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

SLAM在记忆性T细胞、B细胞的一部分、未成熟的胸腺细胞上有结构性表达，但在幼稚T细胞上不表达。然而，当SLAM被激活时，它会迅速被压在幼稚T细胞上。为了分析SLAM的生物学作用，特别是SLAM协同刺激的机制，我们试图确定SLAM的配体。为此，我们从PHA刺激的PBT中提取RNA，通过RT-PCR亚克隆了SLAM的cDNA，并将SLAM胞外部分与人免疫球蛋白I Fc部分的融合蛋白(LTO 717)插入到pCDM8中，构建了融合蛋白(LTO 717)。将pCDM8-SLAM-Ig稳定地转入P3UI细胞，并从P3UI细胞上清液中纯化了大量SLAM-Ig融合蛋白。我们还用类似的技术制备和纯化了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之间的相互作用在抗原提呈细胞介导的T细胞活化中的重要性。虽然CTLA4-Ig或ICAM-L-Ig可通过阻断CD28/CD86或ICAM-L/LFA1的相互作用来抑制T细胞的激活，但SLAM-Ig不影响T细胞的激活。我们还尝试用SLAM-Ig对各种细胞进行染色，没有一个细胞被染色。我们推测SLAM-Ig功能失效的原因是铰链部分的二硫键结合形成了双分子。我们现在正在生成一种在铰链中没有二硫酸盐部分的新结构。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.