Expression of CD175 (Tn), CD175s (sialosyl-Tn) and CD176 (Thomsen-Friedenreich antigen) on malignant human hematopoietic cells.

The expression of the histo-blood group carbohydrate structures T-nouvelle (Tn, CD175), sialylated Tn (CD175s) and the Thomsen-Friedenreich disaccharide (TF, CD176) on human leukemia cell lines was analyzed by their reactivity with specific monoclonal antibodies in flow cytometry, immunohistology and immunoprecipitation. Expression of sialylated CD176 was evaluated by comparative immunostaining with anti-CD176 antibodies before and after sialidase treatment. While only few cell lines expressed unmasked CD176, sialylated CD176 was present on all hematopoietic cell lines and native lymphocytes examined. CD175 and CD175s are preferentially expressed on erythroblastic leukemia cell lines. CD175s expression in these cells is consistent with the transcription of the gene encoding the key enzyme alpha2,6-sialyltransferase (hST6GalNAc1). The staining intensity was reduced after methanol pretreatment of cells, indicating that these glycans are partially expressed as constituents of glycosphingolipids. Immunoprecipitation and subsequent Western blotting revealed a series of distinct high molecular glycoproteins as carriers for these carbohydrate antigens. CD34 was identified as major carrier of CD176 by immunoprecipitation and microsequencing on a KG-1 subline enriched for CD176 expression. Incubation of several CD176-positive cell lines with anti-CD176 antibodies induced apoptosis of these cells, an effect not observed with anti-CD175/CD175s antibodies. Since the presence of naturally occurring anti-CD176 antibodies may represent a mechanism of immunosurveillance against CD176-positive tumor cells, we propose that sialylation of surface-expressed CD176--among other functions--protects against apoptosis.

Detection, isolation and partial characterization of an immunostimulating glycoprotein from Rhodococcus fascians.

In a search for novel immunostimulating substances we detected that culture supernatants of the gram-positive phytopathogenic bacterium, Rhodococcus fascians, were able to induce cytokine release (TNF(alpha)) from mouse peritoneal macrophages. Monoclonal antibodies were generated against the active principle, and were employed for its isolation and partial characterization as a high molecular (MW>100 kDa) glycoprotein. In addition, methods practicable for its biotechnological preparation and several ELISA variants for its determination were developed.

Isolation and characterization of thomsen-friedenreich-specific antibodies from human serum.

The Thomsen-Friedenreich (TF) disaccharide, galactose (Gal)beta1-3GalNAcalpha-, is a blood group-related oncofetal antigen with remarkable tumor specificity. Postpartum, carbohydrate structures on the cell walls of the gastrointestinal flora evoke natural antibodies of presumed TF specificity. These antibodies may provide an early barrier against TF-carrying tumor cells. Their possible role, however, has been difficult to assess, since so far only a multivalent immunosorbent, asialoglycophorin (aGP), has been employed for their preparation, and therefore their fine specificities have been only insufficiently defined. We have used a novel immunosorbent consisting of synthetic TFalpha disaccharides (Galbeta1-3GalNAcalpha-) coupled to polyacrylamide (PAA), which itself was covalently bound to cross-linked sepharose. For specificity analyses, aGP and a panel of PAA-conjugated mono- and oligosaccharides were employed. Binding to the PAA moiety was excluded. The affinity-purified anti-TFalpha antibodies were of the IgM (> or =0.5 mg/100 ml of serum) and IgG (approximately 0.05 mg/100 ml of serum) classes. They did partially cross-react with TFbeta, although we detected a second group of anti-TFbeta antibodies (both IgM and IgG) which did not cross-react with TFalpha. The affinity-purified TFalpha antibodies showed only marginal cross-reactivity with the related antigens lactose, Gal, Tn or the Forssman antigen. Besides TF-specific antibodies, we found antibodies to the carbohydrate antigens Tn, Forssman and beta-D-Gal as well as to noncarbohydrate epitopes of glycophorin in human serum.