Detection of GD3 ganglioside in childhood acute lymphoblastic leukemia with monoclonal antibody to GD3: restriction to immunophenotypically defined T-cell disease

We have recently reported that the disialoganglioside GD3 is found in cellular lipid extracts of T-cell acute lymphoblastic malignancies (T-ALL) but is not detectable by resorcinol staining in extracts of non-T acute lymphoblastic leukemia blasts (non-T-ALL). We have now extended this study to assess the detectability of GD3 in T-ALL vs non-T-ALL utilizing an anti-GD3 antibody, R24. Gangliosides isolated from T-ALL and non-T-ALL blasts by two different methods were separated by thin-layer chromatography and stained with anti-GD3 and a control antibody specific for GM3 and sialosylparagloboside (SPG). Anti-GD3 reactivity was observed in extracts from T-ALL cells in all cases, whereas GD3 was not detected in any of the non-T-ALL samples. The anti-GM3/SPG antibody stained GM3 in all of the leukemic samples analyzed as well as SPG in the non-T-ALL samples. Indirect immunofluorescence was used to assess the expression of GD3 at the surface of leukemic blasts. Fluorescence-activated cell sorting analysis with R24 showed that whereas T-ALL blasts were highly reactive with this antibody, non-T-ALL blasts were totally unreactive. In an analysis of a larger number of leukemia patients by fluorescence microscopy, 20 out of 28 samples with the T-ALL phenotype were positive for R24, whereas zero out of 11 non-T-ALL samples were reactive. These results confirm our earlier finding of the specificity of GD3 to the T-ALL subclass of childhood leukemias and furthermore suggest the potential value of anti-GD3 as an immunological tool for the diagnosis and therapy of T-cell ALL.     

Immune response of chimpanzee to purified melanoma 250 kilodalton tumor-associated antigen

Summary Melanoma high molecular weight tumor-associated antigen (TAA), having a molecular weight of 250 kilodaltons (Kd), was purified from a crude cell membrane extract through a combination of lectin affinity, immunoadsorption, and high performance liquid molecular filtration chromatography. Compared to the starting extract, purified TAA was 600-fold higher in TAA activity per microgram of protein. Purified TAA was used to immunize a chimpanzee and the resulting antiTAA immune response was evaluated. Postimmune chimpanzee serum reacted in solid phase radioimmunoassay against purified TAA with a titer in excess of 100,000. In contrast, preimmune serum had a titer of <100 in the same assay. By immunoprecipitation analysis, we were able to demonstrate reactivity of the chimpanzee immune serum with a 250 Kd TAA in spent culture medium from melanoma cells metabolically labeled with ³⁵S-methionine and with iodinated purified 250 Kd TAA. Reactivity of the chimpanzee antiserum for the 250 Kd TAA was confirmed in blocking and reciprocal immunodepletion studies using murine monoclonal antibody 9.2.27. These studies suggest that the 250 Kd TAA defined by murine monoclonal antibodies may prove to be immunogenic in man and that manipulation of the immune response to this TAA might be used to the clinical benefit of the patient.This work supported in part by Veterans Administration Hospital #821, by National Cancer Institute Grant #CA 32672 and by NIH Grant #RR 00165 from the Division of Research Resources to the Yerkes Regional Primate Research Center. The Yerkes Center is fully accredited by the American Association for Accreditation of Laboratory Animal Care     

Expression of GD2 and GD3 gangliosides in human embryonic neural stem cells

NSCs (neural stem cells) are undifferentiated neural cells endowed with a high potential for proliferation and a capacity for self-renewal with retention of multipotency to differentiate into neurons and glial cells. It has been recently reported that GD3, a b-series ganglioside, is a marker molecule for identifying and isolating mouse NSCs. However, the expression of gangliosides in human NSCs is largely unknown. In the present study, we analysed the expression of gangliosides, GD2 and GD3, in human NSCs that were isolated from human brains at gestational week 17 in the form of neurospheres, which are floating clonal aggregates formed by NSCs in vitro. Employing immunocytochemistry, we found that human NSCs were strongly reactive to anti-GD2 antibody and relatively weakly reactive to anti-GD3 antibody. Treatment of these cells with an organic solvent such as 100% methanol, which selectively removes glycolipids from plasma membrane, abolished the immunoreactivity with those antibodies, indicating that the reactivity was due to GD2 and GD3, but not to GD2-/GD3-like glycoproteins or proteoglycans. The immunoreactivity of human NSCs to antibody against SSEA-1 (stage-specific embryonic antigen-1), a well-known carbohydrate antigen of NSCs, was not decreased by the treatment with 100% methanol, indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in human NSCs. Our study suggests that GD2 and GD3 can be marker gangliosides for identifying human NSCs.     

