 2% BSA in PBS or Tris-buffered saline (TBS) to block non-specific binding sites. Following removal of unbound antibody fractions, specifically bound antibodies were monitored in a gamma counter. Unconjugated Sepharose/agarose beads served as controls. Binding (%) was calculated as follows:
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Evaluation of generation of intracellular reactive oxygen species as an index for effector cell activation through FcR. Intracellular reactive oxygen species (ROS) generation was monitored using a cell-permeable probe 2',7'-dichlorofluorescein diacetate (H2DCFDA, Molecular Probes, Eugene, OR, USA), which is oxidized to a fluorescent compound 2',7'-dichlorofluorescein in the presence of an oxidant.
U937 cells were used to analyze the interaction between FcR present on its surface and the Fc region of anti-9-OAcSGs, resulting in the triggering of a respiratory burst that is considered as an indicator of cell activation. The cells were cultured in medium A in the presence of IFN
(1000 U ml1) for 2 days to induce differentiation and the capacity to generate superoxide.
To pinpoint the binding of the Fc region of anti-9-OAcSGs with FcR present on U937 cells, Fab paratopes of these antibodies (total IgG, IgG1and IgG2) were blocked by incubating them with BSM (1 mg ml1) and/or purified 9-OAcSGs (0.03 mg ml1) at 4°C for 1 h.
These Fab-blocked anti-9-OAcSGs (0.5 µg) were allowed to bind with IFN-sensitized U937 cells (1 x 106 cells) for 1 h at 37°C and incubated with H2DCFDA (2 µg ml1) for 30 min at 37°C in the dark. Following washing, they were analyzed by flow cytometry and also visualized under a confocal microscope as described earlier. In confocal microscopy, slides were analyzed in triplicate and total fluorescence was expressed as fluorescence intensity units (FIU) at 530 nm. Cells incubated only with BSM and/or purified 9-OAcSGs in the absence of antibodies served as controls.
Activation of the classical complement pathway. Purified anti-human C3 chain mAb, SIM 27-49 (36), was iodinated with 125I-Na (35) and used for measuring C3 deposition on the cell surface due to activation of the classical complement pathway triggered by anti-9-OAcSGs. Sera (20%) from normal individuals and/or untreated ALL patients were used as the source of the complement.
Cells (CEM-C7 and REH, 2 x 106 per 100 µl) were suspended in TBS and allowed to bind with purified anti-9-OAcSGs (0.1-1.0 µg) as complement activators at 37°C for 30 min. EDTA (10 mM) was always used as the blocker of alternative pathway of complement activation. Following washing, the cells were incubated with complement (100 µl) for 10 min at 37°C and washed twice in cold TBS, and the amount of C3 deposition on the cell surface was quantitated by incubating further with 125I-anti-C3 mAb (2 x 105 counts per minute) for 1 h on ice. After two washes, the radioactivity incorporated in C3anti-C3 complexes was determined by a gamma counter. Each set was repeated thrice. Cells incubated with complements or complement activators alone or complement activators in the presence of heat-inactivated complement served as different controls.
Evaluation of cytotoxic potential of anti-9-OAcSGs by 51Cr-release antibody-dependent cell-mediated cytotoxicity assay. ALL cell lines (CEM-C7 and REH) were suspended in medium A with human AB serum (10%) and labeled with Na251CrO4 (0.1 mCi per 1 x 106 cells, BARC, Mumbai, India; 37) in the presence and absence of purified anti-9-OAcSGsALL and anti-9-OAcSGsN (IgG1 and IgG2, 1.0 µg) for 3 h at 37°C and 5% CO2. Cells were washed with the same medium and used as target cells. Freshly isolated normal human PBMC were used as effector cells. Target (1 x 104 cells per 100 µl per well) and effector cells were co-incubated in a 96-well U-bottom tissue culture plate for 16 h at 37°C in a 5% CO2 environment at E : T ratios of 5 : 1, 25 : 1, 50 : 1 and 100 : 1. The plate was centrifuged at 500 x g for 5 min, and the supernatants were checked for released 51Cr by a gamma counter. Spontaneous release and maximum release (100% lyses) of 51Cr from labeled target cells were evaluated in the absence of effector cells and on treatment with 5% (v/v) Triton X-100, respectively. Cytotoxicity was calculated as: 100 x [(experimental counts spontaneous counts)/(maximum counts spontaneous counts)]. Specific cell lyses (%) were calculated by normalizing the background cytotoxicity in the absence of added antibody. All assays were done in triplicate wells and three independent experiments were performed for each set.
