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

新型共刺激分子SLAM对T细胞激活的功能作用为基因治疗奠定基础

• 批准号: 09670871
• 负责人: SHIMADA Shinji
• 金额: $ 1.98万
• 依托单位: YAMANASHI MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670871/
• 关键词: SLAM; co-stimulation; T cell activation

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.

SLAM在记忆T细胞、部分B细胞、未成熟胸腺细胞上组成型表达，但在初始T细胞上不表达。然而，SLAM在激活时被快速地按压在初始T细胞上。为了研究SLAM的生物学作用，特别是SLAM的共刺激输入机制，我们尝试鉴定SLAM的配体。为此，我们从PHA刺激的PBT中提取RNA，通过RT-PCR方法亚克隆了SLAM的cDNA，并将SLAM（1 to +717）胞外段与人IgG Ⅰ Fc段的cDNA插入pCDM 8中，制成融合蛋白的新构建体（质粒）。将该质粒（pCDM 8-SLAM-Ig）稳定转染P3 UI细胞，并从细胞上清液中纯化出大量SLAM-Ig融合蛋白。我们还用类似的方法制备并纯化了CTLA 4-Lg和ICAM- 1-IG融合蛋白。我们研究了这些辅助分子之间的相互作用的重要性，在T细胞活化介导的抗原呈递细胞，使用这些融合蛋白。虽然CTLA 4-IG或ICAM-1-IG可通过阻断CD 28/CD 86或ICAM-1/LFA 1的相互作用抑制T细胞活化，但SLAM-IG对本系统T细胞活化无影响。我们还尝试用SLAM-Ig染色各种细胞，没有细胞被染色。我们推测SLAM-Ig功能障碍的原因是铰链部分的二硫键结合使其形成二聚体。我们现在正在产生一种新的构建体，它在铰链中没有二硫键部分。这可能对我们的目的更有用。