Immunocytochemical localization of CDw60 antigens on human peripheral T cells.

About 25-35% of human T cells display the CDw60 ganglioside (9-O-acetyl-GD3) antigen at the cell surface [E.P. Rieber, in W. Knapp, B. Dörken, W.R. Gilks, E.P. Rieber, R.E. Schmidt, H. Stein, A.E.G.K. von dem Borne (Eds.), Leucocyte Typing IV, Oxford University, Oxford, 1989, p. 361.]. Other leucocytes do not express this antigen on the cell surface. This led us to investigate its presence by flow cytometry and immunoelectron microscopy (IEM). Flow cytometric analysis of isolated peripheral T cells showed 26% of the cell population to have the CDw60 antigen expressed on the cell surface whereas 74% did not. Similarly, IEM analysis of 262 random T cells by the preembedding immunogold labeling technique revealed CDw60 surface expression to be tetrapartite: (a) the majority of 63.7% of the T cells did not show any surface associated gold label; (b) 19.5% were of low CDw60 surface exposition, corresponding to a linear density of 0.05-2.0 gold markers per microm; (c) about 13.4% showed a medium surface exposition with a linear density of 2.1-4.5 gold markers per microm; and (d) a high exposition, ranging from 4.6 to 9.0 gold markers per microm, was seen at 3.4% of the T cells. From postembedding label experiments, which additionally make access to the antigen localized within the cytoplasm, it was found that nearly all T cells contained low levels of intracellular CDw60. Most of it was found to be associated with the cytoplasmic membrane or vesicles, derived from the Golgi. Immunogold conjugates associated with the cytoplasmic membrane showed a linear density up to 0.6 gold markers per microm. The asymmetric expression of the CDw60 antigen on human T cells and its occurrence in nearly all T cells suggests that its surface presentation is tightly regulated.

CDw60: an antigen expressed in many normal tissues and in some tumours.

CDw60 is a recently described T-cell antigen, which functionally delivers a costimulatory signal in T-cell activation. In addition, CDw60 has been regarded as a melanoma-associated antigen. To date, only limited information exists on the distribution of CDw60 in other normal and pathologically altered tissues in human. In the present study, the expression of CDw60 was analysed immunohistologically in a large panel of formalin-fixed and paraffin-embedded normal and pathological human tissues. The antigen was detected in several normal tissues, such as epithelia of the reproductive system, exocrine and endocrine glands, glial cells and neurons of the central and peripheral nervous systems, and lymphoid cells. These showed different subcellular distribution patterns, i.e. (1) cell surface labelling of peripheral lymphocytes and lymphocytes of the lymph node and thymus, (2) diffuse cytosolic staining in lymphocytes, subpial glial processes, and the outer plexiform layer of the retina, (3) granular cytoplasmic staining associated with the Golgi apparatus in epithelial cells of certain endocrine and exocrine glands, of the ductus epididymis and deferens, neurons of the peripheral and central nervous system, and lymphocytes and megakaryocytes of the bone marrow. In exocrine glands, e.g. of the prostate and uterine corpus, CDw60-positive Golgi fields were located in the juxtaluminal cell compartment, thus reflecting a polarized distribution. In some malignant tumours, the neoplastic cells contained CDw60-immunolabelled Golgi complexes, which were disorderly distributed throughout the cytoplasm, thus reflecting a loss of epithelial polarity. Only in mammary carcinomas was abnormal cell surface labelling detected. A putative de novo expression of CDw60 was observed in pleomorphic adenoma and mucoepidermoid carcinoma of the parotid gland, seminoma, embryonal and teratocarcinoma of the testis, small cell carcinoma of the lung, and malignant melanoma. These results define the CDw60 determinant as a broadly distributed antigen within a large panel of normal human tissues. The antigen is also detectable in some previously undescribed benign and malignant tumours, which may give importance to CDw60 as a possible diagnostic marker.

Receptor structure for F1C fimbriae of uropathogenic Escherichia coli.

