MOLECULAR ANALYSIS OF HUMAN ANTIGBM ANTIBODIES

该项目的总体目标是更好地了解 在小鼠狼疮性肾炎的肾小球内的细胞事件。 一个自身反应性CD 4 T细胞亚群逃避正常的假设 耐受性，并以Ag特异性方式直接与居民反应， 肾脏内的细胞，将被测试。 实验方法将 强调病理相关细胞的分析。 用于此目的的 预先存在的自身反应性T细胞克隆组（ARTC），肾小管细胞 细胞系和APC，来源于狼疮倾向的MRL-lpr/lpr小鼠，将被 用于实现以下具体目标：1。以确定是否 间质性T细胞代表自身抗原的有限群体， 特异性浸润T细胞或由多克隆（随机）T细胞产生 浸润 将采用两种实验策略： 致肾炎性T细胞的TcR受体使用，以及Ag 对从具有活性的小鼠的肾脏培养的T细胞的要求 肾炎 在前一种方法中，TcRV基因分析的一个子集的T 将从患肾炎的小鼠的肾中获得细胞克隆， 确定，并针对相关TcR序列特异性探针 （寡核苷酸和/或mAb）将用于比较TcR基因 脾内的T细胞库与脾内的T细胞库相比， 病鼠胸腺。 后一种方法将采用管状细胞 细胞系和来自狼疮易感小鼠的专业APC，以检查 致肾炎T细胞活化的Ag/细胞要求。 最终，CD 44 + ARTC将疾病转移到幼稚或 评估疾病前小鼠2.为了检验这个假设， 通过CD 4的信号转导受损导致耐受性缺陷 以及小鼠中自身反应性和组织特异性T细胞的出现 狼疮 这种方法的基本原理来自于观察到的情况， CD 4介导的酪氨酸磷酸化在一个细胞中是异常的， MRL-lpr/lpr衍生的CD 4 + ARTC克隆，并且这与降低的 LCK蛋白的组成型表达。 为了进一步评估这一点 异常，该缺陷的性质将使用 ARTC克隆，这些研究的结果将应用于更多的 MRL-lpr/lpr小鼠中CD 44+细胞的一般检查。 分析将 涉及：通过CD 4-lck评价信号转导，包括 功能性LCK活性和LCK转录水平的测定， 以及通过其他T细胞受体的信号传导的评估，包括 IL-2 R和CD 3。 这一方法代表了联合国具体目标的自然演变， 先前的提议。 重点从分析的作用， ARTC在B细胞活化中能更直接的评价L细胞 以及T细胞介导的间质性 肾炎 隔离促进了方向的转变 疾病相关的细胞系和我的实验室的运动， 环境更有利于分析自身免疫性间质 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.