Thompson L F, Ruedi J M, Low M G, Clement L T
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
J Immunol. 1987 Dec 15;139(12):4042-8.
Human T and B lymphocyte subsets were characterized for ecto-5'-nucleotidase (ecto-5'-NT) expression by two-color immunofluorescence by using polyclonal goat antibodies to 5'-NT and murine monoclonal antibodies to T and B cell subsets. Anti-5'-NT antibodies were prepared by immunizing a goat with purified human placental 5'-NT. Lymphocyte surface 5'-NT was detected with F(ab')2 fragments of immune goat IgG followed by biotinylated F(ab')2 rabbit anti-goat IgG and fluorescein isothiocyanate-avidin. Lymphocyte cell surface antigens were detected with phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD16, and anti-CD19. HB-4, an antigen present on a major subset of human peripheral blood B cells, was detected with murine monoclonal anti-HB-4 and PE-anti-mouse-kappa. Analysis showed that ecto-5'-NT was expressed on 32 +/- 7% of CD3+, 19 +/- 6% of CD4+, and 50 +/- 21% of CD8+ T cells, but not on CD16+ lymphocytes. Ecto-5'-NT was also expressed on 81 +/- 8% of adult peripheral blood B cells as defined by PE-anti-CD19; HB-4 was expressed on 84 +/- 7% of CD19+ cells. The two populations of B cells were not identical, however, because HB-4 was co-expressed on only 79 +/- 18% of ecto-5'-NT+ B cells. Two-color immunofluorescent staining of T cells from a patient with congenital agammaglobulinemia and low T cell ecto-5'-NT activity revealed reduced percentages of ecto-5'-NT+ cells in his CD3+, CD4+, and CD8+ populations. Thus, reduced ecto-5'-NT activity by enzyme assay was paralleled by reduced numbers of 5'-NT molecules on the cell surface. Two-color immunofluorescent staining of B cells from a patient with hypogammaglobulinemia and low B cell ecto-5'-NT activity also revealed markedly reduced expression of 5'-NT. HB-4 expression was normal, however, suggesting that the patient's B cells were blocked in maturation subsequent to the acquisition of HB-4 but prior to that of ecto-5'-NT. These results demonstrate that anti-5'-NT antibodies will be valuable tools for analyzing ecto-5'-NT expression and lymphocyte maturation in patients with immuno-deficiency diseases.
采用针对5'-核苷酸酶（ecto-5'-NT）的多克隆山羊抗体和针对T细胞及B细胞亚群的鼠单克隆抗体，通过双色免疫荧光法对人T和B淋巴细胞亚群的ecto-5'-NT表达进行了表征。用纯化的人胎盘ecto-5'-NT免疫山羊制备抗ecto-5'-NT抗体。用免疫山羊IgG的F(ab')2片段检测淋巴细胞表面的ecto-5'-NT，随后用生物素化的F(ab')2兔抗山羊IgG和异硫氰酸荧光素抗生物素蛋白进行检测。用藻红蛋白（PE）偶联的抗CD3、抗CD4、抗CD8、抗CD16和抗CD19检测淋巴细胞表面抗原。用鼠单克隆抗HB-4和PE-抗小鼠κ链检测人外周血B细胞主要亚群上存在的一种抗原HB-4。分析表明，ecto-5'-NT在32±7%的CD3+、19±6%的CD4+和50±21%的CD8+ T细胞上表达，但在CD16+淋巴细胞上不表达。根据PE-抗CD19定义，ecto-5'-NT在81±8%的成人外周血B细胞上也有表达；HB-4在84±7%的CD19+细胞上表达。然而，这两个B细胞群体并不相同，因为HB-4仅在79±18%的ecto-5'-NT+ B细胞上共表达。对一名先天性无丙种球蛋白血症且T细胞ecto-5'-NT活性低的患者的T细胞进行双色免疫荧光染色，结果显示其CD3+、CD4+和CD8+群体中ecto-5'-NT+细胞的百分比降低。因此，酶法检测到的ecto-5'-NT活性降低与细胞表面5'-NT分子数量减少相平行。对一名低丙种球蛋白血症且B细胞ecto-5'-NT活性低的患者的B细胞进行双色免疫荧光染色，也显示5'-NT的表达明显降低。然而，HB-4表达正常，这表明该患者的B细胞在获得HB-4后但在获得ecto-5'-NT之前的成熟过程中受到了阻碍。这些结果表明，抗ecto-5'-NT抗体将成为分析免疫缺陷疾病患者ecto-5'-NT表达和淋巴细胞成熟的有价值工具。
