van Dongen J J, Krissansen G W, Wolvers-Tettero I L, Comans-Bitter W M, Adriaansen H J, Hooijkaas H, van Wering E R, Terhorst C
Department of Cell Biology, Immunology, and Genetics, Erasmus University, Rotterdam, The Netherlands.
Blood. 1988 Mar;71(3):603-12.
The expression of cytoplasmic CD3 (CyCD3) was analyzed in 45 leukemias, five thymus cell samples, five peripheral blood (PB) samples, and ten cell lines. All T cell acute lymphoblastic leukemias (T-ALL) that did not express surface membrane CD3 (SmCD3) appeared to express CyCD3. Furthermore, the majority of SmCD3+ T-ALL also expressed CyCD3. Analogous results were obtained with thymus cell samples in that about 95% of the thymocytes expressed CyCD3 whereas 60% to 75% of the thymocytes also expressed SmCD3. In normal peripheral blood only prominent SmCD3 expression was found. These data indicate that immature T cells express CyCD3 only, that the combined expression of CyCD3 and SmCD3 is characteristic for intermediate differentiation stages, and that mature T cells express prominent SmCD3. All (precursor) B cell leukemias, acute myeloid leukemias, and non-T cell lines tested did not express CyCD3. On the basis of these data, we conclude that CyCD3 expression is restricted to the T cell lineage and can be used as a diagnostic marker for immature SmCD3- T cell malignancies. Therefore, we evaluated which fixative is optimal for CyCD3 staining, and we determined by immunofluorescence staining and Western blotting which anti-CD3 monoclonal antibody (MoAb) can be used for the detection of CyCD3. In our opinion, acid ethanol was the best fixative for the cytocentrifuge preparations. Furthermore, we demonstrated that CyCD3 can be easily detected by use of MoAbs raised against denaturated CD3 chains such as those of the SP series (SP-6, SP-10, SP-64, and SP-78). In addition we tested 22 anti-CD3 MoAbs of the Oxford CD3 panel that were raised against native SmCD3, and it appeared that only four (UCHT1, VIT-3b, G19-41 and SK7/Leu-4) of them were able to detect CyCD3. In Western blot analysis all four MoAbs recognized the CD3-epsilon chain only.
在45例白血病、5例胸腺细胞样本、5例外周血（PB）样本及10种细胞系中分析了细胞质CD3（CyCD3）的表达情况。所有不表达表面膜CD3（SmCD3）的T细胞急性淋巴细胞白血病（T-ALL）似乎都表达CyCD3。此外，大多数SmCD3+ T-ALL也表达CyCD3。胸腺细胞样本也得到了类似结果，约95%的胸腺细胞表达CyCD3，而60%至75%的胸腺细胞也表达SmCD3。在正常外周血中仅发现明显的SmCD3表达。这些数据表明，未成熟T细胞仅表达CyCD3，CyCD3和SmCD3的联合表达是中间分化阶段的特征，而成熟T细胞表达明显的SmCD3。所有测试的（前体）B细胞白血病、急性髓系白血病及非T细胞系均不表达CyCD3。基于这些数据，我们得出结论，CyCD3表达仅限于T细胞谱系，可作为未成熟SmCD3-T细胞恶性肿瘤的诊断标志物。因此，我们评估了哪种固定剂最适合CyCD3染色，并通过免疫荧光染色和蛋白质印迹法确定了哪种抗CD3单克隆抗体（MoAb）可用于检测CyCD3。我们认为，酸性乙醇是细胞离心涂片制备的最佳固定剂。此外，我们证明使用针对变性CD3链产生的MoAb（如SP系列的MoAb：SP-6、SP-10、SP-64和SP-78）可轻松检测到CyCD3。此外，我们测试了牛津CD3单抗组中针对天然SmCD3产生的22种抗CD3 MoAb，结果发现其中只有4种（UCHT1、VIT-3b、G19-41和SK7/Leu-4）能够检测到CyCD3。在蛋白质印迹分析中，所有这4种MoAb仅识别CD3-ε链。
