MOLECULAR ANALYSIS OF HUMAN ANTIGBM ANTIBODIES

这个项目的总体目标是更好地理解 狼疮性肾炎小鼠肾间质内的细胞事件。 自身反应性CD4T细胞亚群逃脱正常的假设 耐受性，并以特定于Ag的方式与居民直接反应 肾脏内的细胞，将进行测试。试验性的方法将 强调对病理相关细胞的分析。为此目的，一个 预先存在的自身反应性T细胞克隆(ARTC)，肾小管上皮细胞 来自易患狼疮的MRL-LPR/LPR小鼠的品系和APC将被 用来追求以下具体目标：1.确定是否 间质T细胞代表有限的自体Ag群体-- 特异性浸润性T细胞或由多克隆(随机)T细胞引起 渗透。将使用两种实验策略：分析 TCR受体在肾炎T细胞中的应用及抗原检测 活动性肾炎小鼠肾脏对T细胞培养的要求 肾炎。在前一种方法中，对T细胞子集的TCRV基因进行分析 来自肾炎小鼠肾脏的细胞克隆将是 确定并探测相关TCR序列的特异性 (寡核苷酸和/或单抗)将用于比较TCR基因 间质中的T细胞与脾和脾中的T细胞 患病小鼠的胸腺。后一种方法将使用管状细胞 来自狼疮易感小鼠的品系和专业APC来检查 激活肾炎T细胞所需的银/细胞。 最终，CD44+ARTC将疾病转移到幼稚或 我们将对患病前的小鼠进行评估2.检验以下假设 通过CD4的信号转导受损导致耐受缺陷 小鼠体内出现自身反应性T细胞和组织特异性T细胞 狼疮。这种方法的基本原理是从观察中得出的 CD_4介导的酪氨酸磷酸化异常 MRL-LPR/LPR衍生的CD4+ARTC克隆，这与减少 Lck蛋白的结构性表达。为了进一步评估这一点 异常，此缺陷的性质将使用 ARTC克隆，这些研究的结果将应用于更多 MRL-LPR/LPR小鼠CD44+细胞的常规检测分析将 涉及：评估通过cd4-lck的信号转导，包括 功能LCK活性和LCK转录水平的测定， 以及通过其他T细胞受体传递信号的评估，包括 IL-2R和CD3。 这种方法代表了 之前的提议。关注点从分析 ARTC在B细胞活化中能更直接地评价L细胞 T细胞介导的间质中的分子相互作用 肾炎。方向的转变是由孤立的 与疾病相关的细胞系和我的实验室向 更有利于自身免疫间质分析的环境 肾炎。

The overall aim of this project is to develop a better understanding of the cellular events within the interstitium in murine lupus nephritis. The hypothesis that a subset of self-reactive CD4 T cells escapes normal tolerance and reacts directly, in an Ag-specific manner, with resident cells within the kidney, will be tested. The experimental approach will emphasize analysis of pathologically relevant cells. For this purpose a preexisting panel of autoreactive T cell clones (ARTC), tubular cell lines and APC, derived from lupus-prone MRL-lpr/lpr mice, will be utilized to pursue the following Specific Aims: 1. To determine whether interstitial T cells represent either a restricted population of autoAg-- specific infiltrating T cells or result from polyclonal (random) T cell infiltration. Two experimental strategies will be utilized: analysis of TcR receptor use of nephritogenic T cells, and examination of the Ag requirements for T cells cultured from the kidneys of mice with active nephritis. In the former approach, TcR V gene analysis of a subset of T cell clones derived from the kidneys of nephritic mice will be determined, and probes specific for relevant TcR sequences (oligonucleotides and/or mAb) will be used to compare the TcR gene repertoire of T cells within the interstitium to those in the spleen and thymus of diseased mice. The latter approach will employ tubular cell lines and professional APC derived from lupus-prone mice to examine the Ag/cellular requirements for activation of nephritogenic T cells. Ultimately the capacity of CD44+ ARTC to transfer disease to naive or pre-disease mice will be evaluated 2. To examine the hypothesis that impaired signal transduction through CD4 results in a defect in tolerance and the emergence of autoreactive and tissue specific T cells in murine lupus. The rationale for this approach is derived from the observation that CD4-mediated tyrosine phosphorylation is abnormal in a MRL-lpr/lpr-derived CD4+ ARTC clone, and this is associated with reduced constitutive expression of lck protein. To further evaluate this abnormality, the nature of this defect will be further defined using the ARTC clone, and the results of these studies will be applied to a more general examination of CD44+ cells in MRL-lpr/lpr mice. Analyses will involve: evaluation signal transduction through CD4-lck, including determination of functional lck activity and the level lck transcription, and evaluation of signaling through other T cell receptors, including lL-2R and CD3. This approach represents a natural evolution of the specific aims of the previous proposal. The focused is shifted from analysis of the role of ARTC in B cell activation to a more direct evaluation of the L cellular and molecular interactions involved in T cell-mediated interstitial nephritis. The shift in direction has been facilitated by isolation of the disease-relevant cell lines and the movement of my laboratory to an environment more conducive to the analysis of autoimmune interstitial nephritis.