Aubry J P, Shields J G, Jansen K U, Bonnefoy J Y
Glaxo Institute for Molecular Biology S.A., Geneva, Switzerland.
J Immunol Methods. 1993 Feb 26;159(1-2):161-71. doi: 10.1016/0022-1759(93)90154-y.
The interactions between T and B lymphocytes are mediated by several antigen-independent adhesion molecules including LFA-1/ICAM-1 and CD2/LFA-3. Recently new pairs of adhesion molecules involved in T and B interactions have been described: CD28/B7, CD5/CD72 and CD45RO/CD22. In order to study these heterotypic adhesion events, the phenotypes of the subpopulations as well as new potential adhesion molecules involved in conjugate formation, we have developed a flow cytometric method which analyses conjugate formation between T and B cells. The two types of cells were loaded with two vital intracellular dyes: human T lymphocytes purified from blood or tonsils were labelled with BCECF-AM (green fluorescence) and the B lymphoblastoid cell line, RPMI 8866 was labelled with Indo-1-AM (blue fluorescence). The two labelled cell populations were mixed, gently centrifuged for 5 min and then incubated at 37 degrees C in a waterbath for 5 min. The cells were then gently resuspended by inversion and analysed with a double laser flow cytometer. This method permitted us to discover new molecular interactions since preincubation of the two populations with monoclonal antibodies directed against some surface molecules inhibited conjugate formation. As an example, using this technique we found that the low affinity IgE receptor, CD23 and the CR2/EBV receptor are involved in T cell/B cell adhesion and can therefore be considered as a new pair of adhesion molecules. This method also seems to be applicable to recombinant cells bearing a single adhesion molecule such as LFA-1 and ICAM-1. A particular advantage of the two intracellular dyes we used is that they are compatible with the dyes commonly used for classical simultaneous triple colour immunofluorescence (phycoerythrin and Cy-Chrome). We were thus able to determine the subpopulations involved in forming conjugates and we found that T-B conjugates were preferentially formed by CD4, CD45RO positive T cells, which are believed to be the memory T lymphocytes.
T淋巴细胞和B淋巴细胞之间的相互作用是由几种不依赖抗原的黏附分子介导的，包括淋巴细胞功能相关抗原-1/细胞间黏附分子-1（LFA-1/ICAM-1）和CD2/LFA-3。最近，又发现了几对参与T细胞和B细胞相互作用的黏附分子：CD28/B7、CD5/CD72和CD45RO/CD22。为了研究这些异型黏附事件、亚群的表型以及参与共轭体形成的新的潜在黏附分子，我们开发了一种流式细胞术方法，用于分析T细胞和B细胞之间的共轭体形成。这两种细胞分别用两种活性细胞内染料进行标记：从血液或扁桃体中纯化的人T淋巴细胞用2',7'-双（2-羧乙基）-5（6）-羧基荧光素乙酰甲酯（BCECF-AM，绿色荧光）标记，B淋巴母细胞系RPMI 8866用indo-1-乙酰甲酯（Indo-1-AM，蓝色荧光）标记。将这两个标记的细胞群体混合，轻轻离心5分钟，然后在37℃水浴中孵育5分钟。然后通过翻转将细胞轻轻重悬，并用双激光流式细胞仪进行分析。由于用针对某些表面分子的单克隆抗体对这两个群体进行预孵育会抑制共轭体形成，因此该方法使我们能够发现新的分子相互作用。例如，使用该技术我们发现低亲和力IgE受体、CD23和CR2/EB病毒受体参与T细胞/B细胞黏附，因此可被视为一对新的黏附分子。该方法似乎也适用于携带单一黏附分子如LFA-1和ICAM-1的重组细胞。我们使用的两种细胞内染料的一个特别优点是它们与经典同时三色免疫荧光常用的染料（藻红蛋白和Cy-铬）兼容。因此，我们能够确定参与形成共轭体的亚群，并且我们发现T-B共轭体优先由CD4、CD45RO阳性T细胞形成，这些细胞被认为是记忆T淋巴细胞。