F1C fimbriae are correlated with uropathogenic Escherichia coli strains. Although F1C fimbriae mediate binding to kidney tubular cells, their receptor is not known. In this paper, we demonstrate for the first time specific carbohydrate residues as receptor structure for F1C-fimbria-expressing E. coli. The binding of the F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) and purified F1C fimbriae to reference glycolipids of different carbohydrate compositions was evaluated by using thin-layer chromatography (TLC) overlay and solid-phase binding assays. TLC fimbrial overlay analysis revealed the binding ability of purified F1C fimbriae only to glucosylceramide (GlcCer), beta1-linked galactosylceramide 2 (GalCer2) with nonhydroxy fatty acids, lactosylceramide, globotriaosylceramide, paragloboside (nLc(4)Cer), lactotriaosylceramide, gangliotriaosylceramide (asialo-GM(2) [GgO(3)Cer]) and gangliotetraosylceramide (asialo-GM(1) [GgO(4)Cer]). The binding of purified F1C fimbriae as well as F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) was optimal to microtiter plates coated with asialo-GM(2) (GgO(3)Cer). The bacterial interaction with asialo-GM(1) (GgO(4)Cer) and asialo-GM(2) (GgO(3)Cer) was strongly inhibited only by disaccharide GalNAcbeta1-4Galbeta linked to bovine serum albumin. We observed no binding to globotetraosylceramide or Forssman antigen (Gb(5)Cer) glycosphingolipids or to sialic-acid-containing gangliosides. It was demonstrated that the presence of a GalCer or GlcCer residue alone is not sufficient for optimal binding, and additional carbohydrate residues are required for high-affinity adherence. Indeed, the binding efficiency of F1C fimbriated recombinant bacteria increased by 19-fold when disaccharide sequence GalNAcbeta1-4Galbeta is linked to glucosylceramide as in asialo-GM(2) (GgO(3)Cer). Thus, it is suggested that the disaccharide sequence GalNAcbeta1-4Galbeta of asialo-GM(2) (GgO(3)Cer) which is positioned internally in asialo-GM(1) (GgO(4)Cer) is the high-affinity binding epitope for the F1C fimbriae of uropathogenic E. coli.

Differential expression of alpha2-6 sialylated polylactosamine structures by human B and T cells.

We found that human peripheral B and T cells differed in the surface expression of alpha2-6 sialylated type 2 chain glycans. In contrast to B cells, T cells expressed only sialoglycans with repeated N-acetyllactosamine (Galss1-4GlcNAc) disaccharides. This finding was based on the specificity of the monoclonal antibodies HB6, HB9 (CD24), HD66 (CDw76), FB21, and CRIS4 (CDw76) with the alpha2-6 sialylated model gangliosides IV6NeuAcnLc4Cer (2-6 SPG), VI6NeuAcnLc6Cer (2-6 SnHC), VIII6NeuAcnLc8Cer (2-6 SnOC), and X6NeuAcnLc10Cer (2-6 SnDC). We found that, in addition to their common requirement of an alpha2-6 bound terminal sialic acid for binding, the antibodies displayed preferences for the length of the carbohydrate backbones. Some of them bound mainly to 2-6 SPG with one N-acetyllactosamine (LacNAc) unit (HB9, HD66); others preferentially to 2-6 SnHC and 2-6 SnOC, with two and three LacNAc units, respectively (HB6 and FB21); and one of them exclusively to very polar alpha2-6 sialylated type 2 chain antigens (CRIS4) such as to 2-6 SnOC and even more polar gangliosides with three and more LacNAc units. These specificities could be correlated with the cellular binding of the antibodies as follows: whereas all antibodies bound to human CD 19 positive peripheral B cells, their reactivity with CD3 positive T cells was either nearly lacking (HD66, HB9), intermediate (about 65%: HB6, FB21) or strongly positive (CRIS4, 95%). Thus, the binding of the antibodies to 2-6 sialylated glycans with multiple lactosamine units appeared to determine their binding to T-cells.

