Mittler R S, Goldman S J, Spitalny G L, Burakoff S J
Bristol-Myers Co., Pharmaceutical Research and Development Division, Wallingford, CT 06492-7660.
Proc Natl Acad Sci U S A. 1989 Nov;86(21):8531-5. doi: 10.1073/pnas.86.21.8531.
By employing flow cytometric analysis and fluorescence resonance energy transfer (FRET), we examined the physical relationship between the T-cell receptor-CD3 complex (Ti-CD3) and the CD4 molecule on helper T cells. Through the use of an L3T4-negative murine T-cell hybridoma infectant expressing the human CD4 gene and having antigen specificity for HLA-DR, we show that binding of the Ti-CD3 complex with an anti-CD3 monoclonal antibody induces its redistribution proximal to cell-surface CD4. FRET efficiency was 9.4% on cells labeled with rhodaminated anti-CD3 and fluoresceinated anti-CD4. FRET was found to be temperature dependent, since similarly treated cells held at 4 degrees C displayed a FRET efficiency of less than 1%. Energy transfer was evident within 3 min after warming cells to 37 degrees C. Energy transfer was not detected between Ti-CD3 and the abundantly expressed leukocyte common antigen (CD45). Of greater significance was our observation that hybridomas infected with a truncated CD4 gene lacking the cytoplasmic domain failed to transfer energy despite the fact that CD4 was expressed on the cell surface at levels equivalent to or greater than the wild type. These studies suggest that after crosslinking of the Ti-CD3 on CD4+ T cells, a physical association occurs between the antigen receptor complex and CD4 and that the association is dependent upon the presence of the cytoplasmic domain of CD4.
通过采用流式细胞术分析和荧光共振能量转移（FRET），我们研究了辅助性T细胞上T细胞受体-CD3复合物（Ti-CD3）与CD4分子之间的物理关系。利用一种表达人CD4基因且对HLA-DR具有抗原特异性的L3T4阴性小鼠T细胞杂交瘤感染体，我们发现Ti-CD3复合物与抗CD3单克隆抗体的结合会诱导其在细胞表面CD4附近重新分布。在用罗丹明标记的抗CD3和荧光素标记的抗CD4标记的细胞上，FRET效率为9.4%。发现FRET依赖于温度，因为在4℃保存的经类似处理的细胞显示FRET效率低于1%。将细胞升温至37℃后3分钟内能量转移明显。在Ti-CD3与大量表达的白细胞共同抗原（CD45）之间未检测到能量转移。更重要的是我们观察到，感染了缺乏胞质结构域的截短CD4基因的杂交瘤未能转移能量，尽管CD4在细胞表面的表达水平与野生型相当或更高。这些研究表明，在CD4+T细胞上的Ti-CD3交联后，抗原受体复合物与CD4之间发生了物理关联，且这种关联依赖于CD4胞质结构域的存在。
