T cell clones with normal or defective O‐galactosylation from a patient with permanent mixed‐field polyagglutinability

Abstract

To delineate the extent of O-galactosyltransferase deficiency within the lymphoid lineage, monoclonal antibody specific for the Thomsen-Friedenreich (TF) antigen (Gal beta 1----3GalNAc alpha 1-O-Ser/Thr) and its precursor the Tn antigen (GalNAc alpha 1-O-Ser/Thr) were applied to the flow cytometric analysis of peripheral blood lymphocytes from a patient with permanent mixed-field polyagglutinability (PMFP). We show that only a minor population of 4% expressed the Tn antigen which is in contrast to 93% of the patient's erythrocytes carrying the defect. Tn+ lymphocytes mainly belonged to the CD3+ subset, but were also CD19+ or CD16+. Both Tn+ and TF+ T cell clones from patient R. R. were established and shown to belong to the CD4+ or CD8+ antigenic subset. Three glycosyltransferase activities were determined in lysates from these clones: all Tn+ clones were deficient in UDP-Gal: GalNAc alpha 1-O-Ser/Thr beta 1----3 galactosyltransferase (beta 3Gal-T) activity; by contrast this activity was present in all lysates from TF-expressing clones. UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (GalNAc-T) and UDP-Gal: GlcNAc-R beta 1----4 galactosyl-transferase (beta 4Gal-T) exhibited similar activities in both Tn+ and TF+ T cell clones. As a consequence of defective O-galactosylation in Tn+ T cells, cell surface sialic acid of Tn+ clones was reduced by greater than 50% when compared to TF+ clones as demonstrated by sialic acid-specific labeling using fluoresceinated Limax flavus agglutinin(LA) and flow cytometry. The Tn phenotype of T cell clones was stable for more than 1 year of continuous expansion in vitro. These data demonstrate that in PMFP, T cells may also be affected by the O-galactosyltransferase deficiency which is accompanied by a substantial loss of cell surface sialic acid. However, the frequency of Tn+ lymphocytes in peripheral blood from patient R.R. was strikingly low. These T cell clones should be useful to study the defect at a genetic level and the importance of O-linked carbohydrates for proper T cell function.

Persistent repression of a functional allele can be responsible for galactosyltransferase deficiency in Tn syndrome.

“…TKH6 (IgM anti-Tn) and HH8 (IgM anti-TF) mAb were described previously (4,23). DF-Tl, the sialic acid (NeuAc)-dependent mAb anti-CD43 (IgG 1) (24) was purchased from Dakopatts (Copenhagen, Denmark).…”

“…T cell lines and culture. The generation and characterization of the CD4+ PMFP T cell clone used in this study has been described recently (4). Briefly, the Tn+ subpopulation ofthe PBL from patient R.R.…”

“…was enriched by a negative sorting on a fluorescence activated cell sorter. T (4). Protein concentrations of cell lysates were determined with the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL) and BSA as a standard according to the manufacturer's instruction.…”

“…Tn syndrome or permanent mixed-field polyagglutinability (PMFP)' is a human disorder characterized by the exposure of truncated 0-glycans, in particular the Tn antigen (GalNAca 1-O-Ser/Thr) (1) due to a selective deficiency of UDP-Gal:GalNAca l-O-Ser/Thr #1-3 galactosyltransferase (f3Gal-T) activ-ity (2, 3) in a variable proportion of hematopoietic cells (4). These patients, who are also said to have blood group Tn (5), occasionally develop hemolytic anemia, thrombopenia, and leukopenia most likely mediated by anti-Tn natural antibodies present in normal serum (6).…”

“…For this purpose, we took advantage from Tn+ T cell clones recently established in our laboratory from the PBL of a well-characterized patient (R.R.) (4). These Tn+ T cells represented nontransformed cells reflecting an in vivo situation and thus provided a system to investigate whether repression of a functional allele of the f3Gal-T gene may be responsible for the enzyme deficiency.…”