Changed expression of 9-O-acetyl GD3 (CDw60) in benign and atypical proliferative lesions and carcinomas of the human breast.

Expression of gangliosides is affected in various ways by malignant cell transformation. In the present study, we investigated the expression of CDw60, a constituent of O-acetylated disialogangliosides, in benign and atypical proliferative breast diseases, and preinvasive and invasive carcinomas by immunohistochemistry and thin-layer chromatography (TLC). In normal ducts, antibodies to CDw60 (mAb M-T21) reacted to membranes of the Golgi apparatus in the juxtaluminal cell compartment. A similar polarized distribution of Golgi cisterns in epithelial cells was observed in several benign lesions, i.e., fibroadenomas, intraductal papillomas, and gynecomastia. In contrast, blunt duct adenosis and duct hyperplasia exhibited an abnormal cytosolic and cell surface staining, whereas atypical duct hyperplasia showed randomly dispersed immunoreactive Golgi cisterns, indicating loss of epithelial polarity. In mammary carcinomas and in two breast carcinoma cell lines (MCF-7 and EFM-19) the neoplastic cells contained CDw60-immunolabelled Golgi complexes, which were distributed in a disorderly fashion throughout the cytoplasm, thus reflecting a loss of epithelial polarity. Additionally, only well differentiated ductal carcinomas in situ or invasive ductal carcinomas disclosed a strong cell surface labelling, which was absent in lower differentiated carcinomas of the same types. In all carcinomas, the intensity of CDw60 immunostaining decreased with progressing loss of differentiation (grade of dedifferentiation), as demonstrated by staining intensity in paraffin sections and by evaluation of the relative amounts of extracted 9-O-acetyl GD3 by TLC. Our results indicate that abnormal CDw60 expression is already detectable in benign proliferative breast lesions with different risk rates to develop into malignant lesions. Downregulation of CDw60 expression in poorly differentiated invasive carcinomas may be the consequence of loss of cell functions usually associated with poor prognosis.

Glycosphingolipids of skeletal muscle: I. Subcellular distribution of neutral glycosphingolipids and gangliosides in rabbit skeletal muscle.

Membrane vesicles were prepared from rabbit skeletal muscle, separated by sucrose density gradient centrifugation and characterized by their specific marker enzymes, ligand binding, and ion flux activities. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M). Their glycosphingolipid compositions were analyzed by biochemical and immunochemical methods with specific antibodies (TLC immunostaining) and characteristic patterns were obtained from respective membrane fractions, expressed on a protein basis. Glucosylceramide, the main neutral glycosphingolipid of rabbit muscle, was found in SL and TT fractions, whereas SR and Tr/M vesicles lack this compound. Lactosylceramide was selectively recovered in the SR1 fraction. GM3(Neu5Ac), the main ganglioside in rabbit muscle, was found to account for 64% in the SL, 13% in the TT, 7% in the SR1, 3% in the SR2 and 13% in the Tr/M fractions. IV3Neu5Ac-nLcOse4Cer was mostly abundant in SL and decreased in the order SL > TT, Tr/M > SR1, SR2. IV6Neu5Ac-nLcOse4Cer was only detected in the SL and Tr/M fractions in noteworthy quantities. Ganglioseries gangliosides GM1, GD1a, GD1b and GT1b displayed homogeneous distribution patterns in each membrane preparation. They were expressed only in small amounts but mainly in SL, TT and Tr/M vesicles and to less extent in SR1 and SR2 fractions. The presence of GM3(Neu5Ac) in the SL as well as on subcellular level was confirmed in transverse muscle cryosections by means of indirect immunofluorescence microscopy. The SL was brightly stained, but considerable intracellular fluorescence was observed as expected from the biochemical analyses. Thus, the neutral GSL and ganglioside expression of the SL and the intracellular membraneous network is different in skeletal muscle both in terms of quantitative and qualitative GSL composition as demonstrated in details by means of biochemical and immunochemical techniques. The modulatory functions of GM3 and gangliosides of the neolacto- and ganglio-series towards the voltage dependent Ca(2+)-channel, largely preponderant in the triads-containing Tr/M fraction, is the subject of the accompanying paper (J. Müthing, U. Maurer, and S. Weber-Schürholz, Carbohydr. Res., 307 (1998) 147-157).

