The functional role of SLAM,a new co-stimulatory molecules, on T cell activation for the basis of gene therapy

SLAM在记忆T细胞、B细胞的一部分、未成熟胸腺细胞上组成性表达，但在幼稚T细胞上不表达。然而，当激活时，SLAM会迅速地作用于初始T细胞。我们也知道，SLAM对T细胞具有共刺激活性，通过交联刺激与激动单抗。为了分析SLAM的生物学作用，特别是SLAM的共刺激输入机制，我们试图鉴定SLAM的配体。为此，我们利用从pha刺激的PBT中纯化的RNA，用RT-PCR法亚克隆了SLAM的cDNA，并将SLAM的胞外部分（lto+717）与人IgG的Fc部分cDNA组成融合蛋白的新构建物（质粒）插入pCDM8中。这些质粒（pCDM8-SLAM-Ig）被稳定地转染到P3UI细胞中，我们从这些细胞的上清液中纯化了大量SLAM-Ig融合蛋白。我们也用类似的技术生成和纯化了CTLA4-Lg和ICAM- 1-Ig融合蛋白。我们利用这些融合蛋白研究了这些辅助分子在抗原提呈细胞介导的T细胞活化中相互作用的重要性。虽然CTLA4-Ig或ICAM-l- ig可以通过阻断CD28/CD86或ICAM-l /LFA1的相互作用来抑制T细胞的活化，但SLAM-Ig在我们的系统中不影响T细胞的活化。我们也尝试用SLAM-Ig染色各种细胞，但没有细胞被染色。我们推测我们的SLAM-Ig功能失调的原因是通过铰链部分的二硫结合来制造聚合物。我们现在正在产生一种新的结构，它在铰链中没有二硫部分。这可能对我们的目的更有用。

SLAM is constitutively expressed on memory T cells, a part of B cells, immature thymocytes, but not on naive T cells. However, SLAM is rapidlyex pressed on naive T cells when activated. It is also known that SLAM on T cells has a co-stimulation activity by crosslinking stimulation with agonistic mAb.To analyze the biological role of SLAM, especially for mechanism of the input of co-stimulation by SLAM, we tried to identify the ligand of SLAM.For the purpose, we subcloned the cDNA of SLAM by RT-PCR from RNA purified from the PHA-stimulated PBT, and made the new construct (plasmid) of fusion protein composed with extracellular portion of SLAM(lto+717) and the cDNA of Fc portion of human IgG I by inserted into pCDM8. These plasmid (pCDM8-SLAM-Ig) was stably transtected into P3UI cells, and we purified much of SLAM-Ig fusion protein from supematants ofthese cells. We also generated and purified CTLA4-Lg and ICAM- 1-Ig fusion proteinswith similar techniques. We investigated the importance of the interaction between these accessory molecules in T cell activation mediated by antigen presenting cells, using these fusion proteins. Although CTLA4-Ig or ICAM-l-Ig could inhibit the T cell activation by blocking the interaction of CD28/CD86 or ICAM- l/LFA1, SLAM-Ig could not affect on T cell activation in our system. We also tried to stain various cells with SLAM-Ig, none of cells were stained. We speculated the cause of the disfunctionof our SLAM-Ig as making bimer by the disulfide-binding in the portion of hinge. We are now generating a new construct which does not have disulfidate portion in hinge. This might be more useful for our purposes.