Antitumor effects of a novel monoclonal antibody with high binding affinity to ganglioside GD3

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface human tumors of neuroectodermal origin, has been studied as a target molecule for passive immunotherapy. We established ten kinds of anti-GD3 monoclonal antibodies (mAb) of the mouse IgG3 subclass by immunization with purified GD3 and melanoma cells. One of the established mAb, KM641, showed major reactivity with GD3 and minor reactivity with GQ1b out of 11 common gangliosides in an enzymelinked immunosorbent assay. Immunostaining of gangliosides, separated on thin-layer chromatography plates, using KM641 revealed that most of the melanoma cell lines contained immunoreactive GD3 and GD3-lactone at a high level, but only the adrenal gland and the urinary bladder out of 21 human normal tissues had immunoreactive GD3. In immunofluorescence, KM641 bound to a variety of living tumor cell lines especially melanoma cells, including some cell lines to which another anti-GD3 mAb R24, established previously, failed to bind. High-affinity binding of KM641 to a tumor cell line was quantified by Scatchard analysis (K _d = 1.9×10^–8 M). KM641 exerted tumor-killing activity in the presence of effector cells or complement against melanoma cells expressing GD3 at a high level. Not only natural killer cells but also polymorphonuclear cells were effective as the effector cells in antibody-dependent cellular cytotoxicity. Intravenous injection of KM641 markedly suppressed the tumor growth of a slightly positive cell line, C24.22 (7.2×10⁵ binding sites/cell), as well as a very GD3-positive cell line, G361 (1.9×10⁷ binding sites/cell), inoculated intradermally in nude mice. KM641, characterized by a high binding affinity for GD3, has the potential to be a useful agent for passive immunotherapy of human cancer.     

Monoclonal antibody Leo Mel 3, which inhibits killing of human melanoma cells by anomalous killer cells, binds to a sugar sequence in GD2 (II3(NeuAc)2-GgOse3Cer) and several other gangliosides

Human anomalous killer (AK) cells lyse freshly isolated human melanoma cells which are insensitive to human natural killer cell-mediated lysis. Monoclonal antibody Leo Mel 3, an IgM (k), produced by a hybridoma obtained from a mouse immunized with human melanoma cells, binds to melanoma cells and inhibits their conjugate formation with AK cells as well as their AK cell-mediated lysis. Other IgM antibodies from the same fusion that bind melanoma cells do not inhibit (Werkmeister, J. A., Triglia, T., Andrews, P., and Burns, G. F. (1985) J. Immunol. 135, 689-695). Leo Mel 3 binds several different gangliosides from melanoma cells, as determined by immunostaining thin layer chromatograms. Binding is abolished by treatment of the gangliosides with neuraminidase. In solid-phase radioimmunoassay, Leo Mel 3 binds strongly to ganglioside GD2 and less strongly to gangliosides GT3, GD3, and GQ1b. It does not bind to other gangliosides including GM1, GM2, GM3, GD1a, GD1b, and GT1b. Thus, the epitope recognized by antibody Leo Mel 3 is found in the sugar sequence of ganglioside GD2, GalNAc beta 1-4[NeuAc alpha 2-8NeuAc alpha 2-3]Gal beta 1-4Glc beta 1 .... This sequence may contain a target in melanoma cells recognized by AK cells.     

