Thurnher M, Clausen H, Fierz W, Lanzavecchia A, Berger E G
Institute of Physiology, University of Zürich, Switzerland.
Eur J Immunol. 1992 Jul;22(7):1835-42. doi: 10.1002/eji.1830220724.
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.
为了确定O-半乳糖基转移酶缺乏在淋巴谱系中的程度，将针对汤姆森-弗里德赖希（TF）抗原（Galβ1----3GalNAcα1-O-Ser/Thr）及其前体Tn抗原（GalNAcα1-O-Ser/Thr）的单克隆抗体应用于对一名患有永久性混合凝集性（PMFP）患者外周血淋巴细胞的流式细胞术分析。我们发现，只有4%的少数细胞群体表达Tn抗原，这与93%携带该缺陷的患者红细胞形成对比。Tn+淋巴细胞主要属于CD3+亚群，但也有CD19+或CD16+。建立了来自患者R.R.的Tn+和TF+ T细胞克隆，并显示它们属于CD4+或CD8+抗原亚群。测定了这些克隆裂解物中的三种糖基转移酶活性：所有Tn+克隆均缺乏UDP-Gal：GalNAcα1-O-Ser/Thrβ1----3半乳糖基转移酶（β3Gal-T）活性；相比之下，表达TF的克隆的所有裂解物中均存在这种活性。UDP-GalNAc：多肽α-N-乙酰半乳糖胺基转移酶（GalNAc-T）和UDP-Gal：GlcNAc-Rβ1----4半乳糖基转移酶（β4Gal-T）在Tn+和TF+ T细胞克隆中表现出相似的活性。由于Tn+ T细胞中O-半乳糖基化缺陷，与TF+克隆相比，Tn+克隆的细胞表面唾液酸减少了50%以上，这通过使用荧光素化的黄蛞蝓凝集素（LA）进行唾液酸特异性标记和流式细胞术得以证明。T细胞克隆的Tn表型在体外连续传代培养1年多的时间里保持稳定。这些数据表明，在PMFP中，T细胞也可能受到O-半乳糖基转移酶缺乏的影响，这伴随着细胞表面唾液酸的大量丧失。然而，患者R.R.外周血中Tn+淋巴细胞的频率极低。这些T细胞克隆对于在基因水平研究该缺陷以及O-连接碳水化合物对正常T细胞功能的重要性应该是有用的。
