Ruiz P, Zacharievich N, Shenkin M
Department of Pathology, University of Miami School of Medicine, Florida 33101, USA.
Clin Diagn Lab Immunol. 1998 May;5(3):362-8. doi: 10.1128/CDLI.5.3.362-368.1998.
Dipeptidyl peptidase IV (DPP IV), also identified as the glycoprotein CD26, is a transmembrane 110- to 120-kDa serine aminopeptidase involved in immune responses by influencing T-cell costimulation and by cleaving cytokines. Additionally, CD26 is a nonintegrin receptor that contains a binding site for extracellular matrix and other molecules. In order to further define the expression and functional activity of this membrane exopeptidase in human T cells, we developed a nondisruptive, four-color cytofluorogenic assay that utilizes three separate antibodies to cell-surface molecules (e.g., CD4/CD8/CD26 and CD19/CD56/CD26) along with a rhodamine 110-conjugated dipeptide substrate that allows the measurement of DPP IV activity in phenotypically defined cells. We found normal human thymi to have notable differences in time-dependent DPP IV activity among the thymocyte subsets defined by their CD4/CD8 phenotype, with CD4-/CD8- thymocytes containing less DPP IV activity than cells expressing CD4 and/or CD8 (i.e., maturing). CD26 positivity was moderately intense in thymocytes and tended to identify cells with higher DPP IV activity. The four-color technique was also used to examine mature peripheral blood lymphocytes, along with an assortment of leukemias and transformed T-cell lines. These experiments revealed that while DPP IV was consistently evident in normal T cells, neoplastic T cells could vary in their expression patterns. Furthermore, the presence (or intensity) of surface CD26 in some abnormal T cells and certain normal peripheral blood mononuclear cells was separable from the level of DPP IV measured intracellularly. Our results established that multicolor cytofluorographic analysis can be a practical means to measure DPP IV activity in various human cell populations. Furthermore, we found that DPP IV activity could vary in T cells according to their differentiation status and that under certain circumstances surface CD26 expression can be disassociated from the level of measured enzyme (i.e., DPP IV) activity.
二肽基肽酶IV（DPP IV），也被鉴定为糖蛋白CD26，是一种110至120 kDa的跨膜丝氨酸氨基肽酶，通过影响T细胞共刺激和裂解细胞因子参与免疫反应。此外，CD26是一种非整合素受体，含有细胞外基质和其他分子的结合位点。为了进一步确定这种膜外肽酶在人T细胞中的表达和功能活性，我们开发了一种非破坏性的四色细胞荧光检测法，该方法利用三种针对细胞表面分子的单独抗体（例如，CD4/CD8/CD26和CD19/CD56/CD26）以及一种罗丹明110偶联的二肽底物，从而能够测量表型明确的细胞中的DPP IV活性。我们发现，正常人类胸腺中，根据其CD4/CD8表型定义的胸腺细胞亚群在时间依赖性DPP IV活性方面存在显著差异，CD4-/CD8-胸腺细胞所含的DPP IV活性低于表达CD4和/或CD8的细胞（即成熟细胞）。CD26阳性在胸腺细胞中中等强度，并且倾向于识别具有较高DPP IV活性的细胞。四色技术还用于检查成熟的外周血淋巴细胞以及各种白血病和转化的T细胞系。这些实验表明，虽然DPP IV在正常T细胞中始终明显，但肿瘤性T细胞的表达模式可能会有所不同。此外，一些异常T细胞和某些正常外周血单核细胞表面CD26的存在（或强度）与细胞内测量的DPP IV水平是可分离的。我们的结果表明，多色细胞荧光分析可以作为一种实用手段来测量各种人类细胞群体中的DPP IV活性。此外，我们发现DPP IV活性在T细胞中可能因其分化状态而异，并且在某些情况下，表面CD26表达可能与测量的酶（即DPP IV）活性水平无关。