Monoclonal anti-GD3 antibodies selectively inhibit the proliferation of human malignant glioma cells in vitro

The frequently occurring alteration of ganglioside expression in tumor cells has been implicated to play a role in the uncontrolled growth of these cells; antibodies to such gangliosides might affect tumor cell growth. We have studied the effect of IgM monoclonal antibodies to two glioma-associated gangliosides, GD3 and GM2, on cell proliferation of four human glioma cell lines and one renal tumor cell line. Of the two anti-ganglioside antibodies tested, only the anti-GD3 antibody resulted in a significant (p<0.005) inhibition of cell proliferation as measured by thymidine incorporation and Brd-U labeling, after 24[emsp4 ]h incubation. The effect was not dependent on any serum factor and no increased cell death was observed. All cell lines contained higher or similar amounts of GM2 than GD3, and both antigens were shown to be expressed on the cell surface and accessible to antibodies. The selective effect of anti-GD3 antibodies as contrasted to the inactivity of anti-GM2 antibodies suggests a possible role for ganglioside GD3 in tumor cell proliferation.     

Isolation and characterization of gangliosides from Trypanosoma brucei

Gangliosides were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated by TLC. G-1 ganglioside had the same TLC migration rate as GM3. In contrast, G-2, G-3, and G-4 gangliosides migrated a little slower than GM1, GD1a, and GD1b, respectively. To characterize the molecular species of gangliosides from T. brucei, G-1, G-2, G-3, and G-4 gangliosides were purified and analyzed by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside showed the reactivity to the monoclonal antibody against ganglioside GM3. G-2 was recognized by the anti-GM1 monoclonal antibody. G-3 showed reaction with the monoclonal antibody to GD1a. G-4 had the reactivity to anti-GD1b monoclonal antibody. Using 4 kinds of monoclonal antibodies, we also studied the expression of GM3, GM1, GD1a, and GD1b in T. brucei parasites. GM3, GM1, GD1a, and GD1b were detected on the cell surface of T. brucei. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-1Cer), GM1 (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), GD1a (NeuAc alpha2-3Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), and GD1b (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-8NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), respectively, and also that they are expressed on the cell surface of T. brucei.     

Glycosphingolipid antigens in neural tumor cell lines and anti-glycosphingolipid antibodies in sera of patients with neural tumors

To characterize biomarkers in neural tumors, we analyzed the acidic lipid fractions of 13 neural tumor cell lines using enzyme-linked immunoabsorbent assay (ELISA) and high-performance thin-layer chromatography (HPTLC) immunostaining. Sulfated glucuronosyl glycosphingolipids (SGGLs) are cell surface molecules that are endowed with the Human Natural Killer-1 (HNK-1) carbohydrate epitope. These glycosphingolipids (GSLs) were expressed in all cell lines with concentrations ranging from 210 to 330 ng per 2 x 10(6) cells. Sulfoglucuronosyl paragloboside (SGPG) was the prominent species with lesser amounts of sulfoglucuronosyl lactosaminyl paragloboside (SGLPG) in these tumor cell lines as assessed by quantitative HPTLC immunostaining. Among the gangliosides surveyed, GD3 and 9-O-acetylated GD3 (OAc-GD3) were expressed in all tumor cell lines. In contrast, fucosyl-GM1 was not found to restrict to small cell lung carcinoma cells. In addition, we have analyzed serum antibody titers against SGPG, GD3, and OAc-GD3 in patients with neural tumors by ELISA and HPTLC immunostaining. All sera had high titers of antibodies of the IgM isotype against SGPG (titers over 1:3,200), especially in tumors such as meningiomas, germinomas, orbital tumors, glioblastomas, medulloblastomas, and subependymomas. Serum in a patient with subependymomas also had a high anti-SGGL antibody titer of the IgG and IgA types (titers over 12,800). The titer of anti-GD3 antibody was also elevated in patients with subependymomas and medulloblastomas; the latter cases also had a high titer of antibody against OAc-GD3. Our data indicate that certain GSL antigens, especially SGGLs, GD3, and OAc-GD3, are expressed in neural tumor cells and may be considered as tumor-associated antigens that represent important biomarkers for neural tumors. Furthermore, antibody titers in sera of patients with these tumors may be of diagnostic value for monitoring the presence of tumor cells and tumor progression.     

High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization

Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.     