Interaction of antibodies with Staphylococcal protein A and Streptococcal protein G. 125I-Labeled anti-9-OAcSGs (IgG1 and IgG2, 0. 2 µg) purified from ALL and normal sera were separately incubated with SepharoseStaphylococcal protein A (SpA) and Streptococcal protein G (SpG) (20 µl) overnight at 4°C and specific counts were analyzed as described earlier. Experiments were performed in triplicate, and unconjugated beads served as controls.
Statistical analysis
Results are reported as mean ± SD. Statistical analyses were performed using the Graph-Pad Prism statistics software (Graph-Pad Software Inc., San Diego, CA, USA). Student's unpaired t-tests were used. Reported values are two tailed and P values <0.05 were considered as statistically significant at 95% confidence interval.
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Results |
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Anti-9-OAcSGs in ALL patients (IgGALL) differ in the nature and content of glycosylation from those of normal individuals (IgGN)
The degree of glycosylation (Fig. 3A and B) and specifically sialylation (Fig. 3C) of 9-OAcSA-specific purified IgGs was demonstrated using the DIG glycan detection kit. A 2.1-fold increase in overall glycosylation was observed in IgGALL as compared with IgGN, the mean ± SD of densitometric score in arbitrary units being 80 651 ± 8210 versus 38 232 ± 5100, respectively, P < 0.0006 (Fig. 3B).
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To trace the content of individual neutral sugars present in anti-9-OAcSGs purified from ALL patients and normal individuals, GLC analysis was performed. The gasliquid chromatogram peaks revealed a significantly higher amount of N-acetyl glucosamine (GlcNAc) in IgGALL (Fig. 4A) and galactose (Gal) in IgGN (Fig. 4B). As compared with normal controls, IgGALL had a higher amount of GlcNAc (relative percent area of the peak being 6.86 versus 29.88), GalNAc (4.81 versus 9.04%) and mannose (10.24 versus 13.28%), whereas the amount of Gal was marginally higher in IgGN than IgGALL (32.002 versus 21.64%, respectively, Fig. 4A and B).
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Binding of 125I-anti-9-OAcSGs with several Neu5Ac-binding lectins, e.g. WGA (GlcNAc/Neu5Ac), LPA (Neu5Ac), SNA and MAA, also indicated a higher degree of sialylation in IgGALL (Table 3). Other lectins, namely RCA, DBA, HPA, UEA and Achatinin-H, showed no marked difference in their binding. In contrast, a 5.9-fold higher ConA (-Man,
-Glc) binding with IgGN indicated an abundance of terminal mannose residues in normal antibodies.
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9-OAcSA-specific IgGALL are less potent activators of U937 cells through FcFc
R interaction as compared with IgGN
Following the blocking of Fab regions of purified anti-9-OAcSGs by BSM or purified 9-OAcSGs, the antibodies were incubated with IFN-sensitized U937 cells to compare their ability to activate these cells via interaction with Fc
R. The generation of intracellular ROS was considered as an index for cell activation that was monitored using H2DCFDA, which primarily detects H2O2 and hydroxyl radicals. Flow cytometric analyses revealed that anti-9-OAcSGsALL caused a significantly lower level of Fc
R activation as reflected from 45, 50 and 36% ROS-generating cells induced by total IgG, IgG1 and IgG2, respectively. In contrast, anti-9-OAcSGsN (total IgG and IgG1) demonstrated significantly higher ROS-generating cells (90 and 93%, respectively). Anti-9-OAcSGsN of IgG2 subclass, on the other hand, showed only 41% ROS-generating activity (Fig. 6A).
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Anti-9-OAcSGs from ALL patients (IgGALL) are poor activators of the classical complement pathway
The potential of anti-9-OAcSGsALL (IgG1 and IgG2; 0.1, 0.5 and 1.0 µg) to activate the classical complement pathway was compared with anti-9-OAcSGsN using cell lines CEM-C7 (Fig. 7A) and REH (Fig. 7B) in the presence of 20% ALL sera and/or normal sera as the source of complement. Maximum differences in complement activation were observed with 1.0 lg concentration of anti-9-OAcSGs (complement activator).
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To confirm that this C3 deposition was due to activation of the complement pathway, cells were incubated with antibodies or sera alone, or antibodies along with heat-inactivated sera. All controls consistently demonstrated an inability to trigger C3 deposition on both cell types, the range of C3 deposition being 216% (Table 4, Fig. 7A and B).
