EFFECT OF CYCLOSPORIN A ON TCR ONTOGENY AND AUTOIMMUNE DISEASE

最近的观察表明，环孢菌素 A (CsA) 可以阻断 TCR 启动 信号转导，从而导致 TCR 库选择异常。 CsA 治疗可导致两种不同的明显自身免疫性疾病 实验模型。 在这两种模型中，疾病的发展 需要来自 CsA 的自身反应细胞的存在 治疗的胸腺和外周 T 细胞的免疫调节异常。 初步数据表明 CsA 抑制 TCR2 的胸腺选择 (alphabeta TCR) 全部功能，但不是 TCR1 的胸腺成熟 （gammadelta TCR）亚系。 这些目标的广泛、长期目标 研究的目的是确定这些不同 T 细胞的生理功能 亚谱系并阐明外周免疫调节的本质 涉及耐受性的活动。 具体目标是测试三个 假设。 第一个假设是 CsA 同时抑制正向和负向 胸腺中TCR2库的选择，但不影响胸腺 TCR1 细胞成熟。 该假设将通过确定 利用一组识别 CsA 的 mAb 对胸腺个体发育的影响 不同的 TCR 和相关分子。 这些研究将涉及 小鼠和鸡 T 细胞发育分析。 重点将是 小鼠中表达特定 Vbeta 基因的细胞的选择 以及 TCR3 子集（一种新型的类似 Alphabeta 的 TCR）的成熟 鸡。 第二个假设是 TCR1 库是在外设中选择的 组织。 这个假设将通过检查发展来检验 以及表达特定 Vgamma 和 TCR1 细胞的组织归巢模式 Vdelta基因。 将使用两种实验方法：1）确定是否 CsA 治疗可抑制特定组织归巢模式 利用某些 Vgamma 基因的 TCR1 细胞的特征。 2） 胸腺内注射 FITC 来识别最近的胸腺迁移 外周组织和确定 Vgamma 在这些组织中的使用模式 细胞。 第三个假设是，在 CsA 诱导的自身免疫性疾病模型中， TCR2 细胞介导自身反应，而 TCR1 细胞则调节疾病 活动。 将研究两种不同的实验模型：1) 新生儿 CsA 加上胸腺切除术和 2) 用同基因 BM 拯救受致命辐射的成年人 移植加钙治疗。 这些模型将被建立并 不同 T 细胞亚系的频率和组织学定位 将通过免疫组织化学染色和 FACS 分析来表征。 在这两种模型中，“正常”T 细胞的存在阻碍了这种能力 自身反应性 T 细胞会诱发实际的组织损伤。 表型为 自身反应性效应细胞和“正常”调节细胞都会 可以使用磁激活细胞分选和过继来确定 传输协议。

Recent observations indicate that cyclosporin A (CsA) blocks TCR initiated signal transduction and thus leads to abnormal TCR repertoire selection. CsA treatment can lead to overt autoimmune disease in two different experimental models. In both these models, the development of disease requires both the presence of autoreactive cells derived from the CsA treated thymus and an immunoregulatory abnormality in peripheral T cells. Preliminary data suggest that CsA inhibits thymic selection of the TCR2 (alphabeta TCR) repertoire, but not thymic maturation of the TCR1 (gammadelta TCR) sublineage. The broad, long-term objectives of these studies are to determine the physiologic function of these different T cell sublineages and to elucidate the nature of the peripheral immunoregulatory activity involved in tolerance. The specific aims are to test three hypotheses. The first hypothesis is that CsA inhibits both positive and negative selection of the TCR2 repertoire in the thymus, but does not affect thymic TCR1 cell maturation. This hypothesis will be tested by determining the effect of CsA on thymic ontogeny utilizing a panel of mAb which recognize different TCRs and associated molecules. These studies will involve analysis of both murine and chicken T cell development. The focus will be on the selection of cells expressing particular Vbeta genes in the mouse and the maturation of the TCR3 subset, a novel alphabeta-like TCR, in the chicken. The second hypothesis is that the TCR1 repertoire is selected in peripheral tissue. This hypothesis will be tested by examination of the development and tissue homing pattern of TCR1 cells expressing particular Vgamma and Vdelta genes. Two experimental approaches will be used: 1) determining if CsA treatment results in inhibition of specific tissue homing patterns characteristic of TCR1 cells which utilize certain Vgamma genes. 2) intrathymic injection of FITC to identify recent thymic emigrants in peripheral tissues and the determining the pattern of Vgamma usage in these cells. The third hypothesis is that in models of CsA induced autoimmune disease, TCR2 cells mediate autoreactivity, while TCR1 cells regulate disease activity. Two different experimental models will studied: 1) neonatal CsA plus thymectomy and 2) lethally irradiated adults rescued with syngeneic BM transplant plus Ca treatment. These models will be established and the frequency and histologic localization of the different T cells sublineages will be characterized by immunohistochemical staining and FACS analysis. In both these models, the presence of "normal" T cells blocks the ability of autoreactive T cells induce actual tissue lesions. The phenotype of both the autoreactive effector cells and the "normal" regulatory cells will be determined using magnetic activated cell sorting and an adoptive transfer protocol.