Mechanisms for the apoptosis of small cell lung cancer cells induced by anti-GD2 monoclonal antibodies: roles of anoikis

Anti-GD2 ganglioside antibodies could be a promising, novel therapeutic approach to the eradication of human small cell lung cancers, as anti-GD2 monoclonal antibodies (mAbs) induced apoptosis of small cell lung cancer cells in culture. In this study, we analyzed the mechanisms for the apoptosis of these cells by anti-GD2 mAbs and elucidated the mechanisms by which apoptosis signals were transduced via reduction in the phosphorylation levels of focal adhesion kinase (FAK) and the activation of a MAPK family member, p38, upon the antibody binding. Knock down of FAK resulted in apoptosis and p38 activation. The inhibition of p38 activity blocked antibody-induced apoptosis, indicating that p38 is involved in this process. Immunoprecipitation-immunoblotting analysis of immune precipitates with anti-FAK or anti-integrin antibodies using an anti-GD2 mAb revealed that GD2 could be precipitated with integrin and/or FAK. These results suggested that GD2, integrin, and FAK form a huge molecular complex across the plasma membrane. Taken together with the fact that GD2+ cells showed marked detachment from the plate during apoptosis, GD2+ small cell lung cancer cells seemed to undergo anoikis through the conformational changes of integrin molecules and subsequent FAK dephosphorylation.     

Engineering anti-GD2 monoclonal antibodies for cancer immunotherapy

Ganglioside GD2 is highly expressed on neuroectoderm-derived tumors and sarcomas, including neuroblastoma, retinoblastoma, melanoma, small cell lung cancer, brain tumors, osteosarcoma, rhabdomyosarcoma, Ewing’s sarcoma in children and adolescents, as well as liposarcoma, fibrosarcoma, leiomyosarcoma and other soft tissue sarcomas in adults. Since GD2 expression in normal tissues is restricted to the brain, which is inaccessible to circulating antibodies, and in selected peripheral nerves and melanocytes, it was deemed a suitable target for systemic tumor immunotherapy. Anti-GD2 antibodies have been actively tested in clinical trials for neuroblastoma for over the past two decades, with proven safety and efficacy. The main limitations have been acute pain toxicity associated with GD2 expression on peripheral nerve fibers and the inability of antibodies to treat bulky tumor. Several strategies have been developed to reduce pain toxicity, including bypassing complement activation, using blocking antibodies, or targeting of O-acetyl-GD2 derivative that is not expressed on peripheral nerves. To enhance anti-tumor efficacy, anti-GD2 monoclonal antibodies and fragments have been engineered into immunocytokines, immunotoxins, antibody drug conjugates, radiolabeled antibodies, targeted nanoparticles, T-cell engaging bispecific antibodies, and chimeric antigen receptors. The challenges of these approaches will be reviewed to build a perspective for next generation anti-GD2 therapeutics in cancer therapy.     

Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H](-)ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.     

Ganglioside modulation regulates epithelial cell adhesion and spreading via ganglioside-specific effects on signaling

Gangliosides are implicated in regulating cell adhesion and migration on fibronectin by binding with the alpha(5) subunit of alpha(5)beta(1) integrin. However, the effects of gangliosides on cell spreading and related signaling pathways are unknown. Increases in gangliosides GT1b and GD3 inhibited spreading on fibronectin, concurrent with inhibition of Src and focal adhesion kinase. Although antibody blockade of GT1b or GD3 function and gene-modulated ganglioside depletion stimulated spreading and activated Src and focal adhesion kinase, the augmented spreading by disruption of GT1b function, but not by disruption of GD3 function, was inhibited by blockade of Src and focal adhesion kinase activation. In contrast, inhibitors of protein kinase C prevented the stimulation of spreading by GD3 functional inhibition, but not by GT1b functional blockade. Modulation of either GT1b or GD3 content affected phosphoinositol 3-kinase activation, and inhibition of this activation reversed the stimulation of cell spreading by anti-GD3 antibody, anti-GT1b antibody, and ganglioside depletion, suggesting that phosphoinositol 3-kinase is an intermediate in both the FAK/Src and protein kinase C pathways that lead to cell spreading. These studies demonstrate that epithelial cell ganglioside GT1b modulates cell spreading through alpha(5)beta(1)/FAK and phosphoinositol 3-kinase signaling, whereas GD3-modulated spreading appears to involve phosphoinositol 3-kinase-dependent protein kinase C signaling.     