Anti-9-OAcSGsALL (IgG1 and IgG2) are weak mediators for cell-mediated cytotoxicity
To confirm differential functional potential of anti-9-OAcSGs, 9-OAcSA-specific IgG1 and IgG2 from ALL patients and normal individuals were used as mediators for cytotoxic activity of effector cells toward both T- and B-ALL cell lines. Optimum cytotoxicity was observed at 50 : 1 effector/target cell ratio. The 9-OAcSA-specific IgG1ALL and IgG2ALL were found to be poor mediators of antibody-dependent cell-mediated cytotoxicity (ADCC) as evidenced from 610% and 45% specific cell lyses in REH and CEM-C7 cell lines, respectively. Anti-9-OAcSGsN (IgG2) also showed a similar pattern of cytolysis (25%). In contrast to anti-9-OAcSGsN (IgG2), anti-9-OAcSGsN (IgG1) was capable of mediating a high degree of cytotoxicity in both the cell lines, mean specific cell lyses being 62% (P < 0.0006) and 42% (P < 0.0007) for REH and CEM-C7, respectively. Again, anti-9-OAcSGsN (IgG1) was found to be 6- (P < 0.0009) and 7-fold (P < 0.001) more effective in lysing REH and CEM-C7, respectively, than anti-9-OAcSGsALL (IgG1).
Both IgGALL and IgGN showed similar interactions with SpA and SpG
The interaction of 125I-anti-9-OAcSGs (IgG1 and IgG2) purified from ALL patients and normal individuals with SpA and SpG was similar. The specific binding (counts per minute, mean ± SD) of IgG1ALL and IgG1N to SpA was 14 609 ± 673 versus 13 711 ± 541, and that to SpG was 17 021 ± 796 versus 17 935 ± 982, respectively. A similar pattern of binding was observed between IgG2ALL and IgG2N to SpA (11 323 ± 716 versus 13 010 ± 460) and SpG (14 709 ± 683 versus 12 907 ± 557).
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Discussion |
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The major findings of this investigation include the demonstration of (i) a shift in subclass distribution of anti-9-OAcSGsALL toward IgG2 (Fig. 1), (ii) an alteration in total content and pattern of glycosylation including variation in the linkage-specific terminal Neu5Ac residues in anti-9-OAcSGs in ALL as compared with their normal counterparts (Figs 35) and (iii) defective triggering of a few Fc-glycosylation-sensitive effector functions in anti-9-OAcSGsALL (Figs 68
), while their antigen-binding property remained unaffected (Fig. 2).
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Both IgG1 and IgG2 of purified anti-9-OAcSGsALL showed strong binding (8799%) with PBMCALL, suggesting their fully functional antigen-binding capacity. The total absence of binding of anti-9-OAcSGsALL with PBMC from various cross-reactive disorders like chronic myelogenous leukemia, acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), adult ALL, aplastic anemia and thalassemia reiterated its disease specificity. Additionally, weak binding (814%) of anti-9-OAcSGsALL (IgG1 and IgG2) with PBMCN reconfirmed a basal level of expression of 9-OAcSGs on PBMCN as indicated by our earlier observation (12).
Although differential distribution in levels of IgG subclasses is reported in several diseases, disease-specific distinct pattern is restricted to only a few. Accordingly, we attempted to analyze the status of 9-OAcSA-specific IgG subclasses in ALL. A predominance of IgG2 among all four subclasses in these children was demonstrated as evidenced by a 2.78-fold higher amount of IgG2ALL as compared with IgG1ALL (158.1 ± 5.57 versus 56.67 ± 3.63 µg ml1, respectively, Fig. 1). Interestingly, the level of 9-OAcSA-specific IgG2 was much higher (3-fold) in ALL patients than normal controls (158.1 ± 5.57 versus 51.26 ± 4.80 µg ml1, respectively), indicating the high immunogenicity of 9-OAcSGs in disease state and their efficient modulation for disease-specific alteration in isotype subclass switching toward IgG2. Patients with gynecological malignancies (40) and squamous cell carcinomas of head and neck (41) exhibited a characteristic decrease in IgG1 and an increase in IgG2 levels, relative to total IgG.
IgG, a multifunctional glycoprotein, comprises two distinct functional domains, expressing an antigen-specific binding site (IgG-Fab), while the IgG-Fc is responsible for triggering effector mechanisms through its interaction with specific ligands, e.g. cellular receptors (FcR) and the C1 component of complement (42). The IgG-Fc is a homodimer comprising inter-chain disulfide-bonded hinge regions, glycosylated CH2 domains and non-covalently paired CH3 domains (42). Glycosylation of IgG-Fc has been shown to be essential for efficient activation of Fc
R and C1 component of complement (42). The oligosaccharide profiles of the IgG are significantly influenced by disease stress, nutrient depletion or acid pH, resulting in hypogalactosylation or the addition of high mannose forms or failure of glycosylation (42). IgGs isolated from sera of patients with multiple myeloma, CLL and AML showed altered levels of fucose, Gal and bisecting GlcNAc as compared with normal (4345). Based on these observations, we endeavored to analyze the glycosylation profile of different subclasses of monospecific polyclonal anti-9-OAcSGs specifically induced in sera of patients with childhood ALL and compared them with normal individuals.