Subcellular localization of glycosphingolipids in human neutrophils.

The subcellular distribution of five major glycosphingolipids (GSLs) in human neutrophils was analyzed. The neutrophils were isolated from the blood of six donors and subdivided in three fractions containing the cell membranes, and the primary and the secondary granules, respectively. The separation was confirmed with antibodies detecting established subcellular fraction-specific molecules. The two main neutral GSLsGalbeta1-4Glcbeta1-1'Cer (lactosylceramide, LacCer) and nLc4Cer (paragloboside, PG) and the three gangliosides IV3NeuAcnLc4Cer (2-3SPG), IV6NeuAcnLc4Cer (2-6SPG), and VI3NeuAcnLc6Cer (2-3SnHC) were quantitated using the immunochemical digoxigenin (DIG) staining procedure. Secondary granules contained the highest amount of these GSLs. They are followed by the primary granules and the cell membranes. Based on this quantitation, we conclude that the majority of the GSLs of neutrophils occur intracellularly. These findings are in striking contrast to the general assumption of GSLs being mainly concentrated in the cell membrane.

The 9-O-acetylated disialosyl carbohydrate sequence of CDw60 is a marker on activated human B lymphocytes.

Gangliosides with a terminal 9-O-acetylated disialosyl group (CDw60 structures) show a restricted surface expression on human leukocytes. Hithereto, they have only been detected on subpopulations of human T lymphocytes. Using the defined CDw60 antibody UM4D4 and two new antibodies with preferential CDw60 activities, F6 and Z17, we demonstrate for the first time that CDw60 is an activation marker on human B lymphocytes. In vitro phorbol ester-stimulated human peripheral blood B lymphocytes as well as in vivo activated tonsillar B lymphocytes became CDw60 positive. CDw60 expression of these cells exceeds that of resting and activated T-lymphocytes.

The CDw65 monoclonal antibodies VIM-8 and VIM-11 bind to the neutral glycolipid V3FucnLc8Cer.

At the IVth and Vth Workshop on Human Leukocyte Differentiation Antigens a group of monoclonal antibodies recognizing myeloid cells was found to bind to the ganglioside X3-NeuAcVII3FucnLc10Cer (VIM-2 dodecasaccharide). These antibodies were given the provisional cluster of differentiation designation CDw65. Three antibodies of this cluster (VIM-2, VIM-8, and VIM-11) have now been studied in detail at the molecular and the cellular level. Binding of VIM-2 is abolished after treatment of cells with Vibrio cholerae neuraminidase, whereas VIM-8 and VIM-11 show enhanced binding to neuraminidase-treated cells. We investigated binding of the three mAbs to glycolipid antigens with shorter carbohydrate chains. Distinct differences were observed in the binding of CDw65 antibodies to VIII3-NeuAcV3FucnLc8Cer (VIM-2 decasaccharide). VIM-2 strongly bound to this antigen, whereas no binding was observed with the other two mAbs. Conversely, the asialoganglioside of the VIM-2 decasaccharide, V3FucnLc8Cer, was not recognized by VIM-2, but this antigen bound strongly VIM-8 and VIM-11. Thus, VIM-2 and the other CDw65 antibodies represented two different antigen specificities.

7-O-acetyl-GD3 in human T-lymphocytes is detected by a specific T-cell-activating monoclonal antibody.