Immunoglobulin G-class mouse monoclonal antibodies to major brain gangliosides

Mice genetically engineered to lack complex gangliosides are improved hosts for raising antibodies against those gangliosides. We report the generation and characterization of nine immunoglobulin G (IgG)-class monoclonal antibodies (mAbs) raised against the four major brain gangliosides in mammals. These include (designated as ganglioside specificity-IgG subclass) two anti-GM1 mAbs (GM1-1, GM1-2b), three anti-GD1a mAbs (GD1a-1, GD1a-2a, GD1a-2b), one anti-GD1b mAb (GD1b-1), and three anti-GT1b mAbs (GT1b-1, GT1b-2a, GT1b-2b). Each mAb demonstrated high specificity, with little or no cross-reactivity with other major brain gangliosides. Enzyme-linked immunosorbent assay (ELISA) screening against 14 closely related synthetic and purified gangliosides confirmed the high specificity, with no significant cross-reactivity except that of the anti-GD1a mAbs for the closely related minor ganglioside GT1a alpha. All of the mAbs were useful for ELISA, TLC immunooverlay, and immunocytochemistry. Neural cells from wild-type rats and mice were immunostained to differing levels with the anti-ganglioside antibodies, whereas neural cells from mice engineered to lack complex gangliosides (lacking the ganglioside-specific biosynthetic enzyme UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase) remained unstained, demonstrating that most of the mAbs react only with gangliosides and not with related structures on glycoproteins. These mAbs may provide useful tools for delineation of the expression and function of the major brain gangliosides and for probing the pathology of anti-ganglioside autoimmune diseases.     

The addition of exogenous gangliosides to cultured human cells results in the cell type-specific expression of novel surface antigens by a biosynthetic process

After the observation that human mAb 32-27M reacts only with melanoma and astrocytoma cells cultured in the presence of fetal bovine serum, a novel pathway for the uptake of exogenous gangliosides, their further biosynthesis, and expression at the cell surface as novel Ag has been elucidated. The addition of fetal bovine serum to melanoma and astrocytoma cells growing in synthetic medium (insulin-transferrin-selenium) resulted in reactivity with Ab32-27M. As antibody 32-27M detects N-glycolylneuraminic acid (NeuGc)-containing gangliosides, the effect of adding a number of different gangliosides to melanoma and astrocytoma cells cultured in the synthetic medium was studied. Only the addition of NeuGc-GM3 resulted in the development of Ab32-27M reactivity. The identity of the antigenic structures developed after addition of fetal bovine serum or NeuGc-GM3 was determined by analysis of the gangliosides from both samples. The major component detected in melanoma cell lines was shown to be N-acetylneuraminic acid-NeuGc-GD3. Another, slower moving component, present in some melanomas and in astrocytomas may be N-acetylneuraminic acid-NeuGc-GD2. The cell type specificity for these processes can be most readily explained by postulating that all cells can take up exogenous gangliosides but only melanoma and astrocytoma cells have sufficiently high levels of GM3 alpha 2----8-sialyltransferase for the conversion of added NeuGc-GM3 to disialogangliosides to be effective. These results demonstrate a novel pathway for exogenous glycolipid processing that can lead to novel Ag expression but may also play a role in normal glycolipid metabolism and function.     

Synergism between membrane gangliosides and Arg-Gly-Asp-directed glycoprotein receptors in attachment to matrix proteins by melanoma cells

The identification of specific cell surface glycoprotein receptors for Arg-Gly-Asp-containing extracellular matrix proteins such as fibronectin has focused attention on the role of gangliosides in this process. Is their involvement dependent or independent of the protein receptors? In attachment assays with cells from a human melanoma cell line, titration experiments with an antibody (Mel 3) with specificity for the disialogangliosides GD2 and GD3, used together with a synthetic peptide containing the cell binding sequence Arg-Gly-Asp, show that their joint effect is synergistic. Both the Mel 3 antibody and the synthetic peptide individually cause rapid detachment of melanoma cells from fibronectin substrate but, when used together, much smaller concentrations of both are required to achieve the same effect. The Mel 3 antibody was not nonspecifically reducing receptor binding to the Arg- Gly-Asp sequence since, in binding assays with radiolabeled peptide performed with cells in suspension, very little peptide is bound by the melanoma cells under these conditions but addition of Mel 3, an antibody of IgM isotype, causes a two- to threefold increase in specific binding. The simplest interpretation of these data is that the Mel 3 antibody is causing sufficient clustering of membrane gangliosides in local areas and producing a favorably charged environment to facilitate peptide binding by specific glycoprotein receptors.     