A wide variation in the degree of total glycosylation and specifically sialylation was observed in anti-9-OAcSGs as reflected by 2.1- and 3.6-fold higher level of glycosylation and sialylation in ALL patients as compared with their normal counterparts (Fig. 3AC). This was confirmed by fluorimetric analysis that showed a similar increase in sialylation of IgGALL as compared with IgGN (Fig. 3D). A significantly high proportion of SNA binding with IgGALL (IgG1 and IgG2) as compared with MAA binding implies an enhanced induction of terminal Neu5Ac26Gal/GalNAc with concomitant reduction in terminal Neu5Ac
23Gal (Fig. 5). IgGN showed a similar type of specificity toward SNA and MAA binding; however, the degree of binding was much lower than that of IgGALL. The conspicuously lower amount of terminal mannose both in IgG1ALL and IgG2ALL, as reflected from GNA and Con A binding, could be attributed to their being masked by
26-linked Neu5Acs, possibly a characteristic feature of this disease (Fig. 5, Table 3).
Taken together, it has been convincingly demonstrated that 9-OAcSA-specific IgGALL differ both in their content and nature of glycosylation as compared with IgGN. Although a distinct disease-specific pattern of terminal and subterminal sugar moieties has emerged, a chemical analysis of total glycan structure of Igs from individual patients would be of utmost importance.
Glycoproteins are key components of the immune system effectors even though the attached sugar moieties often deviate greatly from their appropriate homogeneous geometrical array, thus affecting the activation of proper immune response. The ubiquity and diversity of protein glycosylation is not a paradox, but consistent with functional rules (46). Human IgG is predominantly glycosylated with N-linked carbohydrates and conserved glycosylation of the Fc region occurs at asparagine 297 (Asn297), from which two opposing bi-antennary oligosaccharide chains protrude and interact. Crystallographic studies have shown that the two CH2 domains do not interact by proteinprotein contacts, but instead through oligosaccharides attached at the conserved site of Asn297 on each heavy chain. Proteinoligosaccharide and oligosaccharideoligosaccharide interactions play a major role in maintaining the relative geometry of the CH2 domains, consistent with the biological role of IgG (47).
As mediators of the humoral immune response, antibodies bind to specific antigens through Fab and then trigger biological responses by interacting through their Fc region with both cellular and soluble effector systems. It has been reported that heavy chain-linked conserved glycans of IgGs at Asn297 significantly contribute to their effector properties by influencing the complement activation via the classical pathway, which begins with the binding of C1q component of the complement to the CH2 domain of the IgG molecule, binding of IgGs to FcR and subsequent cellular function, induction of ADCC and also rapid elimination of the antigenantibody complexes from the circulation (42, 48, 49).
Analysis of effector function of anti-9-OAcSGs revealed that FcR-activating capacity was much lower in IgG1ALL as evidenced from a decrease in the percentage of ROS-generating cells, when U937 cells, known to contain a high amount of Fc
RI on their surface, were targeted with Fab-blocked antibodies, suggesting their functional impairment (Fig. 6A). Whereas IgG1N was fully capable of activating 91% U937 cells for ROS generation. This was corroborated with confocal microscopy (Fig. 6B and C). Similarly, the capacity of Fc
R triggering was decreased both in IgG2ALL and IgG2N, ROS positivity being 35 and 39%, respectively (Fig. 6A). The efficiency of normal human IgG2 in binding to Fc
R is reported to be the lowest among all the subclasses of IgG, the efficiency decreases in the following order IgG1 = IgG3 > IgG4 >>> IgG2 (49). The predominance of 2.8-fold increased production of 9-OAcSA-specific IgG2 (Fig. 1) along with low amounts of non-functional IgG1 in ALL patients suggests the inappropriate immune surveillance.
The impaired functioning of 9-OAcSA-specific IgG1ALL and IgG2ALL has further been demonstrated by their diminished complement activation as reflected from less C3 deposition (Fig. 7, Table 4). Notably, IgG1N was found to be the only potential activator of classical complement pathway. Considering the fact that antibody binding to C1q does not guarantee the activation of C1 (50), we checked the C3 deposition, a downstream component, to get the assurance of activated complement cascade. Nature of the terminal sugars on Fc-glycans is known to influence the activation of C1 (42).