The monoclonal antibody U5, which is a potent inducer of proliferation in human T-cells, was found to bind to an alkali-sensitive derivative of ganglioside GD3. Using immunochemical and spectroscopic methods, the structure of the U5 antigen was determined as 7-O-acetyl-GD3. The antibody U5 did not react with 9-O-acetyl-GD3 and bound severalfold more stronger to 7-O-acetyl-GD3 than to GD3. U5 is the first antibody known to detect preferentially 7-O-acetyl-GD3. Flow cytometric analysis showed that each major class of human leukocytes contained a significant fraction of cells binding the U5 antibody.

Association of the GPI-anchored leucocyte surface glycoproteins with ganglioside GM3.

The major glycolipid co-immunopurifying with the glycosylphosphatidylinositol-anchored leucocyte surface glycoprotein CD59 from detergent lysates of human T cell lines HPB ALL, Jurkat and myeloid line HL-60 was identified as the glycosphingolipid GM3. Monoclonal antibodies to GM3 immunoprecipitated the same large detergent-resistant, protein-tyrosine kinase containing "GPI-complexes" as antibodies to several GPI-anchored proteins. Therefore GM3 is another component of these large membrane complexes potentially involved in signalling through GPI-anchored receptors or through some glycolipids.

CDw60 glycolipid antigens of human leukocytes: structural characterization and cellular distribution.

Monoclonal CDw60 antibodies recognize glycolipid antigens with restricted surface expression on human leukocytes. They allow us to define new functional subpopulations of T lymphocytes and are able to induce costimulatory signals. In this report, we describe the molecular composition of CDw60 glycolipid antigens derived from different human leukocyte subpopulations. The glycolipids were isolated and their structures were identified by immunochemical methods. All molecules containing the CDw60 determinant were found in the disialoganglioside fraction. They were O-acetylated derivatives of the gangliosides II3 (Neu5Ac)2-LacCer (GD3), IV3 (Neu5Ac)2-nLc4Cer (DSPG), and VI3 (Neu5Ac)2-nLc6Cer (DSnHC), respectively. The most common CDw60 glycolipid antigen in human leukocytes was 9-O-acetyl GD3. In a comparison of various cell types, the highest concentration of 9-O-acetyl GD3 on a per cell basis was determined in granulocytes and in blood T lymphocytes, whereas B lymphocytes, thymus cells, and monocytes contained considerably smaller amounts of this molecule. Polar CDw60 antigens such as 9-O-acetyl DSPG and 9-O-acetyl DSnHC were only detected in granulocytes.

Monoclonal antibodies to the carbohydrate structure Lewis(x) stimulate the adhesive activity of leukocyte integrin CD11b/CD18 (CR3, Mac-1, alpha m beta 2) on human granulocytes.

Several carbohydrate structures on human granulocytes have been discussed as potential ligands for C-type lectins (selectins) on endothelial cells. Among them are the lacto-series type II chain antigens sialyl-Lewis(x) (SLe(x), Lewis(x) (Le(x)), and VIM2. We demonstrated in this study that monoclonal antibodies (mAbs) to Le(x) and to SLe(x), but not other anticarbohydrate mAbs (VIM2, CDw17, CD24), can stimulate granulocytes to form homotypic aggregates. This effect was particularly noticeable with three distinct anti-Le(x) mAbs (3C6, 4D1, 6C7). Much less impressive effects were also seen with nine other anti-Le(x) mAbs and with the anti-SLe(x) mAb CSLEX1. Aggregation was shown to be an active process. It is temperature and energy dependent, requires divalent cations, and is selective in terms of mAb specificity. Anti-Le(x)-induced homoaggregate formation could be inhibited with CD11b mAb JML-H11 and CD54 (ICAM1) mAb LB-2 and thus seems to be associated with activation of the beta 2-integrin cytoadhesion pathway.

Gangliosides: differentiation markers for murine T helper lymphocyte subpopulations TH1 and TH2.