Isolation and analysis of monoclonal antibodies against various gangliosides

Female BALB/c mice were immunized with human melanoma (Mewo) cells containing ganglioside GD3 as a surface antigen. Immune splenocytes were fused with syngeneic P3-X63.Ag 8 myeloma cells. Antibodies produced by hybrid clones were analyzed by solid phase immunoassay. B, C, D and Q clones producing antibodies against Raja clavata brain gangliosides were obtained. Monoclonal B and C antibodies bound monosialogangliosides. Monoclonal D antibody bound a number of gangliosides but reacted predominantly with GD1a. Monoclonal Q antibody reacted selectively with GQ1c. It is assumed that ganglioside GQ1c is expressed on the melanoma cell surface and may be found only in the early stage of ontogenesis of high vertebrates.     

Stimulation of T lymphocyte proliferation by monoclonal antibodies against GD3 ganglioside

Cell-surface gangliosides are presumed to play a role in cell growth and differentiation. With the use of monoclonal antibodies directed against GD3, a disialoganglioside expressed predominantly by cells of neuroectodermal origin, we have found that GD3 is expressed by a subpopulation of cells of the immune system including: 1) fetal thymocytes in subcortical regions and near vessels, 2) lymph node lymphocytes in interfollicular areas and near vessels, and 3) a small subset of T cells in the peripheral blood. Mouse monoclonal antibodies (two IgGs, one IgM, and F(ab')2 fragments) reacting with GD3 were found to stimulate proliferation of T cells derived from peripheral blood. Proliferation of T cells was observed even in cultures depleted of macrophages, suggesting that activation by anti-GD3 was not dependent on the presence of accessory cells. T cell proliferation was maximum between days 5 and 7 of stimulation and was preceded by expression of interleukin 2 receptors. No stimulation was observed with control antibodies of the identical isotype or with monoclonal antibodies recognizing the gangliosides GD2 or GM2. During stimulation by anti-GD3 monoclonal antibodies, there was an expansion of the GD3+ pool of T cells, but depletion of GD3+ T cells prior to stimulation abrogated the response. Proliferation induced by binding to GD3 could be augmented by exogenous interleukin 2 and phytohemagglutinin. Anti-CD3 (T3) monoclonal antibodies had little or no effect. These results demonstrate that binding to GD3 on the surface of T cells can elicit signals for T cell proliferation.     

Differential distribution of gangliosides in adult rat ovary during the oestrous cycle

Gangliosides are ubiquitous membrane components in mammaliancells and are suggested to play important roles in various cellfunctions, such as cell-cell recognition, differentiation andtransmembrane signalling. Rat ovary contained GM3, GD3 and GD1aas major gangliosides, and GM1 as a minor one. In order to studytheir distribution in the rat ovary and its possible changesduring the oestrous cycle, frozen sections were stained withspecific monoclonal antibodies against 11 ganglio-series gangliosidesincluding those mentioned above. GM3, GM1 and GD1a were expressedin a spatiotemporally different manner during the oestrous cycle,but GD3 and other gangliosides were not immunohistochemicallydetected. In primary and secondary follicles, GM3, GM1 and GDlawere expressed in theca cells, but not in granulosa cells. Theoocyte in primary, but not secondary, follicles was positiveto the anti-GD1a antibody. In Graafian follicles, GM1 and GD1awere similarly expressed as in secondary follicles, however,the expression of GM3 spread gradually from theca cells to granulosacells. In early Graafian follicles, only GM3 was expressed toa detectable extent from the outer part of the granulosa layerto the inner part Shortly before ovulation, all granulosa cellsand cumulus cells became positive to anti-GM3 antibody. Afterovulation, differential distribution of GM3, GM1 and GD1a wasalso observed in luteal cells. GD1a was localized in thread-likestructures, while GM3 was distributed throughout the cytoplasm,but not in the nucleus. GM1 was localized only in the plasmamembrane and/or its close vicinity. Other ganglio-series gangliosides,including GD3, were not detected to an appreciable extent inthe ovaries by immunohistochemistry ganglioside oestrous cycle rat ovary     

Gangliosides as a potential new class of stem cell markers: the case of GD1a in human bone marrow mesenchymal stem cells

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically ³H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.