ADCC experiments using IgG1 and IgG2 subclasses of anti-9-OAcSGsALL and anti-9-OAcSGsN as mediators clearly demonstrate the inability of anti-9-OAcSGsALL to elicit any specific cytolysis (Fig. 8), thereby further strengthening the observed impairment of their proper biological functioning as evidenced from their low FcR-activating capacity (Fig. 6AC) and diminished complement activation potential (Fig. 7, Table 4).
SpA and SpG are known to bind to IgG-Fc at the interface between the CH2 and CH3 domains of the Fc through hydrophobic interactions (51). X-ray crystallographic and NMR studies clearly illustrated the negligible role of differences in IgG-Fc glycosylation in their SpA and SpG binding (42). Interestingly, our observation also demonstrates the similar binding profile of anti-9-OAcSGs (IgG1 and IgG2) with Protein A and G, irrespective of the source of antibodies, i.e. patient or normal sera.
In spite of several attempts by using glycosylation inhibitor, site-directed mutagenesis and enzymatic addition of particular sugar to analyze the direct influence of distinct glycosylation profile on functional activity of IgG, the underlying mechanism still remains unclear (49). However, dramatic differences in functional activity of IgGs were observed between fully glycosylated and molecular variants with selectively modified glycosylation or aglycosylated forms (42). If receptor/ligand interactions are modulated by glycosylation differences, the protective effector mechanisms activated by an oligoclonal-specific antibody population could be dependent on the predominant glycoforms present (49). This suggests an interesting possibility since IgG antibody responses can be subclass and clonally restricted and the glycosylation profiles of these antibodies may also be restricted as observed for IgG myeloma proteins (49). Surprizingly, passive immunization with anti-tumor antibodies did not protect against tumor growth, in many cases they actually enhanced the growth of the tumor (52).
In summary, this report established significant differences in glycosylation (mainly sialylation) and several functional properties between the 9-OAcSA-specific Igs from ALL patients and normal individuals and suggested anti-9-OAcSGs-mediated impairment of proper immune clearance in ALL. In the future, we hope to address the direct correlation between observed structural heterogeneity in glycosylation and the impaired functions of anti-9-OAcSGs in ALL by analyzing the conformational changes of Fc-carbohydrates, changes in topography of ligand-binding sites and subsequently their functional properties using different truncated glycoforms and also considering an individualistic approach. Studies with soluble recombinant FcRI or Fc
R knockouts would also provide confirmatory evidence.
Considering the phenomena of subclass switching from IgG1 to IgG2, their heterogeneous glycosylation, preferential sialylation along with the impairment of function (activation of FcR and complement) in ALL patients, the generation of customized antibody constructs bearing functional Fc domain of anti-9-OAcSGs-IgG1, having a homogenous glycoform and a predetermined profile of functional potential, might lead to their effective functioning. Such customized antibodies might be used in conjugation with cytokine therapy to activate in vivo anti-cancer pathways for proper immune surveillance in pediatric ALL.
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Acknowledgements |
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Abbreviations |
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ADCC | antibody-dependent cell-mediated cytotoxicity |
ALL | acute lymphoblastic leukemia |
AML | acute myelogenous leukemia |
Asialo BSM | de-sialylated BSM |
BSM | bovine submaxillary mucin |
CLL | chronic lymphocytic leukemia |
de-OAc BSM | de-O-acetylated BSM |
DBA | Dolichos biflorus agglutinin |
DIG | digoxigenin |
DSA | Datura stramonium agglutinin |
FIU | fluorescence intensity unit |
Gal | galactose |
GalNAc | N-acetyl galactosamine |
GLC | gasliquid chromatography |
GlcNAc | N-acetyl glucosamine |
GNA | Galanthus nivalis agglutinin |
HA | hemagglutination |
H2DCFDA | 2',7'-dichlorofluorescein diacetate |
HPA | Helix pomatia agglutinin |
LPA | Limulus polyphemus agglutinin |
MAA | Maackia amurensis agglutinin |
Neu5Ac | sialic acid |
NHL | non-Hodgkin's lymphoma |
9-OAcSa | 9-O-acetylated sialic acid |
9-OAcSGs | 9-O-acetylated sialoglycoconjugates |
PNA | peanut agglutinin |
ROS | reactive oxygen species |
SNA | Sambucus nigra agglutinin |
SpA | Staphylococcal protein A |
SpG | Streptococcal protein G |
TBS | Tris-buffered saline |
UEA | Ulex europaeus agglutinin |
WGA | wheat germ agglutinin |
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Notes |
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Received 19 May 2004, accepted 17 November 2004.
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References |
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