On the basis of the pattern of lymphokines they secrete, murine T helper clones can be divided into two subsets, TH1 and TH2. This concept of two different T helper effector cells helps to explain the diversity of immune reactions occurring in different parts of the body. The in vivo localization of T helper subtypes is of great interest, but up to now no biochemical or surface markers were available to distinguish between them. We analyzed the glycolipids from altogether 12 murine TH1 and TH2 cell lines or clones. A comparison of the gangliosides by thin-layer chromatography showed differences between the TH1 and TH2 cells. Binding studies with specific antibodies to asialo backbone structures after degradation by neuraminidases showed that the main gangliosides from these lymphocytes shared a common GgOse4 backbone and thus differed only in their degree or position of sialylation. Two disialogangliosides appeared to be characteristic. They were isolated from the D10.G4.1 TH2 cell clone and identified by fast atom bombardment mass spectrometry as IVNeuAc,IINeuAc-GgOse4Cer (GD1a) and IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha), respectively. GD1a was characteristically only detected in TH2 cells, whereas GD1 alpha was preferably, but not exclusively, expressed by TH1 lymphocytes. Although GD1a was also found in the lung, heart, kidney, and spleen, its expression within the murine immune cells under investigation was unique to TH2 lymphocytes. Scarcely any GD1a was found in thymocytes, B cells, or CD8 positive (cytolytic) T cells, but significant expression was seen in CD4 positive (helper) T cells which include the TH2 subpopulation.(ABSTRACT TRUNCATED AT 250 WORDS)

CDw 60 antibodies bind to acetylated forms of ganglioside GD3.

Four monoclonal antibodies, M-T21, M-T32, M-T41 and UM4D4, which belong to the new CDw 60 cluster of antibodies specific for a subpopulation of human T-lymphocytes, were found to bind mainly to acetylated forms of ganglioside GD3. After O-deacetylation of the antigen, binding was reduced ("M-T"-antibodies) or abolished (UM4D4).

Immunochemical detection of glycosphingolipids on thin-layer chromatograms.

A sensitive immunochemical method was developed for the detection of glycosphingolipids on thin-layer chromatograms. The procedure involves oxidation of diol groups of glycosphingolipids with sodium periodate, derivatization of the formed aldehyde groups with digoxigenin-hydrazide, and reaction of the bound digoxigenin with an alkaline phosphatase-labeled polyclonal anti-digoxigenin antibody. The latter is detected by an insoluble indigo-like dye as a result of dephosphorylation of 5-bromo-4-chloro-3-indolyl phosphate. The detectability of all glycosphingolipid species was improved over that of the orcinol and resorcinol staining methods. Two nanograms of the standard gangliosides GM1, GD1A, and GT1 was detected, whereas the detection limit for short-chain neutral glycosphingolipids was in the range of 20-50 ng. Long-chain glycosphingolipids were detectable with a particularly high sensitivity. Selective staining of the gangliosides could be achieved by the use of low periodate concentrations.

Monoclonal antibodies against the human lymphocyte differentiation antigen CD 76 bind to gangliosides.

Two monoclonal antibodies, HD 66 and CRIS-4, by which the new CD 76 B-cell-associated cluster was defined, bound to several gangliosides (sialic acid containing glycolipids) of different polarity. One of the gangliosides recognized by HD 66 could be identified as NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-beta 1-1'Cer. This antigen was enzymatically synthesized. Sialidase treatment of the ganglioside antigens abolished binding of HD 66 and CRIS-4.

Neutral glycosphingolipids of the globo-series characterize activation stages corresponding to germinal center B cells.

The neutral glycosphingolipid (GSL) globotriaosylceramide (Gb3) of the globo-series was recently defined as the CD77 antigen. This B cell-associated antigen is characterized by its specific expression on germinal center B cells. In order to study the potential relation of the CD77 antigen and other GSLs to B cell activation we have performed a comprehensive analysis of the synthesis and expression of neutral GSL in tonsillar B lymphocytes. Monoglycosylceramide (GL1) and lactosylceramide (LacCer) comprised the largest portion of GSL in tonsillar B lymphocytes as detected by HPLC analysis. GSLs of the globo-series Gb3 and globotetraosylceramide (Gb4), were found in smaller amounts. Since other GSLs, like gangliotriaosylceramide (Gg3) and gangliotetraosylceramide (Gg4), could only be detected using highly sensitive antibody reactions, we assume that these GSLs occur in B cells only in minor amounts. When tonsillar B cells were density fractionated on Percoll, the light density cells, which correspond to activated cells, contained and expressed more of both globo-GSLs than cells in the higher density fraction. When the dense fraction of tonsillar B cells was activated in vitro by anti-mu/BCGF, synthesis of GL1, LacCer, Gb3, and Gb4 was biphasic, with maxima at 12 and 84 h. Surface expression of the CD77 antigen on the denser cells was strongly induced by anti-mu/BCGF during the first 24 h of cultivation followed by a rapid decline thereafter, mimicking synthesis. PMA treatment of this cell fraction caused an even stronger expression of the CD77 antigen, which lasted over 48 h of cultivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Forssman glycolipid, an antigenic marker for a major subpopulation of macrophages from murine spleen and peripheral lymph nodes.

Three hybridoma clones were isolated after hybridization of a mouse myeloma line with splenocytes from rats immunized with Forssman glycosphingolipid (Fo). Two of these clones produced Fo-specific monoclonal antibodies (MAB) of the IgM class, one MAB of the IgG2c class. In complement-dependent depletion experiments and immunofluorescence studies on the nature of Fo-positive leukocytes in CBA/J mice the following results were obtained: whereas blood monocytes, polymorphonuclear leukocytes, and lymphocytes were Fo negative, 5 to 10% of suspended spleen cells were positive. The majority of these were macrophage-like, glass- and nylon-adherent, nonspecific esterase-positive phagocytizing cells carrying Ia and globoside markers. These cells participated as accessory cells in the mixed lymphocyte culture reaction. In cell suspensions from axillary and inguinal lymph nodes, 2% were Fo positive. They were enriched up to 70% in the glass-adherent, esterase-positive population from this source. In contrast, no Fo-positive cells were detected in mesenteric lymph nodes, and less than 0.1% of the resident peritoneal macrophages bore this marker. The percentage of Fo-positive cells increased to 1% in thioglycollate-elicited peritoneal cells. Immunostaining of cryosections of lung and liver tissue showed alveolar macrophages and Kupffer cells, respectively, to be Fo negative.

Structural characterization of gangliosides from murine T lymphocytes.

Mouse spleen cells were prepared from CBA/J mice, and T lymphocytes were selectively stimulated with the T cell mitogen concanavalin A and further propagated in the presence of the T cell growth factor interleukin-2. The T cells were metabolically labeled with D-[1-14C]galactose and D[1-14C]glucosamine, and the gangliosides were extracted and purified by DEAE-Sepharose column chromatography. Carbohydrate backbone structures of the asialogangliosides, prepared by mild acid hydrolysis, were determined by high-performance liquid chromatography, treatment with exoglycosidases and immunostaining. Monosialylated gangliosides were isolated by gradient elution from DEAE-Sepharose and further separated by preparative high-performance thin-layer chromatography in two solvent systems. Isolated fractions were characterized by preparation of asialogangliosides by mild acid hydrolysis, the action of Vibrio cholerae neuraminidase, and fast-atombombardment mass spectrometry. The following structures were identified: IVNeuAc-GgOse4Cer; IVNeuGc-GgOse4Cer; IVNeuAc-GgOse5Cer; and IVNeu-Gc-GgOse5Cer. The latter two gangliosides were not detected on B lymphoblasts and may be T-cell-specific structures. All gangliosides were heterogeneous in their ceramide moieties, being substituted with C16:0, C24:0, and C24:1 fatty acids. A preliminary study of several other mouse strains showed no strain-specific genetic variations in the T cell gangliosides. The possible role of these gangliosides is discussed.